Comp. Biochem. Physiol. Vol. 72A, No. 4, pp. 623 to 626, 1982 Printed in Great Britain.

0300-9629/82/080623-04$03.00/O 0 1982 Pergamon Press Ltd

SUBSTRATE CONCENTRATION DEPENDENCE OF CONTRACTION CHARACTERISTICS IN

GLYCERINATED HORSESHOE CRAB SKELETAL MUSCLE FIBRES

SHIGERU CHAEN and HARUO SUGI*

Department of Physiology, School of Medicine, Teikyo University, Itabashi-ku, Tokyo 173, Japan

(Received 9 December 1981)

Abstract-l. The MgATP concentration dependence of isometric force, ATPase activity and unloaded shortening velocity was studied on glycerinated horseshoe crab muscle fibres.

2. The isometric force and the ATPase activity reached their maxima at 1O-5 M MgATP and lo-“ M MgATP, respectively.

3. The unloaded shortening velocity continued to increase with increasing MgATP concentration up to 3 x 1O-3 M.

4. The differences in MgATP concentration dependence of contraction characteristics between horse- shoe crab and rabbit skeletal muscle fibres are discussed in relation to the current sliding filament hypothesis.

INTRODUCTION

The molecular mechanism of muscle contraction has been studied by both physiological and biochemical methods. Physiological studies have established the sliding filament mechanism in which muscle contrac- tion is caused by a relative sliding between the thick and thin filaments (Huxley & Nidergerke, 1954; Hux- ley & Hanson, 1954), while biochemical studies have revealed detailed enzymatic kinetics of actomyosin ATPase (Lymn & Taylor, 1971; Tonomura, 1972). From these studies, the sliding between the filaments is thought to result from a cyclic reaction between the projections on the thick filament, e.g. the cross- bridges, and sites on the thin filament. It is not clear, however, which step of the ATP hydrolysis cycle is directly associated with force generation or shorten- ing in muscle. It also remains to be determined whether or not the biochemical schemes for the ATP hydrolysis cycle of the actomyosin system in solution can be applied to the events taking place in the three- dimensional lattice of the thick and thin filaments.

To correlate the biochemical results on the acto- myosin system with the physiological contraction in muscle, the effect of MgATP concentration on glyceri- nated vertebrate muscle fibres has been studied by several investigators (Arata et al., 1977; Cooke & Bialek, 1979; Chaen et aI., 1981). From the standpoint of comparative muscle physiology and biochemistry, it is of interest to compare the MgATP concentration dependence of glycerinated fibres between vertebrate and invertebrate skeletal muscles. The experiments to be described in this paper were undertaken to study the MgATP concentration dependence of the ATPase activity and the contraction characteristics in glyceri- nated invertebrate striated muscle fibres, using horse- shoe crab skeletal muscle as material.

* To whom correspondence should be addressed.

MATERIALS AND METHODS

Preparation

Adult horseshoe crabs (Tachpleus tridentatus) were co1 lected in the vicinity of Fukuoka, Japan, and fed in aeratec sea-water at 18°C until use. Telson depressor muscles were isolated from the animals and glycerinated by the method of Tanaka et al. (1979). Small bundles consisting of 2-5 muscle fibres (2-4 mm in length) were dissected from gly- cerinated muscles in a 50% glycerol solution and mounted horizontally between the lever of a servo motor (General Scanning G-108) and the extension of a force transducer (Aksjeselskapet AE801, compliance 0.4 pm/mN, resonance frequency 3 kHz), using a fast-setting glue. Bathing sol- utions were contained in an array of rectangular wells (2 ml) made of lucite. The array of wells could be moved both vertically and horizontally, so that the solution sur- rounding the preparation was exchanged by lowering the array of wells, positioning a different well under the prep- aration and then again elevating the array of wells (Hellam & Podolsky, 1969). The temperature of the bathing sol- ution was controlled at 15°C with a thermoelectric device (Yamato kagaku Coolnix). The length and force changes were simultaneously displayed on an oscilloscope (Tektro- nix 565) and recorded with a 35 mm camera for analysis.

Solutions

Relaxing solutions contained 88-l 10 mM KCl, 20 mM Tris-maleate (pH 7.0). 4 mM MgCl,, 20 mM phosphocrea- tine, 170 U/ml creatinekinase and 1.6 PM-4 mM ATP. Contracting solutions were prepared by adding 5.3 mM CaCl, to relaxing solutions. The concentration of KC1 was varied to make the ionic strength of the solutions constant. The concentrations of essential ionic species such as MgATP*-, ATP4-, Ca’+ and Mg2+ were calculated by a computer iteration procedure following the method of Per- rin & Sayce (1967). ATP and phosphocreatine were pur- chased from Sigma Chemical Co. Other chemicals were of analytical grade. Creatine kinase was prepared from rabbit skeletal muscle bv the method of Noda et al. (1955). Its activity was 85 U/mg protein in a solution containing 1OOmM KCl, 4mM MgCI,, 4mM ADP, 20 mM phos- phocreatine and 20 mM Tris-maleate (pH 7.0) at 25°C.

623

624 SHKMII CHACN and HAKO SI,GI

OTSC 02;nW

Fig 1. Records of length (upper traces) and force (lower traces) changes during and after a quick release on a slow (A) and fast (B) time base. Downward deflection of length and force traces indicates decrease

in length and in force, respectively. L,, 2.4 mm. MgATP, 11 pm. For further explanation see text,

Measurement of’ unloaded shortening velocit) RESULTS

The preparation was first made to contract isometrically at its slack length, L,. After development of full isometric force, P,, a quick release (about 307; of L,) was applied with the servo-motor, so that the preparation was first slackened and then shortened to redevelop isometric force at a new length. The time, t, was measured from the onset of release to the point at which the force started to redeve- lop (Fig. 1A). The velocity of unloaded shortening, V,, was calculated by the following equation:

l’, = [(L, - L’) - AL]/Af,

where L’ is the length after a quick release and AL is the amount of the early part of the quick release required to drop the isometric force to zero (Fig. IB). The sarcomere length of the fibres at L,,, as measured by the diffraction of He-Ne laser light, was about 9 pm in all preparations examined.

Measurement 01 ATPase activity during isometric contrac- tion

About twenty fibre bundles (about 1 cm in length), each consisting of 223 fibres, were mounted horizontally between two glass rods fixed at the bottom of a lucite trough (1.4ml) and made to contract isometrically with contracting solutions. There was another trough contain- ing only contracting solutions to serve as control. The tem- perature of the troughs was also kept at 15°C with the thermoelectric device. The ATPase activity of the fibre bundles during a sustained isometric contraction was esti- mated by measuring the amount of creatine liberated in the contracting solution. The concentration of creatine was de- termined by the method of Eggleton et al. (1943). The pro- tein content of the preparation was determined by means of the biuret reaction,

Determination qf’amount of‘ ATP regenerating system

Since the rate of ATP hydrolysis by contractile pro- teins within glycerinated fibres is higher than the rate of diffusion of ATP from the bathing solution into glycerinated fibres, the MgATP concentration in the interior of the fibre may be much lower than that of the surrounding contracting solution, especially at low MgATP concentrations. This problem can be solved by the addition of an ATP regenerating sys- tem. Skeletal muscles of invertebrates are known to contain arginine kinase and phosphoarginine as an ATP feeder system. In the present experiments, we used the phosphocreatine-creatine kinase system instead of the phosphoarginine-arginine kinase sys- tem because of commercial availability. The amount of this feeder system necessary for the present study was examined by the method of Cooke & Bialek (1979) which is based on the idea that the velocity of unloaded shortening is sensitive to MgATP concen- tration, decreasing with decreasing MgATP concen- tration. Figure 2 shows the dependence of unloaded shortening velocity on the amount of ATP regener- ating system in the contracting solution containing 34pM ATP. The results indicate that, to obtain the maximum unloaded shortening velocity, more than 85 U/ml of creatine kinase is necessary at 20mM phosphocreatine (Fig. 4A), while more than 10mM phosphocreatine is required at 170 U/ml creatine kinase (Fig. 4B). In this study, we used 170 U/ml crea- tine kinase and 20 mM phosphocreatine.

(b)

0 IO 20

Creatine kinase cancentration, x 85 unit/ml

Phosphocreatine concentration, mM

Fig. 2. Dependence of unloaded shortening velocity on the amount of ATP regenerating system at a low MgATP concentration (34 PM). In (A), creatine kinase concentration was varied at a constant phospho- creatine concentration (20 mM), while phosphocreatine concentration was varied at a constant creatine

kinase concentration (170 U/ml) in (B).

Contraction in horseshoe crab muscle 625

6 5 4 3

MgATP concentration -Log EMgATPl

Fig. 3. Relation between isometric force and MgATP con- centration. Isometric force values are expressed relative to the value at 2.3 mM MgATP (1.2-1.4 kg/ems). In each preparation, the isometric force was measured at 2.3 mM MgATP and at another chosen MgATP concentration

below 2.3 mM.

Dependence of isometric force on MgATP concen- tration

The relation between the magnitude of isometric force developed and the MgATP concentration in the contracting solution is shown in Fig. 3. The isometric force increased steeply with increasing MgATP con- centration up to about lO-‘j M, reaching a maximum value at lo-’ M MgATP. No significant change in isometric force was observed when the MgATP con- centration was further increased up to 3 x 10e3 M, in contrast with glycerinated rabbit psoas muscle fibres in which isometric force development was maximum at lo-‘M MgATP but further increases in MgATP concentration caused a decrease of isometric force (Chaen et al., 1981).

Dependence of ATPase activity during isometric con- traction on MgATP concentration

The ATPase activity of glycerinated fibres during sustained isometric contraction was examined at various concentrations of MgATP. As shown in Fig. 4, the ATPase activity increased with increasing MgATP concentration and reached a plateau value above 10Y4M MgATP. The ATPase activity in gly- cerinated horseshoe crab muscle fibres at 2.3 mM MgATP was about 0.03 ~mols/minjmg protein, amounting to only one sixth of the corresponding ATPase activity in glycerinated rabbit psoas muscle

u 0

I I I

6 5 4 3 -I

MgATP concentration -log I MgATPl

Fig. 4. Relation between ATPase activity and MgATP con- centration. Data points were obtained from different

preparations.

6 5 4 3

MgATP concentration -\og E MgATP 3

Fig. 5. Relation between unloaded shortening velocity and MgATP concentration. Data points were obtained from

different preparations.

fibres (Chaen et al., 1981). The dependence of the ATPase activity on the MgATP concentration was much less marked in horseshoe crab muscle fibres than in rabbit psoas muscle fibres; the increase in the ATPase activity when the MgATP concentration was increased from 11 PM to 2.3 mM was about three-fold in horseshoe crab fibres (Fig. 5) and about seven-fold in rabbit psoas fibres (Chaen et al., 1981).

Dependence of velocity of unloaded shortening on MgATP concentration

The relation between the unloaded shortening vel- ocity and the MgATP concentration is shown in Fig. 6. The unloaded shortening velocity also increased with increasing MgATP concentration, as with the ATPase activity. The unloaded shortening velocity in horseshoe crab fibres at 2.3 mM MgATP was about 3 pm/set/half sarcomere, while that in rabbit psoas fibres was about 9 pm/set/half sarcomere. The depen- dence of the unloaded shortening velocity on the MgATP concentration in horseshoe crab muscle fibres was also less marked than that in rabbit psoas muscle fibres. The increase in the unloaded shortening velocity when the MgATP concentration was in- creased from 11 PM to 2.3 mM was about five-fold in horseshoe crab muscle fibres (Fig. 7) and about nine- fold in rabbit psoas muscle fibres (Chaen et al., 1981).

DISCUSSION

The present experiments have shown the simiiari- ties and the differences in the substrate concentration dependence of contraction between glycerinated horseshoe crab and rabbit psoas muscle fibres. Unlike rabbit psoas fibres, the isometric force in horseshoe crab fibres was a monophasic function of MgATP concentration (Fig. 3). A monophasic relation between isometric force and MgATP concentration has also been reported for skinned crayfish muscle fibres (Kawai & Brandt, 1977). On the basis of Hux- ley’s contraction model (Huxley, 1957), the magnitude of isometric force, P, is expressed as

p = f/U + 9) wherefand g are rate of formation and that of break- ing of the cross links between the filaments, respect- ively.

626 SHI~;ER~ CHMN and HARI:O SLIGI

Thus, the decrease of isometric force at high MgATP concentrations in rabbit psoas fibres can be explained as being due to an increase in 9 (Chaen et al., 1981). Meanwhile, the monophasic relation between isometric force and MgATP concentration in crustacean muscle fibres can be accounted for by assuming that y is much smaller than,/; or by assum- ing that bothf‘and g increase in proportion (Kawai & Brundt, 1977). In the case of vertebrate smooth muscle, it has been shown that isometric force is also a monophasic function of MgATP concentration (Filo et al., 1965; Saida & Nonomura, 1978) and that the dissociation constant of actomyosin is much smaller than that in vertebrate skeletal muscle (Mar- ston & Taylor, 1978; Takeuchi & Tonomura, 1978). This may be taken to indicate that, in vertebrate smooth muscle, the monophasic relation between iso- metric force and MgATP concentration results from a small value of g.

The ATPase activity in horseshoe crab muscle fibres was only a fraction of that in rabbit psoas muscle fibres over the whole range of MgATP con- centrations studied (Fig. 4), suggesting a small rate constant for breaking cross links. Thus, the monopha- sic dependence of isometric force on MgATP concen- tration is likely to result from a small value of g rather than a proportional increase of ,f and g with high MgATP concentrations.

As with rabbit psoas muscle fibres, the relation between unloaded shortening velocity and MgATP concentration in horseshoe crab muscle fibres (Fig. 5) was nearly the same as that between ATPase activity and MgATP concentration (Fig. 4), although the values of both ATPase activity and unloaded shorten- ing velocity in horseshoe crab fibres were smaller than theacorresponding values in rabbit psoas fibres. In general, these results are consistent with the finding of BBr6ny (1967) that, in various types of muscle, ATPase activity increases roughly in proportion to the velocity of shortening. This indicated that the unloaded shortening velocity is primarily determined by the rate of ATP splitting, coupled with the alter- nate formation and breaking of cross links between the filaments. Thus, all the results obtained on horse- shoe crab muscle fibres can at present be explained on the basis of a small value of g, since the rate of ATP splitting decreases with decreasing g value according to the Huxley model.

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