Myocardial Failure
Editors: G. Rieeker, A. Weber, J. Goodwin Co-Editors: H.-D. Bolte, B. Liideritz,
B.E. Strauer, E. Erdmann
International Symposium, Rottach-EgernlTegernsee, Germany, June 17-19, 1976
Under the auspices of the "European Society of Cardiology"
ISBN-13: 978-3-540-08225-5 e-ISBN-13: 978-3-642-46352-5 DOl: 10.1007/978-3-642-46352-5
Library of Congress Cataloging in Publication Data. Main entry under title: Myocardial failure. (International Boehringer Mannheim symposia) "International symposium,
Rottach-EgernlTegernsee, Germany, June 17-19,1976, under the auspieces of the "European Society of Cardiology". Includes index. 1. Heart failure-Congresses.
2. Heart-Muscle-Diseases-Congresses. 3. Heart-Muscle-Congresses. 4. Muscle contraction­ Congresses. I. Riecker, G., 1926 -. II. European Society of Cardiology. III. Series.
(DNLM: 1. Heart failure, Congestive-Congresses. W3 IN1242KJ v. 11976/WG370 M997 1976) RC682.M93 616.1'2 77-5159.
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Table of Contents
Session I. Molecular Basis of Myocardial Function
Part 1. Regulatory and Contractile Proteins Chairmen: A. Weber and S. V. Perry
A. Weber
Introductory Remarks
The structural Basis of Contraction in Muscle and Its study
2
K. C. Holmes
J. Kendrick-Jones and R. Jakes
Myosin- Linked Regulation: A Chemical Approach 28
J. W. Herzig and J. C. RUegg
Myocardial Cross-Bridge Activity and Its Regulation by Ca++, Phosphate and stretch . . . . . . . . . . . . . . . . . . 41
J. Wikman-Coffelt and D. T. Mason
Myosin Characteristics and Immunological Properties of Myocardial Tissue . . . . . . . . . . . . . . . . . . 52
V
W. Hasselbach
Introductory Remarks
A. M. Katz, D. I. Repke, J. Dunnett, and W. Hasselbach
Relation of Calcium Permeability to the Ca++ Concentration Gradient Across the Sarcoplasmic Reticulum . . . . . . .
N. Briggs, J. Shiner, N. Gleason, F. Bruni, and J. Solaro
Calcium Binding and Cardiac Myofibril Activation. . . . .
S. E. Mayer
Part 3. Membrane-Bound Receptors Chairman: R. J. Lefkowitz
R. J. Lefkowitz
65
70
72
80
90
102
E. Erdmann, W. Krawietz, and P. Presek
Receptor for Cardiac Glycosides. . . . .
H. Glossmann, C.J. Struck, C. Konrad, W. Krawietz, D. Poppert, E. Erdmann, and L. -B. Veil
Adenylate Cyclase Regulation and ~-Adrenergic Receptors in Guinea- Pig Myocardial Tissue. . . . . . . . . . . .
VI
120
132
M. Klingenberg
The Role of the Mitochondrial Adenine Nucleotide Transport in Heart . . . . . . . . . . . . . . . . . . . . . . . . 153
Session ll. Clinical Aspects of Myocardial Failure
Part 1. New Diagnostic Procedures Chairmen: J. - F. Goodwin and G. Riecker
J. -F. Goodwin
Introductory Remarks . . . . . . . . . . . . . . . . . . . 164
R. J. Richardson
V. J. Ferrans
Ultrastructure of Degenerated Muscle Cells in Patients With Cardiac Hypertrophy . . . . . . . . . . . . . . . . . . 185
S. E. Read, M. A. Engle, and J. B. Zabriskie
Humoral and Cellular Studies in Diseases With Heart-Reactive Antibodies . . . . . . . . . . . . . . . . . . . . . . . . 201
P. Hanrath, W. Bleifeld, S. Effert, H. Nowack, and W. Kupper
Relationship Between Pulmonary Artery Pressure and Echo­ cardiographic Mitral Valve Closure in Patients With Acute Myocardial Infarction . . . . . . . . ~ . . . . . . . . . . 209
G. Autenrieth, Ch. Angermann, F. Goss, and H. -D. Bolte
Echocardiographic Evaluation of Myocardial Performance During Infusion of Angiotensin and Handgrip-Exercise . . . 220
Vll
Part 2. Problems of Etiology and Classification Chairman: J. P. Shillingford
J. P. Shillingford
230
R. T. Bulloch and M. B. Pearce
Myocardial Lesions in Cardiomyopathies
Cardiomyopathies Related to Immunological Processes
L. H. Opie
251
266
Session m. Clinical Pharmacology
W. Klaus
Introductory Remarks
K. Greeff
Contraction and Relaxation of Heart Muscle as Influenced by cAMP, Isoproterenol, Glucagon, Ouabain, and Calcium
B. Liideritz, C. Naumann d' Alnoncourt, and G. steinbeck
Direct Effects of Diuretic Drugs on the Myocardium
B. E. strauer and W. Schulze
Circulatory and Contractile Effects of Thyroid Hormones
VIII
292
293
298
311
H. Reuter
Introductory Remarks. . . . . . . . . . . . . . . • • . . 330
R. W. Tsien, R. Weingart, W. J. Lederer, and R. S. Kass
On the Inotropic and Arrhythmogenic Effects of Digitalis.
C.M. Oakley
D. C. Harrison and W. G. Irwin
The Hemodynamic Effects of Antiarrhythmic Drugs on the Depressed Myocardium .............•.. 353
Subject Index. . . . . . . . . . . . . . . . . . . . . . . 369
IX
Introduction
More than 10 years have passed since the memorable symposium on "Myocardial Contractility," edited by R. D. Tanz, F. Kavaler and J. Roberts (New York and London, Academic Press, 1967). PathogeneSis of myocardial failure still involves many questions. The latest scien­ tific findings on fundamentals of myocardial contraction encouraged us to organize this international symposium held in Rottach- Egern at Tegernsee (Germany), June 17 to 19, 1976 sponsored by the European Society of Cardio~ogy. It seemed appropriate to assemble prominent workers in this field in an attempt to correlate their respective in­ formation on cardiac function.
In this connection it must be remembered that our present understand­ ing of the cardiovascular system and today's therapeutic and preventive measures are the fruits of yesterday's research. Further progress in this field will be conditioned by various circumstances: to win highly motivated creative people for clinical research, to mediate time and contacts for their learning new methods, and to provide adequate faci­ lities for scientific work in our hospitals.
Therefore, the aim of the conference was to discuss those aspects of myocardial failure, that are believed to require further studies in the future by integrated efforts of research workers in several disciplines, especially to promote the pertinent exchange of ideas between basic and clinical research.
This book contains all parts of the proceedings of this meeting. The papers have been grouped into different sections:
(1) Molecular Basis of Myocardial Function (2) Sarcoplasmatic Reticulum (3) Membrane-Bound Receptors (4) New Diagnostic Procedures (5) Problems of Etiology and Classification (6) Clinical Pharmacology, and finally (7) Drugs Influencing Myocardial Contractility,
The advice and cooperation of the presidents and chairmen for the planning and the performance of the symposium are gratefully acknowl­ edged. I also express my sincere gratitude to the editorial staff for
x
their part in the conduct of this symposium and in preparing this book. Concerning the generous support of this conference we express our gratefulness to the organizer of the symposium, Boehringer-Mannheim, who brushed aside all economic obstacles to promote this meeting. It is obvious, that Macaenas, the patron, has not become extinct. I should like to thank all contributors, busy people who nevertheless promptly submitted their manuscript, answered many queries and kindly accepted suggested changes. We are particularly grateful to Springer-Verlag who so efficiently made all the necessary arrangements for this edition.
G. Riecker
Autenrieth, G., Dr. med. Medizinische Klinik I, Klinikum GroBhadern, MarchioninistraBe 15, D-8000 Miinchen 70
Bolte, H. -D., Prof. Dr. med. Medizinische Klinik I, Klinikum GroBhadern, MarchioninistraBe 15, D- 8000 Miinchen 70
Briggs, N., M. D. Prof. Department of Physiology, Medical College of Virginia Hospital, MCV station, Richmond, VA 23298/USA
Bulloch, R. T., M. D. Prof. Section of Pathology, National Heart and Lung Institute, Building 10A/ Room 3E30, National Institutes of Health, Bethesda, MD 20014/USA
Dengler, H. J., Prof. Dr. med. Medizinische Universitats-Klinik, Venusberg, D-5300 Bonn 1
ErdmalUl, E., Dr. med. Medizinische Klinik I, Klinikum GroBhadern, MarchioninistraBe 15, D- 8000 Miinchen 70
Ferrans, V.J., M.D. Prof. Section of Pathology, National Heart and Lung Institute, National Insti­ tutes of Health, Bethesda, MD 20014/USA
Glossmann, H., Prof., Dr. med. Pharmakologisches Institut der Universitat, SchubertstraBe 1, D-6300 GieBen
Gold, H. K., M. D. Prof. General Hospital, I. S. A. Phillips 2, Boston, MA 02114/USA
Goodwin, J. F., M.D., F. R. C. P. Prof. Royal Postgraduate Medical School, University of London, Hammersmith Hospital, Ducane Road, London W12, England
Hanrath, P., Dr. med. Abteilung llUlere Medizin I, Rheinisch-Westfalische Technische Hoch­ schule, GoethestraBe 27/29, D-5100 Aachen
XII
Harrison, D. C., M. D. Prof. Cardiology Division, School of Medicine, stanford University, stanford, CA 94305/USA
Hasselbach, W., Prof. Dr. med. Max-Planck-Institut fUr Medizinische Forschung, Abteilung Physiologie, JahnstraBe 29, D-6900 Heidelberg 1
Herzig, J. W., Dr. med. II. Physiologisches Institut der Universitat, 1m Neuenheimer Feld 326, D-6900 Heidelberg
Holmes, K. C., Prof. Dr. med. Max- Planck- Institut fUr Medizinische Forschung, Abteilung Biophysik, JahnstraBe 29, D-6900 Heidelberg
Huxley, H. E., M. D., Prof. MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, England
Katz, A. M., Dr. med., Prof. Division of Cardiology, Department of Medicine, Mount Sinai School of Medicine, 100th and Fifth Avenue, New York, NY 10029/USA
Kendrick-Jones, J., M. D., Prof. MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, England
Klaus, W., Prof. Dr. med. Pharmakologisches Institut der Universitat, Gleueler StraBe 24, D-5000 KOln 41
Klingenberg, M., Prof. Dr. med. Institut fUr Physiologische Chemie und Physikalische Biochemie der Universitat, GoethestraBe 33, D-BOOO Miinchen 2
Lefkowitz, R. J., M. D. Prof. Department of MediCine, Duke University Medical Center, P. O. Box 3325, Durham, NC 27710/USA
Loogen, F., Prof. Dr. med. 1. Medizinische Klinik B der Universitat, MoorenstraBe 5, D-4000 DUs­ seldorf
LUderitz, B., Priv. -Doz. Dr. med. Medizinische Klinik I, Klinikum GroBhadern, MarchioninistraBe 15, D- BOOO Miinchen 70
Mayer, St.E., M.D. Prof. Division of Pharmacology UCSD, 2042 BSB, La Jolla, CA 92093/USA
Oakley, C. M., M. D. Prof. Royal Postgraduate Medical School, Hammersmith Hospital, London Wl2, England
xm
Olsen, E. G. J., M. D., Prof. National Heart Hospital, Westmoreland Street, London WIM 8BA, England
Opie, L. H., M. D. Prof. Department of Medicine, Groote Schuur Hospital, Cape Town, South Africa
Perry, S.V., M.D. Prof. Department of Biochemistry, University of Birmingham, P. O. Box 363, Birmingham B15 2TT, England
Read, S. E., M. D., Ph. D., Prof. The Rockefeller University, 1230 York Avenue, New York, NY 10021/ USA
Reuter, H., Prof. Dr. med. Pharmakologisches Institut der Universitat, FriedbUhlstraBe 49, CH-3008 Bern
Richardson, P. J., M. D. Prof. King's College Hospital, Denmark Hill, London SE5 9RS, England
Riecker, G., Prof. Dr. med. Medizinische KIinik I, Klinikum GroBhadern, MarchioninistraBe 15, D- 8000 MUnchen 70
Schoner, W., Prof. Dr. med. Institut fUr Biochemie und Endokrinologie des Fachbereichs Veterinar­ medizin, Frankfurter StraBe 100, D-6300 GieBen
Shillingford, J.P., M.D. Prof. Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12, England
Strauer, B. E., Priv. -Doz. Dr. med. Medizinische Klinik I, KIinikum GroBhadern, MarchioninistraBe 15, D- 8000 MUnchen 70
Tsien, R. W., M. D., Prof. Yale School of Medicine, Department of Physiology; 333, Cedar Street, New Haven, CT 06510/USA
Weber, A., M. D., Ph. D., Prof. University of Pennsylvania, School of Medicine, Department of Bio­ chemistry, Philadelphia, PA 19174/USA
Wikman-Coffelt, J., Ph.D., Prof. University of California, Davis Section of Cardiovascular Medicine, School of Medicine, Department of Internal Medicine, Davis, CA 95616/ USA
XIV
Molecular Basis of Myocardial Function
Part 1. Regulatory and Contractile Proteins Chairmen: A. WEBER and S. V. PERRY
Introductory Remarks
A. WEBER
Molecular biologists have been assembled at this meeting together with clinical cardiologists because of our desire to gain a complete under­ standing of heart disease. As yet there is still a great gap in our knowledge of clinical manifestations and therapy and our information about changes in the molecular biology of the proteins involved in the contractile process.
During the first part of the conference we shall hear about the mole­ cular biology of muscle in general and heart muscle fibers and proteins in particular.
Dr. H. E. Huxley will remind us that muscle contracts without any length change in the filamentous substructure as a result of the sliding of the myosin and actin filaments past each other, a process driven by the energy of ATP hydrolysis. Although this much has now been known for some time, evidence concerning many of the details of the reactions has eluded us so far. For instance, we have assumed that myosin bridges move the actin filaments by attaching to them at right angles and then swinging towards the center of the sarcomer over a distance of about 70-100 A before letting go again of the actin filament. While such 11 rowingl1 along of the actin filament seems plausible nobody ever had demonstrated that myosin can bind to actin at right angles. Dr. K. Holmes will discuss some recent data derived from electron microscopy and X- ray diffraction, using A TP analogues rather than A TP, which show just that: myosin attachment to actin at 90 0 • Dr. Huxley will describe to us certain new technical developments which should allow us to learn more about the movements of the myosin bridges during contraction. For the first time X-ray diffraction is being used to follow rapid structural changes as fast as they occur during muscle contraction. This is possible now because X-ray photons are registered by position­ sensitive counters rather than film, and the power of X-ray beams has been greatly increased. During contraction there occur changes in the X-ray diffraction pattern that have been assigned to a movement of the bridges towards the actin filament and away from the myosin filament. The first question that can be answered as a result of the new technical advances is: are these bridges that apparently move out in fact attached to the actin filament? Dr. Huxley expects an answer from a comparison of the time course of tension development and the change in the diffraction pattern.
2
Dr. Kendrick-Jones addresses himself to the control of contraction by calcium with special attention to mechanisms built into the myosin mole­ cule. Myosin-linked control was first discovered by him and A. G. Szent-Gyorgyi in scallop muscles. Later it was found that many muscles possessed a double control mechanism: during rest, in the absence of calcium inactivation by troponin as well as inactivation of myosin by the regulating myosin light chains. Although there is no direct evidence yet for myosin-linked control in vertebrate skeletal and cardiac muscle the tantalizing fact exists that these muscles possess calcium-binding light chains that are similar to the invertebrate-regulating light chains, and that these vertebrate light chains can substitute for the invertebrate ones in excercising control over invertebrate myosin. Dr. Kendrick Jones has obtained a great deal of information about the invertebrate regulatory light chains, including their primary sequence and is viewing them in comparison with the vertebrate calcium binding myosin light chains and other regulatory calcium binding proteins.
With Dr. Herzig we move a more or less direct viewing of myosin bridges to that of more physiological parameters such as tension devel­ opment, stiffness and A TP hydrolysis in cardiac fibers. By using fibers with permeable membranes rather than living fibers the physio­ logical response to calcium and phosphate ions could be explored.
Lastly, Dr. Wikman-Coffelt presents data where she studied myosin A TPase activity, calcium binding and light chain content in myosin from left and right ventricles of dogs with surgically induced pulmonary and aortic stenosis. She observed a number of changes in thz myosin mole­ cule in response to stress, some of them quite remarkable, such as an increase in the number of calcium-binding light chains per myosin molecule.
3
The Structural Basis of Contraction in Muscle and Its Study by Rapid X-Ray Diffraction Methods
H. E. HUXLEY and J. C. HASELGROVE
By way of introduction to this part of the symposium, we think we should first describe very briefly the basic features of the contr.actile structure of muscle, as far as we know them at present. These features are virtually the same throughout all types of striated muscle including heart muscle. For convenience of experimentation, a considerable amount of the structural work has been carried out using certain skeletal muscles of the frog or rabbit, but there are of course very good reasons to believe that the conclusions about the mechanisms derived from such studies will be of general application.
The contractile myofibrils are built up from alternating and partially overlapping arrays of longitudinally oriented actin and myosin filaments, and it is now generally accepted that changes in muscle length, whether active or passive, take place by a process in which the filaments remain virtually constant in length, but change their extent of overlap. This basic model was originally proposed in 1954, independently, by A. F. Huxley and R. Niedergerke (4) and by H. E. Huxley and the late Jean Hanson (15). The sliding force between the actin and myosin filaments is believed to be generated by cross-bridges projecting outwards from the myosin filaments, attaching in a cyclical fashion to actin, as suggested by Hanson and Huxley in 1955, (23) splitting ATP as they do so and thereby releasing the energy for contraction. These cross-bridges represent the enzymatically active parts of the myosin molecule.
Myosin is a molecule having a very remarkable structure (17). Basically it contains two very large polypeptide chains of molecular weight about 200,000 daltons each (the "heavy chains") and four smaller polypeptide chains having molecular weights in the 20,000 daltons range. Along part of their length, the two heavy chains are coiled around each other to form a 2-chain a-helical coiled-coil structure about 1400 A in length and 20 A in diameter. About one-half of each heavy chain is involved in this structue. The rest of each heavy chain is folded up separately in a globular form, together with some or all of the light chains. The two heavy chains are of very similar aminoacid sequence, and are arranged in parallel to each other with the same polarity so that the two globular regions are located at the same end of the molecule. The a-helical portions of the myosin molecules are involved in forming the
4
I I I I I I I I I I II -----One so rcomere----~I I II I I I I I I
I II I I I I I I I II I I I I I II I I- I bond -------0 ·1 • A bond • 1-- I bon d -.-I I " I I I I I , I I I I I I I I I I II I I II I I I I I I i I I I II I I I I II I I I I I , ..... H zone ~, I I : I II I I I I I I II I II I I I I I I I I
~ M Ii", ~ II I I
Z line
Fig. 1. Electron micrograph and diagrammatic representation of con­ struction of striated muscle from overlapping arrays of thick (myosin) and thin (actin-tropomyosin-troponin) filaments. Sliding force between filaments is generated by repetitve cyclic movement of cross-bridges. Attachment of cross-bridges to thin filaments is blocked by regulatory system when muscle is switched off
backbone of the thick filaments, by side-to-side bonding along part of their length with their neighbours, whereas the globular regions - known as the 81 - subunits -, which have on them the sites for splitting A TP and combining with actin, project out sideways from the thick filaments and form the cross-bridges. Present evidence indicates that a portion of the rod part of myosin (know as the 82- region) can hinge out sideways from the backbone of the thick filaments so as to allow the 'head' or '81' subunits to attach to the actin filaments alongside, whose sidespacing from the myosin filament backbone varies somewhat according to muscle length. The thick filaments are about 1. 6 ~ in length and lie about 400 A apart. Each of them contains about 250-300 myosin molecules, which corresponds to a concentration of approximately 10-4 M.
5
The thin filaments contain actin, a protein of molecular weight of about 42,000 which forms globular units approximately 50 A in diameter which in turn assemble into filaments composed of two helically wound strings of the G-actin units. The thin filaments in vertebrate striated muscle also contain the regulatory proteins troponin and tropomyosin, which are involved in switching the actin-myosin interaction on or off in response to changes in calcium concentration.
In a resting muscle, the cross-bridges are not attached to actin, the filaments can slide past each other readily under an external force, and the muscle is plastic and readily extensible. When a muscle contracts, it is supposed that any particular cross-bridge will first attach to actin on one configuration, in an approximately perpendicular orientation and then, while still attached, will undergo some configurational change so that its effective angle of attachment alters, i. e. it 'swings' or 'tilts' in such a direction as to pull the actin filament along to the direction of the centre of the A-band (13). When this movement is complete - the extent of movement probably being 50-100 A - the cross-bridge can be detached from actin by the binding to it of another molecule of A TP (whose first effect is to dissociate actin and myosin). The A TP is then split by the myosin head, while still uncombined with actin (as indicated by the work of Lymn and Taylor (18)), but the reaction products remain attached to the enzyme, with the complex probably in a 'strained' state, until the enzyme once more attaches to actin and releases the stored energy in the form of mechanical work. The combined effect of all the cross-bridges undergoing these asynchronous cycles of attachment, pulling and detachment is to produce a steady sliding force which will continue as long as the muscle is active. It will be realised that this type of mechanism depends on a very specific interaction between actin and myosin molecules and therefore requires that they be built into the structure with very specifiC orientations. It is found in practice, from electron microscope observations (11), that the myosin molecules along one-half of the length of each thick filament (and hence in one-half A-band) are all oriented with their' tails' pointing in one direction (towards the centre of the A-band). In the other half of the filament, this polarity is reversed, so that the tails again point towards the centre. Such an arrangement would ensure that all the elements of force generated by individual cross-bridges will add up in the proper direction. Similarly, aU the actin monomers along each thin filament have the same structural polarity (which reverses at the Z-lines) so that they will all be able to interact with myosin cross-bridges in identical fashion. Indeed, one might regard one of the most essential features of the structure of muscle as being the organisation of all the individual interacting molecules in a large body of tissue so that they act in a concerted fashion.
6
Actin
Myosin
Myosin
Fig. 2. Active change in angle of attachment of cross-bridges (81 sub­ units) to actin filaments could produce relative sliding movement between filaments maintained at constant lateral separation (for small changes in muscle length) by long range force balance. Bridges can act asynchronously since subunit and helical periodicities differ in the actin and myosin filaments
Above: Left hand bridge has just attached; other bridge is already partly tilted
Below: Left hand bridge has just come to end of its working stroke; other bridge has already detached, and will probably not be able to attach to this actin filament again until further sliding brings helically arranged sites on actin into favourable orientation
The central problem in understanding the mechanism of muscular con­ traction - and of many other motile processes, too, which employ actin and myosin-like proteins - is to understand the detailed functioning of the cross-bridge mechanism. To do this, we need to know a good deal
7
about the structure of the cross-bridges and about how that structure changes during their operation. Because of the submicroscopic size of the bridges, it is a considerable technical challenge to obtain this kind of information.
The existence of some form of lateral cross-connection between separated filaments of myosin and of actin was originally proposed (7) in order to account for the greatly increased resistance to stretch exhibited by a muscle in rigor, and this argument still remains a very powerful one. The visualisation of cross-bridges in electron micrographs of muscle was first cescribed in 1953 (8) and their appearance was shown rather more clearly a few years later (16, 10); the actual images of cross­ bridges in muscle have not improved much since that time. They appear as projections originating on the thick filaments, having a diameter of about 50 A and a length of about 100-150 A, extending out towards the thin filaments and attached to them in muscles in rigor. In insect in­ direct flight muscle in rigor they can be seen to be attached to the actin filaments in a characteristically tilted configuration, the tilt being in such a direction as to move the attached end of the cross-bridge towards the centre of the A-band. The attachment of 'free' myosin heads can be examined in the electron microscope by using the negative staining technique to examine actin filaments' decorated' with myosin subfragment 1 (Sl) in absence of nucleotide (11; 19) and a tilted form of attachment is again apparent. It is very likely, therefore, that this corresponds to the configuration adopted by the cross-bridge at the end of its working stroke, when ADP and P1 have been released, and when no further force is being exerted. The X-ray and EM evidence indicate that bridges in relaxed muscle are approximately perpendicular to the filament axiS, so the simplest supposition would be that they attached to actin in this configuration at the beginning of their working· stroke. The extent of movement per stroke that this would imply (based simply on geometric considerations) is about 70-80 A, which accords well with the values obtained by Huxley and Simmons (5, 6) from observations on rapid mechanical transients in muscle. The return stroke of the cycle does not necessarily require much energy to be expended - it could simply correspond to return to the equilibrium configuration adopted by a cross-bridge when carrying ATP or its immediate split products. It should be recalled that even in the absence of actin (in a very stretched muscle) the cross-bridges lose their regular arrangement in the absence of ATP and it seems likely, but has not been established, that under these conditions they no longer adopt a constant and approxi­ mately perpendicular angle of tilt.
One of the best, though by no means straightforward methods of investigating the nature and behaviour of the cross-bridges is by low­ angle X-ray diffraction, since a large part of the diagram comes from the cross-bridges themselves, and since it is possible to study a muscle by this technique under almost normal working conditions. The disad­ vantage of this technique - besides the inherent and well-known ambiguities of X-ray diagrams! - is that the reflections from muscle are rather weak (about one millionth as strong a§ the direct beam) -
8
and it has therefore been necessary to invest a considerable amount of time and effort into the technical innovations required to record the patterns suffiCiently rapidly.
In the diagram from a resting muscle there is a well-developed system of layer lines with a 429 A axial repeat and a strong third order meridional repeat at 143 A (14, 1). This pattern arises from a regular helical arrangement of cross-bridges on the thick filaments, with groups of cross-bridges occurring at intervals of 143 A along the length of the filaments with a helical repeat of 429 A. The number of cross-bridges in each group is not yet absolutely certain, but it is more likely to be three than two (21).
Diagrams from contracting muscle may be recorded on film using a shutter so as to transmit the X-ray beam only when the muscle is being stimulated. A long series of tetani is necessary, with· intervals in between them for recovery - usually 1- s tetani and 2- min intervals. Such diagrams show that the whole pattern becomes very much weaker during contraction, indicating that the cross-bridges are much less regularly arranged, as would be expected if they were undergoing asyn­ chronous longitudinal or tilting movements (and possibly lateral ones too) during their tension- generating cycles of attachment to actin. If this is indeed the case, then the very rapid development of the active state of a muscle following stimulation should be accompanied by an equally rapid decrease in intensity of the layer line pattern.
The equatorial part of the X-ray diagrams from muscles is also very informative. It is generated by the regular side-by- side hexagonal lattice in which the filaments are arranged, and the relative intensity of the two principle reflections is strongly influenced by the lateral position of the cross-bridges. Large changes occur as between resting muscle and muscle in rigor (7, 9, 12) when a high proportion, if not all, of the cross-bridges will be attached to actin. These have been interpreted as indicating that in a resting muscle, the cross-bridges lie relatively closer to the backbone of the thick filaments, whereas when they attach to actin they hinge further out and lie with their centres of mass nearer to the axes of the actin filaments at the trigonal positions of the hexagonal lattice.
Similar changes have been observed in contracting muscles by us, though the extent of change is less and would correspond to about half the cross-bridges being in the vicinity of the actin filaments at anyone time. Again, the observations are consistent with a model in which the cross-bridges are undergoing a mechanical cycle of attachment to and detachment from the actin filaments during contraction. However, it should be appreciated that the parameter that is being measured is the average lateral position of the cross-bridges and this provides no direct evidence concerning the proportion attached, or even indeed whether any are attached at all. It does not follow - indeed it is very unlikely - that the average angle of attachment of the cross-bridges to actin is the same during contraction as it is in rigor. and this angle will affect the posi­ tion of the centre of mass of the cross-bridge relative to the axis of
9
the actin filament. So the proportion attached may be more than 50%; or it may be less if some cross-bridges lie near to actin but are not themselves attached. Nevertheless, the changes do indicate that a substantial lateral movement of the cross-bridges takes place during contraction and, as in the case the layer-line changes, it is important to establish whether the movement occurs at a sufficiently rapid rate for it to arise from a force-generating attachment of cross-bridges.
From the above discussion, it can be seen that the X-ray diffraction technique does offer a method by which structural information can be obtained while the tissue is still intact and functioning. In order to exploit this possibility to the full, it would be desirable to make the measurements with a time resolution which matches the speed at which the structural changes take place. In a frog sartorius muscle near OOC, the active state seems to become fully developed in considerably less than 100 ms after stimulation, and so to follow its onset with reasonable accuracy a time resolution at least as good as 10 ms is necessary. A.gain, if the 'working stroke' of a cross-bridge is 75 A and if a muscle is shortening at a rate of one muscle length cEer second (again a typical figure for frog sartorius muscle at 0 C) then a given cross­ bridge would be engaged in one cycle for about 7 ms. Thus, to follow structural 'transients' associated with changes in the number of attached cross-bridges would again call for a time resolution of better than 10 ms.
This does not mean that the X-ray diagram has to be recorded within such a short interval of time. It is perfectly practical to repeat the stimulation of such a muscle up to 1000 times before significant fatigue has developed, so that the actual available time to record the signal is 1000 x 10 ms = 10 s. Nevertheless, until recently even the strongest parts of the muscle X-ray diagram took 15-20 min to record on film, so a gain by at least two orders of magnitude was required before the time-course experiments could be attempted. This gain in speed has now been achieved, as a consequence of a numher of changes and improve­ ments in the X-ray cameras. It would be inappropriate to discuss the technical details of these here, but basically there has been one major change and a number of significant improvements.
The X-ray patterns are now recorded by X-ray photon counters, rather than film, a technique first used on muscle by Tregear and Miller (22). In a typical experiment, about 40,000 counts might be recorded in a 10 ms time (repeated 1000 times) interval from an equatorial reflection, using slit geometry so that the counter was recording from an area about 15 mm x O. 3 mm. This number of counts would give a statistical accuracy of 1/2%. However, to record the same pattern on film with an optical density of unity would require approximately 100 times as many counts (one developed grain per square micron). The use of counters - both proportional and position sensitive - also leads to an enormous increase in the ease and efficiency of data handling, since the counts can be fed into and stored within successive time channels in a multichannel scalar, and all the data generated during the whole
10
period of activity of the muscle can be utilized - for example, 128 channels of 10 ms, covering a period of 1. 28 s. The scalar is syn­ chronized so that the sweep through the time channels always begins at exactly the same length of time before the stimulus of the muscle. Thus during the recording 0 f the pattern during one single twitch, each channel might accumulate 40-80 counts, which would be insufficient to detect, let alone accurately measure, the change in the intensity of the X-ray reflection (which is superimposed on a rather high background, so that the percentage change in the total X-ray signal is small, often of the order of 5-10%). However, after 1000 twitches synchronized to the time scan, the intensity of the X-ray signal at that particular moment in the contractile cycle can be determined with an accuracy of the order of 0.5-0.35% and the intensity of the X-ray reflection itself with an accuracy of a few percent.
Other improvements which have contributed substantially to the gain in speed have included the development of a 18" diameter rotating anode X-ray tube which is used in a pulsed mode so that the electron beam is kept at a moderate intensity during most of the 1- or 2-min cycle time (which provides resting periods between the intervals at which the muscle is stimulated) and is switched up to high intensity during the experimental period.
Since our initial studies have been either of the equatorial or meridional reflections, it has been possible to record these using slit geometry (i. e. as opposed to 'pinhole-type' or 'double-focussing' collimation). The use of long slits perpendicular to the direction in which higher resolution is required (whether it be along the meridian or the equator) further increases the total number of counts available. Moreover, the use of slit geometry means that the focal spot on the X- ray tube needs to be very small in one direction only, so that the total power loading on it can be increased to the point where the limiting factor is not the instantaneous loading of the surface, but the total power that can be fed through the anode. Under these conditions, it is advantageous to reduce the duty cycle of the X-ray tube.
A further gain in intensity has been obtained by using, in the normal mirror-monochromator configuration, a double mirror (14) with 20-cm­ long reflecting elements, not to produce a focussed beam in a direction at right angles to the monochromator focussing direction but to collect X-rays over a wider range of angles from the X-ray tube focus and to reflect them into the aperture of the counter.
In these ways a counting rate of 5 x 108 counts per second in the direct beam has been achieved, and by collecting the reflected beam on both sides of the origin simultaneously, counting rates of up to 10,000 counts per second, for example, on the (10) equatorial reflection from frog sartorius muscles, have been recorded.
Other teclmical developments have been concerned with the employment of position-sensitive X-ray detectors to collect data more efficiently. These are devices which can record the whole of a one-dimensional (or even a two-dimensional in recent developments) X-ray pattern
11
simultaneously with a spatial resolution of 100 or 200 !.I.. Thus, on the muscle X- ray diagram, both the (10) and (11) reflections and the back­ ground can be recorded simultaneously. By suitable electronic means (2) the entire equatorial diagram (for example) can be recorded during chosen phases of the contraction cycle of a muscle - for example at rest before stimulation, then during isometric contraction, then during a controlled amount of shortening against a chosen load, and then during isometric contraction at the shortened length. A similar procedure, together with some results, has been described by Podolsky, st. Onge, Yu and Lymn (20). The use of multichannel scalars with larger storage capacity now also makes it possible to record the entire X- ray diagram from a position-sensitive counter at 10-ms intervals during contraction.
The purpose for which these rapid recording techniques have been developed is to put our knowledge of cross-bridge behaviour during contraction on a much surer and more detailed footing. Whilst a great many lines of evidence support the hypothesis that muscle tension is developed by cross-bridges attaching to actin in a cyclical fashion and developing a longitudinal sliding force as a result of structural changes in the myosin-actin complex accompanying certain stages of the bio­ chemical cycle in which A TP is split, many of these arguments are somewhat indirect ones (see for example Huxley, (13». That is, while there is very good evidence from enzyme kinetics that actin activates myosin A TPase by combining with the myosin at one stage of the enzy­ matic cycle, and while there is very good structural evidence that myosin heads can indeed physically attach to actin filaments in the absence of ATP, the structural evidence concerning attachment of cross-bridges to actin during the contraction of a muscle is rather less decisive. The axial X-ray pattern during contraction shows only an increased lOngitudinal disorder of the cross-bridges - compatible with but not proving an asynchronous attachment of cross-bridges to actin filaments having different axial periodicities to those of the myosin filaments. The equatorial pattern during contraction does indeed show positive new features, in particular a large increase in the intensity of the (11) reflection, but these provide evidence for lateral movement of the cross­ bridges rather than for actual attachment to actin.
This type of evidence would be very much strengthened if it could be shown that the changes in X- ray patte:rn which have been observed are not merely present in a fully active muscle, but that the time course with which they occur follows what we would expect from the observed time course of tension development and decay, given reasonable models of cross-bridge action. Accordingly, we have investigated the behaviour of both the equatorial reflections and the 143 A meridional reflection during contraction, in particular during the early stages of tension development during isometric twitches.
In the case of the equatorial reflections, the problem is complicated by the fact that internal shortening of the muscle takes place, even at constant overall muscle length, due to stretching of series elastic ele­ ments as tension is developed. This shortening takes place predominantly very early during tension development, because of the non-linear
12
behaviour of the series elasticity. Increases in the extent of overlap of the filaments even in relaxed muscles produce changes in the equatorial reflections which are in the same direction as those presumed due to attachment of cross-bridges to actin, i. e. decrease of (10) and increase and increase of (11). At the same time, the decrease in muscle length (of the order of a few percent.) increases the amount of material in the X- ray beam and so leads to an increase in the intensity of both reflections and of the continuous background scattering produced by the muscle. These two effects are additive in the case of the (11) reflection but tend to cancel out in the case of the (10), which, when it is recorded together with the background using a slit camera, always increases in intensity very slightly with decreasing muscle length, by an amount which is small (..., 10% or less) compared to the decrease in intensity accom­ panying activation itself.
Thus measurements of the time course of the change in the (10) intensity will give values which are slightly slower than the changes produced by activation itself (aside from internal shortening) whilst measurements of the (11) time course will tend to overestimate the speed of the change.
In practice, we find that large changes do indeed take place in both reflections very early on in a twitch, of the character expected for cross-bridge attachment, and of a much greater magnitude than those associated with internal shortening, and that these changes are reversed as the muscle relaxes. In one series of experiments on frog sartorius muscles at 20 C in which the time to half maximum tension was on average 49.5 ms, the decrease in intensity in the (10) reflection was first detectable about 20 ms after stimulation, and reached its half maximum value at an average time of 38.5 ms, i. e. considerably ahead of the tension half-maximum time. In another series of experiments using the oosition- sensitive counter in which the time courses of the (10) and (11) reflections were compared. it was found that the time at which the increase in intensity of the (11) reflection was half complete was on average about 5 ms earlier than that of the (10) change, which in turn was approximately 15 ms ahead of the tension half time.
The conclusions which we draw from these experiments are that the structural changes in muscle evidenced by the changes in the low-angle equatorial X-ray diagram following stimulation do indeed occur fast enough for them to be associated directly with the development of the capacity of the muscle to exert tension. The changes do not take place abruptly at the start of activity, and indeed during the latent period (say up to 15 ms after stimulus) the pattern remains unchanged. However, the changes do occur at a rate appreciably greater than the rate at which tension is developed. These are several possible explanations for this effect and we cannot distinguish between them at present. If we assume (as seems most likely) that there is no direct activating effect of calcium on the myosin filaments themselves, and that the changes we observe are associated with cross-bridges (which presumably are always moving about under Brownian motion) attaching to actin and so altering their average position, then:
13
(1) There may be a significant delay between the attachment of a cross­ bridge and the production of tension by it.
(2) During the early stages of tension development, when quite rapid internal shortening is taking place, the proportion of cross-bridges carried into the negative-tension developing region (3) may be larger than envisaged in that model, so that the deficit between proportion of maximum tension developed and proportion of bridges attached is larger than expected.
(3) Since bridges which remain attached but have ceased to develop tension may be attached at a higher degree of tilt and with their centres of mass nearer to the actin on average than tension-devel­ oping ones, the former may contribute disproportionately to the changes in the X-ray pattern.
(4) The attachment of some bridges to actin or the activation of actin itself may alter the ionic environment between the filaments in such a way as to allow the remaining unattached ones to move nearer to the actin filaments.
(5) The attachment of some bridges to actin may stabilize the actin filaments in positions closer to the trigonal positions of the lattice.
(6) CrOSS-bridges which have already been through one cycle of action may remain near to the actin filaments.
Several of these possibilities are quite interesting ones, but a great deal more work will be necessary to distinguish between them. For the present, we can only re-iterate that the X- ray changes take place rapidly enough to be associated with cross-bridge movement producing tension.
Observations of the time course of the changes in the 143 A meridional reflection from frog sartorius muscle show that these too occur very early on in a twitch. For technical reasons, we carried out these experiments at lOoC. The decrease in intensity of this reflection (which falls to about half its resting value at the peak of contraction) begins about 10-15 ms after stimulation and the change is half complete in approximately 20 ms. At this temperature, the time to reach half maximum tension is approximately 35 ms. Thus here too the changes in structure certainly occur early enough to reflect changes in the position and ordering of the cross-bridges as they attach to actin and develop tension. But once again, the results indicate that the extent of change exceeds that expected, on a simple model, from the proportion of cross-bridges generating tension.
In the case of the meridional 143 A reflection, the return to the resting configuration is appreciably slower than the decay of isometric tension and indeed the change from the resting intensity is often at its greatest value part way through relaxation. This suggests that there is a signi­ ficant delay between the detachment of a cross-bridge and its return to its appropriate position in the myosin filament helix. Such an effect might also account for the excess of 'disordered' bridges during the onset of tension, since the number detected would correspond to the cumulative total of bridges which had interacted, not the number attached to actin at the time in question. 14
Thus we see that while this approach provides quite dramatic confirmation of the general features of the moving cross-bridge model of muscle contraction, the detailed interpretation of the results is still in its early stages, and, like the earlier X-ray studies on muscle (7, 9) will probably require a good deal of evidence from other fields before 'we begin to understand it properly.
References
3. Huxley,
4. Huxley,
5. Huxley,
G. F., Lowy, J., Millman, B. M.: J. molec. BioI. 25, 31
W.: Proc. 2nd ISPRA Nuclear Elect. Symp. pp. 199-204,
A. F.: Progr. biophys. & biochem. CytoI. 1, 255 (1957)
A. F., Niedergerke, R.: Nature (Lond.) 173, 971 (1953)
A. F., Simmons, R. M.: Nature (Lond.) 233, 533 (1971)
6. Huxley, A. F., Simmons, R. M. Cold Spr. Harb. Symp. quant. BioI. 37 (1972)
7. Huxley, H. E.: Ph. D. TheSiS, University of Cambridge. (1952)
8. Huxley, H. E.: Biochim. biophys. Acta 12, 387 (1953a)
9. Huxley, H. E.: Proc. roy. Soc. B141, 59 (1953b)
10. Huxley, H. E.: J. biophys. biochem. CytoI. ~, 631 (1957)
11. Huxley, H. E.: J. molec. BioI. 1, 281 (1963)
12. Huxley, H. E.: J. molec. BioI. 37, 507 (1968)
13. Huxley, H. E.: Science 164, 1356 (1969)
14. Huxley, H. E., Brown, W.: J. molec. BioI. 30, 383 (1967)
15. Huxley, H. E., Hanson, J.: Nature (Lond.) 173, 973 (~954)
16. Huxley, H. E., Hanson, J.: Proc. 1st Europ. Regional Conf. Elect. Micro. Stockholm, pp. 260 (1956)
17. Lowey, S., Slater, H. S., Weeds, A. G., Bakev, H.: J. molec. BioI. 42, 1 (1969)
18. Lymn, R. W., Taylor, E. W.: Biochemistry~, 2975 (1970)
19. Moore, P. B., Huxley, H. E., DeRosier, D. J.: J. molec. BioI. 50, 279 (1970)
20. Podolsky, R. J., St. Onge, R., Yu, L., Lymn, R. W. Proc. nat. Acad. Sci. (Wash.) 73, 813 (1976)
21. Squire, J. M.: J. molec. BioI. 72, 291 (1973)
22. Tregear, R. T., Miller, A.: Nature (Lond.) 222, 1184 (1969)
23. Hanson J. Huxley, H. E.: Symp. Soc. Exp. BioI. ~ 228 (1955)
15
K. C. HOLMES *
Introduction
Since the discovery of the myosin cross-bridge (10) it has been widely assumed that such a structure is responsible for generating force between the thick and thin filaments (7, 13). In order to accommodate
Fig. 1. A possible four-state cross-bridge cycle (18) based on the kinetic studies of Lymn and Taylor and the structural studies summarized by H. E. Huxley (13). This cycle was first suggested by Lymn and Taylor (17). The objec~ on the left of each part of the diagram represents the S2 tail and S1 head of a heavy meromyosin molecule which is joined to the myosin filament at the base of the S2 rod. The S1 head depicted as a single head contains the ATPase. The actin filament is shown on the right. The rotation of the bridge while attached to actin moves the actin filament along. Probably ADP is released during this part of the cycle. The binding of ATP to the now empty nucleotide binding site releases the bridge and restores it to the right-angled conformation. During this process ATP is hydrolysed: concomitantly the affinity for actin is raised leading to a rebinding and repeat of the cycle
* I am obliged to my colleagues, particularly Drs. Barrington Leigh, Goddy, Mannherz, and Tregear for frequent helpful discussions. I am also very grateful for the opportunity to quote some of their inpublished work.
16
this idea in the sliding filament hypothesis (15, 8) the cross-bridges must attach and detach from actin in a cyclical fashion (11, 13). Kinetic studies on the soluble fragments of myosin, heavy meromyosin (HMM) and S1 and on their interaction with actin led Lymn and Taylor (17) to propose the four-state cross-bridge cycle which is depicted in Figure 1. Two of the states are states of attachment and two of the states are free. The present working hypothesis of cross-bridge action is that the bridge alters its angel of attachment to actin thereby generating a relative sliding between the filaments (rotating bridge) (13). In the second part of the cycle the bridge detaches from actin and returns to its original position ready to rebind to actin. During this process A TP is hydrolysed.
One of the major conceptual problems in accepting the rotating bridge hypothesis is how to accommodate the variations of distance between the actin and myosin filaments which arise during the shortening of the sarcomere. The solution proposed by H. E. Huxley (13) is depicted in Figure 2. The S2 fragment of heavy meromyosin is tought to be a 400 A long relatively inextensible link between the S1 and the myosin filament. The S2 is fastened to the myosin anchor-point by a hinge and is joined to the cross-bridge by a second hinge. This structure provides the necessary degree of freedom for the rotating bridge. The notion of the rotating bridge is supported by two lines of evidence: a) structural information from X-ray diffraction and electron microscopy. b) Interpretation of the response of stimulated frog muscle to rapid
length changes (9).
The systems mostly studied under a) are intact frog skeletal muscle, glycerinated rabbit psoas muscle, and glycerinated insect flight muscle. In both systems the actin filaments and myosin filaments are regularly arranged on a hexagonal lattice. The myosin filaments sit on the lattice points and the actin filaments respectively on the threefold and twofold positions. In the case of insect muscle the organisation of the cross­ bridges is crystalline at low resolution.
Below we survey the evidence pertaining to a) and report briefly on some new results obtained by the group in Heidelberg on insect flight muscle making use of ATP analogues.
HMM 52 HMM 51~
LMM Backbone of Myosin filament
Fig. 2. In order that the cross-bridge (S1) may bind to actin over a range of filament spacings Huxley (13) envisages the S2 part of the heavy meromyosin acting as a stiff light rod with hinges at both ends. The S1 is thereby allowed to be closer to the myosin filament or closer to the actin filament depending upon the state of the muscle
17
Structural Information Showing That the Cross Bridge Can Exist in a Number of Conformations
The evidence is taken from the following classes of investigation:
a) Changes in the low angle X-ray diffraction patterns of frog muscle occurring between relaxed and rigor or relaxed and activated and muscle (14; H. E. Huxley, this volume). In the relaxed state the myosin cross-bridges are regularly ordered around the myosin molecule, the axial spacing between cross bridges being 143 A, and give rise to a series of layer lines with a spacing of 429 A in the low angle fibre diffraction pattern. The layer lines become very weak on stimulation of the muscle showing that the regular ordering has been disrupted. Concomitantly, large changes of the intensities of the low order equatorial Bragg reflexions may be interpreted to mean that the cross-bridges move away from the myosin filaments towards the actin filaments on activation (H. E. Huxley, this volume).
Fig. 3. Rigor insect muscle fibre diagram - vertical fibre axis. Note the strong crystalline 388 A layer lines of which three orders are visible. Between the second and third order layer lines is the weak 145 A meridional reflexion. Obtained from a bundle of glycerinated fibers immersed in a buffered salt solution (5) from the longitudinal flight muscle from Lethocerus cordefanus. Film Ilford G, specimen­ film distance 1200 mm. Exposure 9 h, 1. 5 A radiation obtained from the DESY electron synchrotron, Hamburg, running at 6 GeV and 6 m average injection current. The focussing camera and remote controlled optical bench is described in Barrington Leigh and Rosenbaum (2)8 (Photograph obtained by J. Barrington Leigh and K. C. Holmes, muscle kindly provided by Dr. C. Rodger, University of Oxford)
18
Fige 4. Relaxed insect muscle - the fibres are bathed in 15 mM ATP and an EGTA buffer ensuring very low free Ca++ levels (see (5), for details). Note the weakening of the 388 A I?eries of layer lines and the
two strong meridional reflexions at 1!5 A -1 and 1;5 A -1 respectively.
Technical details as for Fig. 3 expect that a germanium 111 mono­ chromator was used. DESY parameters 7 GeV, 9 mAo Specimen-film distance 80 cm, exposure time 5 hs. (Photograph obtained by R. Goody, w. Hofmann, H. G. Mannherz, J. Barrington Leigh, and G. Rosenbaum)
b) The appearance of cross-bridges in ultra-thin sections of rabbit and frog muscle (11). The cross-bridges appear as structures which connect the actin and myosin filaments. Some of the bridges appear to be angled in a manner similar to that seen in insect flight muscle (see below) whereas others connect the thick and thin filaments at right angles. However, the strongly angled conformation characteri­ stic of insect rigor muscle is not seen. Furthermore, clear diffe­ rence between a II rigorll bridge and a II relaxedll bridge, which can be seen in insect flight muscle, has not been observed in rabbit muscle.
19
-z-
-M-
+- z -
Cross bndge :axIG! spaCing 145/..
Fig. 5. A diagram of the rigor (left), and relaxed (right) states of insect flight muscle (24). In rigor the cross-bridges bind streospecifically to the actin every 388 A (380 A in electron micrographs of thin sections) in a strongly angled con­ figuration. In relaxed muscle the cross­ bridges stick out at right angles to the filament axis and manifest their natural 145 A periodicity. The apparently shorter
bridges in the drawing of the relaxed muscle represent cross-bridges at an angle to the plane of the drawing. It is not certain if the difference in length of cross-bridge between rigor and relaxed can be accounted for by the hinged S2 hypothesis of Huxley (13) or wheter an actual change in length of the cross-bridge takes place between rigor and relaxed muscle (see text)
c) F-Actin may be 'decorated' with the soluble fragments of myosin, HMM or Sl (12). Negatively stained electron micrograph images show a steep helical array of the elongated HMM or S1 molecules each attached at an angle to the actin filament so as to give the appearance of arrow heads. Three-dimensional image reconstruction (21) shows that the Sl molecules which comprise the myosin cross-bridges, are boomerang-shaped molecules binding at about 450 to the axis of the actin filament. This is taken to be typical rigor conformation for rabbit muscle myosin. Images obtained with HMM, or whole myosin at high ionic strength (12), appear similar to those obtained with Sl. As far as one can see the tail of a myosin molecule extends outwards in a straight line at 450 to the filament axis to reinforce the arrow­ like configuration. Only one SI of the pair within a myosin molecule binds to actin. Moreover, one complete HMM (or myosin) doublet is able to bind per actin monomer.
d) Ultra-thin sections of insect flight muscle (Lethocerus maximus or cordefanus) fixed in rigor and in the presence of A TP (24; 23). Characteristic of rigor are strongly angled bridges binding regularly every 388 A along the actin helix (the so-called chevrons). Fixed
20
relaxed muscle does not show chevrons nor any tendency to angled cross-bridges. Most of the cross-bridges are detached from the actin as judged from measurements of stiffness. The median direction appears to be at right angles to the filament axes. The bridges are spaced every 145 A along the myosin helix.
e) Low angle X-ray diffraction from insect flight muscle in the relaxed and rigor state s and in intermediate states which can be induced with A TP analogues. The diffraction patterns obtained from rigor and relaxed are characteristic and different (Figs. 3 and 4). In rigor the system of 388 A spacing layer lines is strong and arises from a uniquely crystalline arrangement of rigor cross-bridges (23; 6). In 5-15 mM ATP the 388 A period layer lines become 20 times weaker (20) and are essentially replaced by a system of strong meridional reflexions with an axial spacing of l45 A. These changes are approximately what one would expect from the electron micro­ graphs.
The deductions from d and e are summarized in Figure 5. The coupling of b with c leads to a similar conclusion. H. E. Huxley's experiments on activated muscle clearly demonstrate that the cross-bridges move around during activation and that a considerable transfer of mass from the neighbourhood of the myosin filament to the actin filament accompanies stimulation. This can be interpreted as a movement of the cross-bridge from the neighbourhood of the myosin to the neighbourhood of the actin (Fig. 2).
The two states depicted in Figure 5 are:
(1)
(2)
The relaxed state, produced in glycerinated fibres by a high concentration of ATP (5-15 mM) and low Ca ++. Under these condi­ tions the acto-myosin interactions are reduced to a low value. The bridges are largely dissociated from the actin and protrude at right angles, at the same time exhibiting a strong l45 A axial periodicity.
Rigor, obtained in the absence of ATP. The bridges are strongly angled and bind to the actin preferentially every 388 A. The 145 A periodicity is destroyed. It is currently assumed that these two states occur in the cross-bridge cycle in the manner depicted in Figure 1. Although this is a reasonable working hypothesis one should bear in mind that such marked changes of cross-bridge con­ formation have only been observed in insect flight muscle.
The Mechanism of A TP Hydrolysis by Actomyosin
The hydrolysis of Mg ++ ATP by actomyosin (HMM or S1 + f-actin) has been shown by Lymn and Taylor (17) and by Bagshaw et al. (1) to proceed by the following steps:
(1) Binding of ATP to actomyosin causes rapid dissociation of actin and myosin ATP + AM --+ ATPM + A.
21
(2)
(3)
(4)
(5)
The binding of ATP to myosin is a two-step process involving a first order isomerism of the A TP-protein complex to a new form with higher ln~rinsic protein fluorescence MATP -+ MATP. The hydrolysis of A TP without release of the products M*ATP-+M**ADP. Pi, where M** is a further conformation of myosin with high fluorescence. In the absence of actin: Slow release of product in a multistep process with the net result M** ADP. Pi---+- M + ADP + Pi Or, in the presence of actin: Recombination with actin and concomitant fast release of products M** ADP Pi + A---+AM + ADP + Pi.
Reaction (5) is apparently the step present in the power stroke, i. e the A TP is cleaved before recombination of myosin with actin takes place: the binding of actin release of ADP appears to induce a change of shape of the cross-bridge thereby displacing one filament with respect to the other.
Figure 1 results from the combination of this kinetic model with the structural hypothesis that the myosin has two stable conformations (described above as relaxed and rigor). Myosin has a low or high affinity for actin depending on the integrity of the A TP. From Figure 1 one notes that two other states might be identifiable, namely 11 up and onll and 11 down and offll. Our group in Heidelberg has tried to characterize such states with the help of A TP analogues which are not cleaved by myosin or are cleaved with very different rate constants from ATP. We have used low angle X-ray diffraction from glycerinated fibre bundles as our method.
X-Ray Diffraction From Insect Flight Muscle in the Presence of A TP (6. Y )-NH
In the presence of 1 mM ATP (t3, Y )-NH a new kind of fibre diffraction pattern may be obtained from insect flight muscle fibres (Fig. 6) (5; 4). ATP (t3, Y )-NH is not cleaved by myosin but binds strongly and compe­ titively (25). In conjunction with mechanical measurements the diffraction pattern may be interpreted as showing that the cross-bridges have the right-angled conformation typical of relaxed muscle (Fig. 5) but are at the same time joined onto the actin filaments (5; 19). It is possible that the binding of A TP (t3, Y . )- NH induces a state similar to the 11 up and on state 11 shown in Figure 1. This would be the transitory ternary complex between actin, myosin and ADP present at the start of the power stroke. If so, the addition and removal of ATP (~, Y )-NH should allow a controlled simulation of the conformational changes taking place in the power stroke (16).
22
Fig. 68 The ATP (~, y )-NH induced state (5). Note in comparison with Figures 3 and 4 that the 145 A layer lines and meridional reflexions are strong but that the 388 A series of layer lines are not much weaker than in rigor. Stiffness measurements (16; 19) show the cross-bridges to be still attached to actin. However, cross-bridges have apparently rearranged their pattern of attachment to the actin so as to reinforce the 145 A periodicity. Whether or not that part of the cross-bridge which attaches to the actin stereospecificity has a markedly different con­ formation from that in rigor cannot be ascertained from studies of the low angle crystalline part of the fibre diagram because of lack of resolution. Technical details as for Figure 3, exposure time ca. 20 h. DESY synchrotron 6 GeV, 6 mAo (Photograph obtained by G. Rosenbaum and K. C. Holmes)
Unfortunately the simple view may be somewhat too naive since it can be shown by an analysis of the low angle X-ray diffraction pattern that a redistribution of cross-bridges accompanies the binding of the nucleotide (Holmes, Tregear, and Barrington Leigh, in preparation). In the rigor state the bridges attach with a 388 A period to each actin filament. The
23
orIgm of the 388 A period is probably the twostart actin helix which makes a half turn in 388 A. Therefore, each actin filament shows apparently the same aspect to the neighbouring myosin filament every 388 A . If the myosin stereospecificity for actin is high then this gene­ rates the 388 A period. This 388 A period is considerably weakened in the presence of A TP (~, y )- NH. Concomitantly a strong meridional 145 A period appears.
The coming-and-going of the two periodicities may be interpreted as being manifestations of two periodic probability functions which we may think of as effective concentrations: the concentration of actin binding sites, and the concentration of bridges along the length of the myosin filament. These spatial concentration functions are, however, rather dependent of the state of the cross-bridge. In binding a nUCleotide, the 388 A period actin binding site function becomes much more uniform and the 145 A period myosin concentration becomes dominant. The concentration functions are clearly empirical functions reflecting funda­ mental and important changes in the nature of the cross-bridge. However, the existence of these effects considerably complicates the interpretation of the low angle X-ray diffraction pattern.
In summary, ATP (~, y )-NH has three main effects: a) It increases the rate of dissociation of myosin from actin thereby
allowing a rapid redistribution of bridges. b) It lowers the stereospecificity of myosin for actin (alters the distri­
bution of the effective concentration of actin binding sites). c) The binding of the bridges to actin appears to be at right angles
rather than 450 •
The third effect is the apparent conformational change we were seeking; however even this may be open to reinterpretation. For example, an analysis of the wide-angle actin-based X-ray pattern (Goody, Tregear, Mannherz, Barrington Leigh, Rosenbaum, and Holmes, in preparation) seems to show that that part of the cross-bridge which binds stereo­ specifically to actin does not alter its conformation between rigor and the ATP W, y )-NH state. However, it is clear that considerable shifts of mass do accompany the binding of the nucleotide (4) so that the problem is really to try to be more specific about the nature of the conformational change. In doing this we may find it necessary to abandon the swinging bridge hypothesis, at least in the simple form depicted in Figure 1.
A Hypothesis for the Changes Occurring on Binding ATP (~, y )-NH
The observations described above could be explained if the cross-bridge in insect flight muscle (and by analogy in other muscles) has the following properties:
a) The cross-bridge consists of a head and a tail. Part of the tail is probably similar to the S2 fragment of rabbit skeletal muscle myosin.
24
The end of the cross-bridge tail next to the myosin rod is anchored in the rod. The anchor points are spaced every 145 A along the rod. The head of the cross-bridge (being one of the myosin pair) attaches firmly to actin. This part of the molecule, which probably has a radial extent of 60-70 A from the actin surface and could be the part up to the bend in the boomerang- shaped S1 (but which could be the whole of the S1 region) does not undergo major conformational changes during the cross-bridge cycle.
b) In rigor the cross-bridge and tail are stiff and colinear and make an angle of about 450 to the filament axis. As seen in Reedy's (23) micrographs the cross-bridge so defined can be up to 220 A long.
c) On adding a nucleotide which produces either the form M*ATP, or M**ADP Pi a large portion of the tail of the molecule melts. The melted tail piles up round the anchor point producing a considerable movement of mass towards the myosin filament. Moreover, the flexibility of the (now) rope-like tail would allow the head considerable freedom to find a conveniently placed actin binding site on activating the muscle.
d) ATP (~, y )-NH produces an interesting intermediate between rigor and relaxation where apparently the tail has been melted but the head has not been released from actin. Thus the pile- up of tail round the anchor points leads to considerable redistribution of mass but is not as total as it is in the case of relaxed muscle. However, the greater flexibility allowed to the head leads to much more uniform filling of the actin binding sites.
If the cross-bridge has the properties of a fixed head and a meltable tail the structural observations (EM and X-ray) on insect flight muscle could be unified and explained. The power stroke of muscle would then presumably consist of a random-coil-helix transition in the tail induced by losing the nucleotide from the binding site on the head. This has similarities with the Davis (3) model for muscular contraction. Such a hypothesis would also provide a ready explanation for the observations of Pollard (22; Fig. 3) on reconstituted myosin filaments. Under certain conditions the myosin heads are relatively small and are connected to the myosin rod by tenuous structures up to 600 A long. Under other conditions this structure collapses onto the myosin rod to produce a tangled blob. The explanation for these phenomena advanced by Pollard himself calls on the rotating bridge hypothesis with the hinged S2 as formulated by H. E. Huxley (13) (Figs. 1 and 2). If, as we presently suspect, it should turn out that the head only has one conformation when bound to actin then the passive role envisaged for the S2 leaves no mechanical degrees of freedom in the myosin cross-bridge and muscle will not be able to contract. In this case we will be obliged to postulate a much active role for the tail of the cross-bridge of the kind I have suggested here.
25
References
1. Bagshaw, C. R., Eccleston, J. F., Eckstein, F., Goody R. S., Gutfreund, H., Trentham, D. R.: The magnesium ion-dependent adenosine triphosphatase of myosin. Biochem. J. 141, 351-364 (1974)
2. Barrington Leigh, J., Rosenbaum, G.: A report on the application of synchrotron radiation to low angle scattering. J. appl. Cryst. 1, 117 (1974)
3. Davies, R. E.: A molecular theory of muscle contraction: calcium­ dependent contractions with hydrogen bond formation plus A TP­ dependent extensions of part of the myosin cross bridges. Nature (Lond.) 199., 1068-1074 (1963)
4. Goody, R. S., Barrington Leigh, J., Mannherz, H. G., Tregear, R. T., Rosenbaum, G.: X-ray titration of binding of ~, y -imido­ ATP to myosin in insect flight muscle. Nature (Lond.) 262, 613- 615 (1976) -
5. Goody, R. S., Holmes, K. C., Mannherz, H. G., Barrington Leigh, J., Rosenbaum, G.: Cross bridge conformation as revealed by X-ray diffraction studies of insect flight muscle with ATP analogues. Biophys. J. 15, 687-705 (1975)
6. Holmes, K. C., Goody, R. S., Mannherz, H. G., Barrington Leigh, J., Rosenbaum, G.: An investigation of the cross bridge cycle using ATP analogues and low angle X-ray diffraction from glyce­ rinated fibres of insect flight muscle. In: Molecular Basis of Motility. 26 th Colloquium Mosbach 1975, Heilmeyer, L. (ed.) Heidelberg: 1976 Springer-Verlag
7. Huxley, A. F.: Muscle structure and theories of contraction. Progr. Biophys. 1, 255-318 (1957)
8. Huxley, A. F., Niedergerke, R.: Interference microscopy of living muscle fibres. Nature (Lond.) 173, 971-973 (1954)
9. Huxley, A. F., Simmons, R. M.: Proposed mechanism of force generation in striated muscle. Nature (Lond.) 233, 533-538 (1971)
10. Huxley, H. E.: Electron microscope studies of the organisation of the filaments in striated muscle. Biochim. biophys. Acta 12, 387-394 (1953)
11. Huxley, H. E.: The double array of filaments in cross striated muscle. J. biophys. biochem. Cytol. ~, 631-648 (1957)
128 Huxley, H. E.: Electron microscope studies on the structure of natural and synthetic protein filaments from striated muscle. J. molec. BioI. 1, 281-308 (1963)
13. Huxley, H. E.: The mechanism of muscular contraction. Science 164, 1356-1366 (1969)
26
14. Huxley, H. E., Brown, W.: The low angle X-ray diagram of vertebrate striated muscle and its behavious during contraction and rigor. J. molec. BioI. 30, 383-434 (1967)
15. Huxley, H. E., Hanson, J.: Changes in the cross- striations of muscle during contraction and stretch and their structural inter­ pretation. Nature (Lond.) 173, 973-976 (1954)
16. Kuhn, H. J.: Transformation of chemical into mechanical energy by glycerol extracted fibres of insect flight muscles in the absence of nucleoside triphosphate hydrolysis. Experientia 29, 1086-1088 (1973)
17. Lymn, R. W., Taylor, E. W.: Mechanism of adenosine triphosphate hydrolysis by actomyosin. Biochemistry 10, 4617-4624 (1971)
18. Mannherz, H. G., Barringt 0 n Leigh, J., Holmes, K. C., Rosenbaum, G.: Identification of the transitory complex myosin ATP by use of a, ~ methylene-ATP. Nature (New BioI.) W, 226-229 (1973)
19. Marston, S. B., Rodger, C. D., Tregear, R. T.: Changes is muscle cross bridges when ~, Y -imido-ATP binds to myosin. J. molec. BioI. 104, 263-274 l1976)
20. Miller, A., Tregear, R. T.: Structure of insect fibrillar flight muscle in the presence and absence of ATP. J. molec. BioI 70, 85-104 (1972)
21. Moore, P. B., Huxley, H. E., DeRosier, D. J.: Three-dimensional reconstruction of F-actin, thin filaments, and decorated thin fila­ ments. J. molec. BioI. 50, 279-295 (1970)
22. Pollard, T. D.: Electron microscopy of synthetic myosin filaments: evidence for cross bridge flexibility and copolymer formation. J. Cell BioI. El, 93-104 (1975)
23. Reedy, M. K.: Ultrastructure of insect flight muscle. 1. Screw sense and structural grouping in the rigor cross bridge lattice. J. molec. BioI. ]1, 155-176 (1968)
24. Reedy, M. K., Holmes, K. C., Tregear, R. T.: Induced changes in orientation of the cross bridges of glycerinated insect flight muscle. Nature (Lond.) 207, 1276-1280 (1965)
25. Yount, R. G., Ojala, D., Babcock, D.: Adenylyl imidophosphate, an adenosine triphosphate analog containing a P-N-P linkage. Bio­ chemistry 10, 2490-2496 (1971)
27
Myosin-Linked Regulation: A Chemical Approach
J. KENDRICK-JONES and R. JAKES
Calcium is know to play a central role in controlling many cellular processes, for example, in secretion, in the visual cycle and in motility and has been implicated together with cyclic AMP in development, and in the control of many metabolic pathways; however, its role in regulating contraction in muscle is probably the best understood. Muscular contraction is controlled by specific calcium receptor proteins located on either the thick or thin filaments which are involved in switching actin-myosin interaction ON or OFF in response to changes in calcium concentration. In vertebrate striated and cardiac muscles, the regulatory
- - - MYOSIn Filament--_
ThIn Filament
Fig. 1. Diagrammatic representation of myosin filament with cross­ bridge and thin filament, showing the relative positions of the calcium regulatory components. The myosin cross-bridge consists of two globular heads containing the A TP hydrolytic and actin combining sites. Associated with each head are two classes of light chains, one of which, termed 'regulatory' light chains, acts as calcium regulatory subunits in molluscan muscles and may serve a regulatory role in all myosins. In the vertebrate thin filament, tropomyosin lies in the groove between the two helical strands of actin monomers and attached to it, at inter­ vals of about 38. 5 nm is the calcium regulatory complex, troponin, which consists of three differont subunits, one of which, troponin C binds calcium with a high affinity
28
proteins troponin and tropomyosin are associated with the thin filaments (Fig. 1). The extensive electron microscope, X- ray and biochemical evidence suggests that calcium induced changes in the troponin complex may be transmitted via a movement of the tropomyosin in such a way that it affects those sites in the actin molecules which are required for interaction with the myosin cross-bridges (see reviews Weber and Murray, 27; Huxley, 13). In molluscan muscles, the troponin complex is absent and instead calcium regulation of contractile activity requires the presence of specific regulatory light chains on the myosin (Fig. 1) (15, 24). However, little is known about how these regulatory light chains, under calcium, control, effect the transition of the myosin cross-bridge from the resting to the active state. Myosin-linked regulation operates in many invertebrate phyla (18) and in vertebrate smooth muscle (2). However all the myosin so far studied, contain two classes of light chains; (16); one class, called regulatory light chains, are capable of 'functionally' replacing the regulatory light chains of scallop myosin (14). An understanding of the mechanism involved in myosin-linked calcium regulation may therefore be of general interest in clarifying the role of these regulatory light chains in all myosins.
Molluscan myosin contains two high affinity calcium binding sites and requires the presence of specific regulatory light chains for its calcium sensitive interaction with actin (Fig. 1) (24). The regulatory function of these light chains (called EDTA light chains) has been established by the selective release of one mole of light chain from scallop myosin, which results in a complete loss of calcium regulation and the loss of one calcium binding site, i. e. the myosin is 'desensitized' and no longer requires calcium for interaction with actin. The isolated light chain readily recombines with the' desensitized' myosin and calcium regulation is completely restored. One rather intriguing question is why the removal of only one mole of regulatory light chain completely' desensitizes' the myosin, since the two regulatory light chains in a myosin molecule appear to be identical by chemical and functional tests (16). The two light chains are also identical by immunological criteria (Fig. 2). Immunodiffusion of antisera prepared against both scallop regulatory light chains produces a single precipitin band when run against the same light chains, and shows no cross-reactivity against' regulatory' light chains isolated from vertebrate and invertebrate sources (which hybridise with' desensitized' scallop myosin) (Fig. 2a). Scallop myosin in the presence and absence of calcium, or in combination with actin or in the 'desensitized' state,. gives a single precipitin line which fuses with that of the isolated light chain suggesting that the light chain when bound to the myosin under these conditions is freely accessible to the rather 'bulky' antibody and indicates that there are no obvious antigenic differences between the light chain in the isolated compared with bound state (Fig. 2b). If the antigenic properties of proteins depend to a large extent on conformation (1; 22) then the conformation of the light chain when isolated and when combined with the myosin heavy chain must be similar, at least around their antigenic sites. The complete loss of regulation, therefore, when only one of the two iden­ tical regulatory light chains is removed indicates that only myosin
29
Cardiac
(a) ( b)
Fig. 2. Immunodiffusion of antiserum to scallop regulatory light chains with a) other' regulatory' light chains b) scallop myosin alone or in combination with actin or in the 'desensitized
state. Specific antiserum to purified scallop regulatory light chains was elicited in rabbits by the procedure described by Holt and Lowey (11). In series a) double diffusion was carried out at 40 C in 0.1 M KC1, 0.01 M phosphate buffer pH 7.2 with the' regulatory' light chains indicated, present at 2 mg/ml except the scallop light chain 0.08 mg/ml. In series b) diffusion was carried out in 0.5 M KCl, 0.02 M phosphate buffer pH 7. 2, 1 mM MgCl2 with scallop myosin, actomyosin and 'desensitized' myosin at concentration of approx. 10 mg/ml and the isolated scallop light chain 0.05 mg/ml. Either 1 mM CaCl2 or 1 mM EGTA included in the antigen wells were indicated. The gels were also stained for protein with Coomassie Brilliant Blue to verify that no other faint precipitin bands were present
molecules that contain both light chains can be regulated by calcium and would suggest that regulation requires cooperation between either the two regulatory light chains of the two myosin heads. However, as yet we have no direct evidence for such a cooperative regulatory mechanism.
Further insight into the regulatory mechanism, especially by identi­ fication of the calcium binding site, may be gained by comparing the sequence of the regulatory light chain with the known sequences of other muscle regulatory and calcium binding proteins, e. g. carp calcium binding protein, troponin C and rabbit' regulatory' light chain (DTNB light chain) which previous comparative sequence studies have indicated are homologuos and contain identifiable calcium binding regions (26; 3, 4; 29). The three dimensional structure of the calcium binding protein established by Kretsinger and Nockolds (17), which clearly shows two basic structural units, each composed of a calcium binding site in a 'pocket' surrounded by helical regions on either side, has served as a general model for these comparative studies. Figure 3 shows the sequence of the scallop regulatory light chain, initially aligned with the sequence of the rabbit DTNB light chain and troponin C (5, 4) and then drawn out to correspond to the main structural features of the carp calcium binding protein. Four regions composed of a pair of helices surrounding a potential calcium binding site are easily recognisible.
30
1
.,E S E D L T E 'K: CD ~ K
H4 t~6~ R
A
S,te 1 Site 2 S,te 3 Site 4
Fig. 3. Amino acid sequence of the scallop regulatory light chain, aligned with the sequence of rabbit DTNB light chain and troponin C (5; 4) and drawn out to correspond to the main structural features of carp calcium binding protein (17) with four pairs of helices (numbered H1-8) surrounding four' potential' calcium binding regions (sites 1-4). Circled residues are the positions of buried hydrophobic groups which form the core of the calcium binding protein and the residues in the 'site' regions in squares the positions of residues possibly ligated to calcium. The N-terminus, residue 1, is blocked presumably by an acetyl group and the C-terminus is at residue 153; the areas in black denote regions where identical residues are observed in the sequence of the rabbit 'DTNB' light chain and troponin C. For convenience, the one letter amino acid code has been used, where A = ala, R = arg, N = asn, D = asp, Q - gIn, E = glu, G = gly, I = ile, L = leu, K = lys, M = met, F = phe, P = pro, S = ser, T = thr, Y = tyr and V = Val
The positions of buried hydrophobic residues which form the core of the calcium binding protein are circled and it is remarkable that in virtually every position hydrophobic residues are present in the scallop light chain, which suggests a degree of structural similarity between these proteins. It has been established (17) that in the calcium binding sites of the calcium binding protein, calcium is coOrdinated to oxygen atoms from the six amino acids in the positions indicated by the squares, and within these six coordinating positions four acidic residues are present. In the scallop light chain, 'sites' 2, 3 and 4 contain less than four negatively charged groups and residues such as proline which would distort these sites. These regions can therefore be ruled out as potential calcium binding sites. Comparison of site I of the scallop and gizzard light chains with the identical site in troponin C, calcium binding protein and rabbit' DTNB' light chain shows striking similarities (Table 1). Four negatively charged groups are present within the six coordinating positions in the sites of troponin C, calcium binding protein,
31
W
in
scallop and gizzard light c~ins_ fnd since the binding constant of this site in troponin is about 10 M (21) it is reasonable to assume that this site in the scallop light chai~ is the high affinity calcium binding site present in the myosin (K 10 M-l) (24). It is interesting that the binding site in rabbit DTNB light chain contains only three negatively charged residues within the coordination positions which may explain the lower calcium affinity of this site (binding constant about 105 M- 1) (30; 19) and the failure of this rabbit light chain to restore the high affinity calcium binding site when it binds to 'desensitized' scallop myosin (14; 16).
Comparison of the sequence of the scallop light chain with rabbit regula­ tory (DTNB) light chain and troponin C (Fig. 3) indicates considerable homology (25-30% overall identical residues) especially in the N terminal halves of the molecules: 42% of the residues in this region in the scallop light chain are identical to those in the rabbit light chain and 30% are identical to those in troponin C and in many cases the replacements are conservative. Given the extensive sequence homology that exists between the calcium binding protein, troponin C and light chains, it is reasonable to speculate that they evolved from a common ancestral gene by successive gene duplications (3; 29), however since they have obviously evolved to perform quite distinct functions within the muscle cell and the in vitro evidence indicates they are not interchangeable (10), it may be unwise to believe that their overall three dimensional structures are similar. Calcium binding protein and troponin C are globular proteins probably with rather similar structure, whereas hydrodynamic measurements (23) indicate that the molluscan regulatory light chains are extremely asymmetric, with an axial ratio of about 8 : 1, which would indicate a length ranging from 100-140 A, about the same length as a single myosin head.
Fragmentation of proteins whether with proteolytic enzymes of selective chemical reagents, is a useful technique for the identification of those reg