Studies on adenosine triphosphate transphosphorylases: Isolation and several properties of the crystalline calf ATM-AMP transphosphorylases (adenylate kinases) from muscle and liver

ARCHIVES OR BIOCHEMISTRY AND BIOPHYSICS Vol. 187, No. 1, April 15, pp. 34-52, 1978

Studies on Adenosine Triphosphate Transphosphorylases

Isolation and Several Properties of the Crystalline Calf ATP-AMP Transphosphorylases (Adenylate Kinases) from Muscle and Liver and Some

Observations on the Rabbit Muscle Adenylate Kinase’

STEPHEN A. KUBY,2 MINORU HAMADA, DOROTHY GERBER, WEI-CHONG TSAI, HANS K. JACOBS,3 MICHAEL C. CXESS, GEOK KOON CHUA, GERALD

FLEMING, LILLY H. WU, ANNE H. FISCHER, ASTA FRISCHAT, AND

LYNN MALAND

Laboratory for Study of Hereditary and Metabolic Disorders and Departments of Biological Chemistry and Medicine, University of Utah, Salt Lake City, Utah 84132

Received September 7, 1977; revised November 9, 1977

Detailed procedures are described for the large-scale isolation of crystalline calf muscle and calf liver adenylate kinase isoenzymes applicable to frozen tissue. Proposed modifications in the large-scale isolation of the crystalline rabbit muscle adenylate kinase are also described. Although a great deal of homology exists between the muscle types, calf and rabbit, as revealed by their amino acid compositions (differing in only 10 out of 193 residues), the calf muscle adenylate kinase acts as a potent antigen in the rabbit. A description for the purification (ca. 600-fold) from the rabbit antiserum of the anti-enzyme globulin is given. The anti-enzyme shows a remarkable specificity in that it is largely unreactive as an inhibitor toward the calf liver-type enzyme, but is a powerful inhibitor of the calf muscle type (or human muscle type) with a kinetically evaluated dissociation constant of ca. 3 x lo-l4 r.? (for the case of a stoichiometry of 2 Ag to 1 Ab), a second-order k = 5 x lob; M-I. min-’ at 3O”C, and an Arrhenius energy of activation of ca. 6.3 kcallmol. Dodecyl sulfate polyacrylamide gel electrophoresis revealed only a single heavy chain (M, , -42,000) and a single light chain (M,, -25,000) of the antibody, which together with studies employing 3.5% stacking gels lead to a molecular weight of approximately 134,000 for the native antibody. Similarly, by dodecyl sulfate polyacrylamide gel electrophoreses, only single bands were obtained for the calf liver and rabbit muscle adenylate kinase t&f,, ca. 21,000-23,000) and the calf liver adenylate kinase (M, , ca. 25,000). Sedimentation velocity of the calf muscle-type and calf liver-type proteins yielded single sedimenting boundaries, with .s&,~ = 2.1, and 2.3, S, respectively. Estimates of the molecular weight8 of the native structure of the calf isoenzymes by sedimentation equilibrium gave it& = 21,200 * 200 for the calf muscle enzyme and 25,600 f 200 for the calf liver adenylate kinase. Electrophoresis by liquid boundary of the rabbit muscle enzyme at protein concentrations of 5-8 mg/ml resulted in a nonideal system, as a result largely of charge effect8 and interaction8 with buffer components. Similarly, isoelectric points determined by isoelectric focusing were very dependent on the protein load, but could be extrapolated to zero protein to approximately pl, of 10.6. However, electrophoresis on cellulose acetate at microgram to nanogram levels of protein under conditions where electroendosmosis seemed absent did permit an accurate estimate of the pfO by a plot of pl,,, vs r/2Y2 to a pl, = 10.60. This value is now in good agreement with an estimate made earlier from its amino acid composition (T. A. Mahowald et al., 1962, J. Biol. Chem. 237, 1136-11451. By this

1 This work was supported in part by grants fkom dressed. the National Institutes of Health. This is Paper 12 s Present address: Department of Biochemistry, of the series; Paper 11 is Ref. 41. University of Manitoba, Winnipeg, Canada.

’ To whom all correspondence should be ad-

34

0003~9661/78/1871-0153$02.00/0 Copyright 0 1978 by Academic Press, 1,~. All rights of reproduction in any form remerged.

CRYSTALLINE ADENYLATE KINASE ISOENZYMES

technique, the pl,,, values at 0.05 (I/Z) for the calf isoenzymes were estimated and found to differ only slightly (viz., 9.6 for the liver adenylate kinase vs 10.1 for the muscle myokinase). While inhibition of the rabbit and calf muscle adenylate kinases (which are apparently cytoplasmic enzymes) by ApsA [p1,p5-di(adenosine- 5’)pentaphosphatel was very significant at even lo-* M and similar to that previously reported for the rabbit muscle adenylate kinase (Lienhard and Secemski, 1973, J. Biol. Chem. 248,1121-1123), inhibition ofthe liver-type adenylate kinase (which is apparently a mitochondrial enzyme) required almost lo-” M (Ap,A) for similar percentage inhibi- tions. For the forward reactions (i.e., MgATP*- + AMP- +), Ap,A acts as a competitive inhibitor with respect to either substrate with Ki = (0.5-l) x 1Om8 M for both of the muscle enzymes, but yields a Ki = (3-8) x lo-’ M for the liver enzyme. However, in the reverse direction (MgADP- + ADP3- +), inhibition by ApJA appears to be noncompetitive with respect to either substrate, with a Ki = (0.6-2) x lops M for the muscle enzymes, but only (3-4) x lo-” M for the liver enzyme. Thus, although Ap,A may act in part as a transition state analog, the explanation for its “multisubstrate inhibition” (Lienhard and Secemski, see above) may also lie in the structure of its metal chelates,

35

e.g., of Mgz(Ap5W.

The isolation of crystalline rabbit muscle myokinase (ATP-AMP transphosphorylase) was first described by Noda and Kuby (1; see also Ref. 2), who also determined several of its physicochemical properties (3). Adenylate kinases have since been purified and characterized from a variety of sources, which include porcine (51, carp (5a), and human muscle (6), bovine liver mitochondria (7, 8), rat liver (9, lo), and porcine liver (ll), rat hepatomas (12), rat brain (131, human erythrocytes (141, and baker’s yeast (15).

Adenylate kinase has been reported to exist in two major sets of multiple or polymorphic forms in populations of the human erythrocyte which were presum- ably genetically determined (16,17). These two major genetic variants of human erythrocyte adenylate kinase, which had been designated AK, and AK, (161, were separated and partially purified (18, 19); a species which was designated “AKa” was isolated (14), and one species which was designated AK,’ was sequenced (20). In addition to the genetically determined variants, tissue-determined variations of adenylate kinase in man were described early (21) and these studies were carefully ex- tended by Russell et al. (22).

Thus, the interest generated by the fmd- ings that there exist a number of tissue- determined forms of isoenzymes of adenylate kinase versus the genetically determined species variations, as well as apparently a number of mutational variants within a given species, e.g., man, was the

underlying reason for the present studies. The immature calf was selected for study since developmentally determined forms (if any) of adenylate kinase will be a subject for future studies. Our preliminary studies, coupled with those reported (e.g., Refs. 7-11, 21, and 22), led us to the hypothesis that, at least in mammals, there may be two major forms of isoenzymes of adenylate kinase: the cytoplasmic type, present largely in the skeletal muscle, and the mitochondrial type as repre- sented by the liver. This prompted us to attempt the isolation of the muscle-type and the liver-type adenylate kinase from the same organism, viz., the calf. As we shall see, although there is a very high degree of homology between the muscle types in the mammals, there is apparently a sufficient number of differences between the muscle type and liver type to permit their distinction by immunological means (cf. Ref. 22).

A preliminary report on some aspects given below has been presented (42).

I. EXPERIMENTAL SECTION

Materials

Fresh calf tissues were obtained from the local slaughterhouse or from the Utah Biomedical Test Lab. Frozen rabbit and calf tissues were obtained from Pel-Freeze. The Sephadexes (G-100, G-200, and G-200, superfine) were obtained from Pharma- cia; Bio-Rex 70 resin (200-400 mesh; exchange capacity, 10.2 meq/g) and phosphocellulose (exchange capacity, 0.88-1.00 meq/g) were from Bio-Bad (Cel- lex-P); high-capacity phosphocellulose (exchange capacity, 7.4 meq/g) was from Whatman (P-l, cellu-

36 KUBY 2” ’ - l!al AL.

lose phosphate); DEAE’-cellulose (exchange capacity, 1.0 meq/g) was from Whatman (DE-32, micro- granular). The acid-base cycling of the ion-exchange cellulose and storage were conducted as described for the calf brain ATP-creatine transphosphorylase (231. (NH&SO, was obtained from Schwarz/Mann (ultrapure); disodium EDTA was from Fisher Scientific Co.; Tris, dithioerythritol (DTE), and the sodium salts of ATP, ADP, and AMP were from Sigma or Boehringer. Hexokinase, lactate dehydrogenase, pyruvate kinase, and glucose g-phosphate dehydrogenase were obtained from Sigma; crystalline bovine albumin was from Ar- mour. The lithium salt of Ap,A (diadenosine pentaphosphate) was obtained from Sigma. P-Mercapto- ethanol (Eastman) was redistilled under vacuum. All other reagents were of analytical grade or of the best grade available. Twice-distilled deionized water was used for the preparation of all reagents including the dialysis fluids; in some cases, solutions were prepared in degassed (by boiling) distilled water or saturated with nitrogen. Dialysis was conducted in cellophane tubing with a 12,000 molecular weight maximal cutoff (Arthur H. Thomas), and the concentration of protein solutions was conducted where indicated by evacuation through bundles of hollow cellulose fibers with a nominal cutoff of 5000 molecular weight (Bio-Rad Bio-Fiber No. 50 beaker or Dowex permeator, b/HFD-1).

Methods Measurements of enzymatic activity. Both the pH-

stat method (24, 25) with the titrant (0.0100 N

NaOH) and reaction mixture as given in Ref. 26 (i.e., with final concentrations of 4 mM ADP, 2 mM MgSO+ 50 mM n-glucose, 1 mg/ml of albumin, and 50 U of hexokinase at pH 8.00 and 30°C) and a spectrophotometric [coupled-enzyme assay (27)l procedure [with the following final concentrations in a 3.9ml reaction mixture: 2.1 mM ADP, 2.1 mM Mg(Ac),, 6.7 mM glucose, 0.67 mM NADP, 4 U of hexokinase, 2 U of glucose 6-phosphate dehydrogenase, 50 mM Tris (acetate), pH 8.1,3O”Cl were utilized to follow the purification. Dilutions of the enzyme were made in ice-cold 1 mM DTE, 1 mM EDTA, pH 7.5; toward the end of the purification, 1 mg/ml of albumin was added to stabilize the highly diluted enzyme. The pH-stat unit was approximately twice that of the coupled-enzyme unit.

Protein was determined by the calorimetric biuret procedure of Gornall et al. (28) or, in later stages, by its extinction coefficient at 279 nm. For the calf muscle enzyme, a biuret factor of 3.1 mg/absorbance unit (for a IO-ml volume of 540 nm) and an extinc-

4 Abbreviations used: DEAE, diethylaminoethyl; DTE, dithioerytbritol; Ab, anti-calf muscle myokinase; Ag, calf muscle enzyme; I, inhibitor; E, enzyme.

tion coefficient Ey$$ (at 279 nm; 0.05 M phosphate, pH 7.01, of 0.53’were obtained, which were assumed to apply for the calf liver enzyme.

Immunological procedures. Rabbit antisera against calf muscle myokinase were obtained, after a long period of hyperimmunization, by a multiple- sites intradermal injection technique similar to that described by Amon and Shapira (29; see also Ref. 30). The rabbits were immunized initially with several milligrams of the enzyme in 0.2 (I/2), pH 7.7, 0.05 M Tris-NaCl buffer 10.05 M Tris, 0.037 M

HCl, 0.163 M NaCl, 1 mM EDTA, 1 mM DTE (or p- mercaptoethanol)], pH 7.7 at 25”C, in complete Freund’s adjuvant (Difcol, at multiple intradermal sites. Blood was collected biweekly from the mar- ginal ear vein prior to additional intradermal immunizations, but with incomplete Freund’s adjuvant. When the anti-enzyme activity had risen to significant values (several months), intradermal immunizations were discontinued and blood was withdrawn monthly. Boosters by intradermal injec- tions were given when the titers of anti-enzyme activity appeared to decrease sharply.

Antibody (anti-enzyme) purification. Pooled rabbit antisera were diluted with an equal volume of 1 mM EDTA, pH 6.8, and precipitated at 0°C with 55%~saturated (NH&SOa, pH 6.8. The precipitates were washed at 0°C until colorless with 0.1 yol of 55%~saturated (NH,),SO,, pH 6.8, and dissolved in 0.1 vol and dialyzed vs the 0.2 r/2), pH 7.7, buffer (without DTE) described above. After clarification by centrifugation, the anti-enzyme (at concentrations of at least 200 anti-enzyme units/ml at 25°C) was then precipitated by titration over a period of several hours with microaliquots of the antigen (calf muscle myokinase) as monitored by the absorbance increase at 700 nm. After the equivalence point was reached, the antigen-antibody precipitates were allowed to stand overnight at 3°C and were then collected by centrifugation; the precipitates were washed exhaustively with the 0.2 (r/2) pH 7.7 buffer without DTE until the A,,, of the washes decreased to a minimal value.

The pellets were dissolved at 0°C in a minimum volume of 10 mM glycine (HCl), 0.2 M NaCl, 1 mM EDTA, pH 2.0, and the pH was adjusted, if necessary, to 2.0; the mixture was allowed to stand at 3°C for 24 h (cf. Ref. 49). The solution, after clarification if necessary, was then chromatographed at 25°C on a 2.5 x 90-cm column of Sephadex G-200 (superfine) with an internal support of 6-mm (diameter) silicon&d glass beads, packed and prepared as described by Sachs and Painter (31), with a hydrostatic head of 10 mM glycine (HCl), 0.2 M NaCl, pH 2.0, at a flow rate of ca. 5 ml/h [Fig. 1A(P)l.5 The major peak containing the anti-enzyme

5 The miniprint supplement (Supplementary Ma- terial), which appears after the reference list, con-

CRYSTALLINE ADENYLATE KINASE ISOENZYMES 37

activity was pooled, adjusted to pH 7.4, concentrated at 3°C under reduced pressure through collodion bags (Schleicher and Schuell, No. 100 collodion bags), and chromatographed on a 1.5 x 90-cm column of Sephadex G-200 developed at pH 7.4 with 10 mM Tris, 0.3 M NaCl, 1 mM EDTA [Fig. lB(P)l. Again, the major area containing the anti-enzyme activity was pooled and concentrated as before to ca. 0.5 ml in volume. Table I(P) summarizes the purification of the anti-calf muscle myokinase antibody (anti-enzyme), where 1 anti-enzyme unit is defined as that amount of antibody which would inhibit 1 unit (spectrophotometric) of enzyme activity after 6 h of reaction at 37”C, pH 7.7, in 0.2 (I/2) buffer containing also 1 mg/ml of albumin and 1 mM DTE (see above for injection buffer composition). The antibody with a specific activity of ca. 507 anti-enzyme units/mg is obtained after ca. a 600-fold purification with ca. 20% yield. Its estimated molecular weight (ca.134,000) and interaction with its antigen (calf muscle myokinase) will be described below, except that it may be mentioned here that the anti-enzyme also appears to inhibit the human muscle myokinase, but it seems to be relatively unreactive toward the calf liver adenylate kinase. Its ultraviolet spectrum in 50 mM Tris (HCl), 0.2 M NaCl, 1.0 mM EDTA, pH 7.4, revealed the following: A,,, = 278 nm; Amin = 252 nm; shoul- ders at 283 and 290 nm; h278/X252 = 2.59; A,,&,,, = 1.82; @‘ik”7 = 1.50.

Other methods. Physical methods: Sedimentation velocity and sedimentation equilibria measurements and electrophoresis on cellulose acetate are summarized in Ref. 4, and some details are provided in the legends to the appropriate figures; the methodology used for amino acid analyses is also described in Ref. 4. Isoelectric focusing was carried out in a llO-ml LKB 8101 Ampholine column according to the directions of the manufacturer (32); further details will be given below. Sodium dodecyl sulfate a&amide gel electrophoresis was conducted according to the method of Weber and Osbom (35) with the slight modifications indicated (36).

Kinetics: Complete details of the methodology and evaluation of the kinetic parameters will be presented elsewhere.” A brief description is presented here. The initial velocity of the forward reaction (MgATP2- + AMP*- -+ MgADP- + ADP3-) was measured by a spectrophotometric coupled-enzyme procedure for ADP, employing pyruvate kinase and lactate dehydrogenase. The reaction mix-

tains the isolation procedures and several fwres pertinent to the results section. Figures and tables in the parent text are identified by (P) and in the supplement by (S).

s Hamada and Kuby, 1977, manuscript in preparation.

ture (1.0 ml) consisted of 10 mM Tris (Cl-), pH 7.4 at 25”C, 150 mM NaCl, 4 mM dithioerythritol, 0.1 mM EDTA, 0.25 mM phosphoenolpyruvate, 20 mM KCl, 0.5 mglml of bovine serum albumin, 1 mM

MgCl,, 0.2 mM NADH, 20 U of lactate dehydrogenase [purified by repeated precipitation at 55% saturation of (NH&SO, to remove traces of myokinasel,

n l5- 400 300

- -

?

FIG. l(P). Typical chromatographic profiles of dissociated calf muscle ATP-AMP transphosphorylase-specific antibody complex and of purified anti- calf muscle ATP-AMP transphosphorylase antibody. (A) Chromatography on a 2.5 x 96-cm column of silicon&d glass beads (6-mm diameter) packed with Sephadex G-200 (superfine) (cf. Ref. 31) of the antigen-antibody complex dissociated at pH 2.0 and 3°C in 10 mM glycine (HCl) containing 1 mM EDTA, 0.2 M NaCl. Just prior to the chromatography, 3.0 ml of the Ag-Ab solution (containing 25.94 mg of protein) was adjusted to pH 1.9 with 10 ~1 of 2 N

HCl at 25°C. The chromatography was initiated at 25°C with a hydrostatic head of 10 mM glycine (HCD, 1 mM EDTA, 0.2 M NaCl pH 2.0 buffer, at a flow rate of 4.8 ml/h. x---x , absorbance at 280 nm; O-O, anti-enzyme units per fraction (see text); A-A, myokinase enzyme units (spectrophotometric) per fraciton; 2.4 ml/fraction. (B) Chromatog- raphy of the dissociated anti-calf muscle ATP-AMP transphosphorylase antibody on a 1.5 x 90-cm column of Sephadex G-200 (tine) developed with a hydrostatic head of 10 mM Tris (HCl), 0.3 M NaCl, 1 mM EDTA, pH 7.4 and 3”C, and a flow rate of 7.5 ml/h. The applied sample (1.2 ml) contained 8.54 mg of protein (equivalent to 3400 anti-enzyme units) and had been dialyzed against the above buffer; 1.5 ml/fraction. Symbols are the same as in A.

38 KUBY ET AL.

TABLE I(P) PURIFICATION OF RABBIT ANTI-CALF MUSCLE ADENYLATE KINASE GLOBULIN”

Fraction Total ac- Total protein Specific ac- Yield (%I tivity (anti- (mg) tivity (anti-

enzyme enzyme unit@ unita/mg)

I. Serum 11,841 13,256 0.89 ww

II. 0.55 saturation (NH&SO, precipitation 8,563 2,808 3.05 72.5

III. After dialysis vs 0.2 (l/2) (Tris-NaCl) pH 7.7 7,425 2,448 3.13 62.9 buffer

IV. Precipitation with calf muscle myokinase

V. Sephadex G-200 (superfine) gel filtration at pH 2.0 [lo mM glycine (HCl), 0.2 M NaCll

26.9

3,409 8.6 395 28.9

VI. Sephadex G-200 gel filtration at pH 7.4 (10 mM Tris, 0.3 M NaCl, 1 mM EDTA)

2,479 4.9 507 20.5

a Initially, ca. 200 ml of rabbit antiserum. b One anti-enzyme unit = the amount of antibody which will inhibit 1 unit of calf muscle myokinase, at

pH 7.7, 0.2 (l/2) Tris-NaCl, 1 mglml of albumin, 1 mM DTE, 6 h at 37°C.

10 U of pyruvate kinase, and varying and calculated amounts of MgAc,, AMP,,, ATP,, and ApsA when necessary. The reaction was initiated by the addition of the suitably diluted (see above) myokinase. The final ionic strength averaged ca. 0.18. Measure- ments of the decrease in absorbance at 340 nm were made with a Cary 14 spectrophotometer thermo- stated at 25°C; in this case, the activity was expressed as the rate of ATP, disappearance, where l/z x (A,J6.22) = micromoles of ATP, disappearance (and subzero implies total concentration).

Measurements of the initial velocity of the reverse direction (MgADP- + ADP3- + MgATPZ- + AMP*-) were made by a coupled-enzyme procedure for ATP, employing hexokinase and glucose bphos- phate dehydrogenase. The reaction mixture contained 10 mM Tris (Cl-), pH 7.4 at 25°C 150 mru NaCl, 4 mM DTE, 6.6 mM glucose, 0.67 mM NADP+, 0.5 mg/ml of bovine serum albumin, 1 mM MgCl,, 0.1 mM EDTA, 10 U of hexokinase, 5 U of glucose 6- phosphate dehydrogenase, varying but calculated amounts of MgAc,, and ADP,. Final ionic strength averaged ca. 0.16. The reaction was initiated by the addition of the enzyme and the initial velocity in this case was expressed as A,d6.22 = micromoles of ATP, formation. Further details are given in the legends to Figs. 7(S)-9(S).

Calculations of MgATPz- and AMP*- for the forward reaction and of MgADP- and ADPa- for the reverse reaction were made by employing a set of conservation equations applicable to the myokinase reactions and similar to those listed before (46). These will appear in a kinetic description of the adenylate kinases (see Footnote 6), which will also

give details of the methods of calculations of the kinetic parameters and the values assigned for the chelation and dissociation constants (but see Refs. 45-47). Calculations employed a fixed concentration of free magnesium of 1 mM Mg*+ for the kinetic studies on the forward direction.

II. ISOLATION PROCEDURES

To facilitate the description of the isolation of these enzymes, the isolation procedures are divided into three sections: (A) isolation of the isoenzyme from calf muscle tissue; (B) isolation of the isoenzyme from calf liver tissue; and (C) a few notes on the isolation of the rabbit muscle enzyme.

All steps were carried out in a cold room (3-4°C) or in an ice bath unless otherwise specified: pH measurements were made at or near 5°C with a Radiometer ‘PITla meter equipped with scale ex- pander PHA 630 Ta and A. H. Thomas glass-calo- me1 electrodes. The required amounts of solvent and (NH&SO,, as well as the concentrations of these reagents, were estimated by the formulas given, e.g., Ref. 1 (cf., Refs. 2, 33, and 34). The technique of continuous-flow dialysis is described in Ref. 33. A saturated solution of (NH&SO4 was taken to be 3.9 M at 0°C.

The detailed procedures and figures pertinent to the isolation procedures are described in the miniprint supplement (Supplementary Material; see Footnote 5).

A. Isolation of the Muscle Type (Adenylate Kinasel from Calf Skeletal Muscle Tissue

Table II(P) summarizes the purification in terms


TABLE II(P) FRACTIONATION OF CALF SKELETAL MWCLE ATP-AMP TEANBPH~SPHORYLASE (PREPARATION 8)”

Fraction No. Volume Pro- Total Specific activity (ml) tein activ- Recovery of ac-

con- ity (unitsirE;f pro- Purification tivity (%)

ten- (units tra- x lo+) DH stat Snectro- Over- Over Overall Over tion (mg/

photo metric

all pre- ceding

ml) step

I. Supernatant from 13,000 100 mM KC1 homogenate

9.11 32.76” 27.66 12.6 (1)” Wb

II. Acid denaturation 13,288 of inert protein

III. Zn fractionation 1,195

IV. (NH&SO, fraction- 110 ation, followed by dialysis, pH 7.1

2.64 32.98 93.94 44.4 3.4 3.4 100.6 100.6

7.30 23.34 267.5 65.3 9.7 2.9 71.2 71.2

39.84 8.43' 192.3 77.8 7.0 - 25.8 36.1

V. Phosphocellulose 1,450 chromatography, pH 7.1

0.19 5.90 2,146 726 77.7 11.2 18.0 70.0

VI. Concentration with Dow (hollow-ti- her) unit followed by (NH&SOI fractionation

100 2.30 8.35 3,630 1,636 131.5 1.7 25.5 142

VII. Sephadex G-100 gel filtration

60 12.34 5.20 3,716 1,673 134.5 1.0 15.9 62

VIII. Crystallization 1. Crystals

2. Crystals

4.25 17.0 2.86 3,960 1,783 143.2 1.07

4.13 15.85 3.24 4,951d 2,230 179 1.25

3. Crystals 8.41 5.59 2.34 4,982d 2,244 180 1.01

- ing step

(100)b (100)b

8.7 55

9.9 -

- -

a Initially, 4.2 kg of tissue (wet). b One unit = 1 peqlmin by the pH-stat procedure. c Dialysis tubing with unspecified cutoff molecular weight used in this preparation. In succeeding

preparations, dialysis tubing used with 12,000 molecular weight cutoff (A. H. Thomas). d Data from Preparation 10.

of yield and purification for each step in the puriil- zyme units/mg) for the crystallized enzyme, which cation scheme, which is outlined in the first column, is about twice the specific activity cf the rabbit up to the final crystallization stage. The protein muscle enzyme and about 10 times the specific crystallizes from aqueous ammonium sulfate solu- activity of the calf liver enzyme (see below). The tions (see the supplement) at pH 6.9 in the form of procedure is applicable to frozen tissue, but with relatively large rectangular-shaped crystals [see somewhat lower yields than from fresh tissue. Usu- Fig. 2A(P)l. A purification of ca. 180-fold to a final ally, four batches derived from 4-5 kg of tissue each specific activity of ca. 4980 U/mg (pH stat) is ob- are worked up to the point just before the phospho- tained (or ca. 2240 spectrophotometric coupled-en- cellulose step (see the supplement) and stored as

40 KUBY ET AL.

FIG. 2(P). (A) Crystalline ATP-AMP transphosphorylase (adenylate kinase) from calf muscle (Preparation 8). The photomicrographs were obtained with a Zeiss phase-contrast microscope at 3°C. (B) Crystalline ATP-AMP transphosphorylase (adenylate kinase) from calf liver (Preparation 9). (C) Crystalline ATP-AMP transphosphorylase (adenylate kinase) from rabbit muscle (Preparation I-M).

the 80%saturated (NH&SO, pellet. The pellets are then combined and dialyzed together for the chromatography. In this manner, about 300-350 mg of final crystallized material (ca. 15 pmol) is obtained, which is of sufficient purity for many of the chemical studies, e.g., for the amino acid sequence studies.’

B. Isolation of the Liver Type (Adenylate Kinasel from Calf Liver Tissue

Table III(P) contains the outline of the puritica- tion procedure for the calf liver adenylate kinase and a summary of the purification and yield at each step to the point of crystallization (see the supplement). The liver-type enzyme crystallizes from aqueous ammonium sulfate solutions at pH 7.0, in the presence of AMP, in the form of huge, elongated

7 Kuby et al., 1977, unpublished work.

rod-like crystals [Fig. 2B(P)l and is isolated after ca. a 700-fold purification over the initial extract at pH 4.6 (see the supplement). A constant specific activity is reached after essentially one crystallization. For increased yield, fresh liver tissue should be employed with yields obtained which are two to three times the amount derived from the frozen tissue used in Preparation 9 [Table III(P)]. The frozen tissue used in Preparation 9 had been stored for convenience. Compared to the fresh skeletal muscle tissue [cf. Table II(P)], frozen calf liver homogenate yields only 4.4% total enzymatic activity per kilogram wet weight; but the enzymatic activity would rise to ca. 10% for fresh liver tissue. The liver-type adenylate kinase is largely derived from the mitochondria* and qualitatively ca. 6-15% of the muscle type (largely’cytoplasmic) contami-

S Chua and Kuby, unpublished work.


TABLE III(P)

FRACTIONATION OF CALF LIVER ATP-AMP TRANBPHOBPHORYLASE (PREPARATION 9)”

Fraction No. Volume (ml)

Protein Total Specific Purification Recovery of ac- concen- activ- activity tivity (%) tration ity (units/

(mg/ (unite mg of Overall Over Overall Over ml) x 10~‘) protein) pre- preced-

ceding ing step step

I. pH 4.6 supernatant from homogenate in 10 mM Na,HPO,

II. Zn fractionation

III. (NH&SO, fractionation

IV. Acid denaturation of inert protein, followed by precipitation with 90% saturation (NH&SO, and dialysis

V. DEAE-cellulose chromatog-

raphy

VI. High-capacity cellulose phosphate chromatogra-

phy

VII. Low-capacity phosphocellulose chromatography followed by precipitation with saturated (NH&SO,

VIII. Carboxymethyl cellulose chromatography followed by precipitation with saturated (NH&SO,

IX. (NH&SO, fractionation

X. Sephadex G-100 gel flltra- tion

XI. Crystallization 1. Crystals

2. Crystals

57,500 11.97 23.80* 0.345" Wb Wb (100)” (100)b

20,000

2,405

1,750

17.28 14.60 0.422 1.23 1.23 61.5 61.5

105.6 11.40 0.448 1.30 1.06 48.0 78.2

60.8 9.20 0.88 2.55 1.97 38.7 80.7

748 1.11 38.9 100.3

73

108.8 9.24 1.135 2.86

35.04 8.26 32.76 95 28.9 34.7 89.5

42 24.0 6.09 60.4 175 1.85 25.6 73.8

17 31.1 4.27 80.6 234 1.34 18.0 70.3

4.4

84

53.2 2.41 103.2 299 1.29 10.1 56.5

1.63 2.55 186.4 541 1.81 10.7 106

2.54 18.94 1.18b 245.6," 712b 344.9’

1.32b 5.00 46.4b

2.02 20.00 0.93* 230.0,b 668* 360.3'

a Initially, 19 kg (nine livers) of tissue (wet). b One unit = 1 wmol/min by spectrophotometric (coupled-enzyme) procedure. c One unit = 1 peqlmin by pH-stat assay.

nated the early fractions as estimated very approxi- capacity phosphocellulose chromatographies (see mately by anti-enzyme analyses (see below). The the supplement). The enzyme is readily attacked by small amounts of muscle type were largely removed the proteases present in the liver extracts, but these after the high-capacity cellulose phosphate and low- are partially inhibited by phenylmethanesulfonyl

42 KUBY ET AL.

fluoride, and the addition of 5’-AMP further stabi- lizes the enzyme. To isolate sufficient amounts of crystalline protein for chemical studies, Fractions V (see the supplement) are stock-piled and worked up together.

C. Isolation of the Muscle Type (Adenylate Kinase) from Rabbit Skeletal Muscle

The procedure essentially follows that of Ref. 2 with the few changes indicated in the supplement, especially in the use of Bio-Rex 70 in place of the IRC-50 (XE64) used before and the introduction of a phosphocellulose chromatography step with a Na+ gradient at pH 7.2. After concentration with a Dow hollow-fiber permeator (b-HFD-11, operated to col- lect the concentrated protein solution outside the fiber bundles, the protein is then crystallized from aqueous ammonium sulfate at pH 6.4 in the form of huge rhombohedrons [Fig. 2C(P)l, which differ from the needle-like crystals reported earlier (Ref. 1). The final specific activity of four-times-crystallized material appears to be ca. 2700 Ulmg (by the pH- stat procedure), compared to ca. 5000 U/mg for the crystalline calf muscle adenylate kinase [Table II(P)].

Kress et al. (37) were apparently among the first to introduce phosphocellulose as a suitable chromatographic support for adenylate kinase, and it appears to function largely as if it were an affinity chromatographic adsorbent. Thus, 5’-AMP may be used to elute the adenylate kinases from the phosphocellulose columns; however, its use invariably leads to long tailing, prolonged periods of time for the chromatography and relatively large elution volumes which must then be concentrated. How- ever, 5’-AMP is essential to add near the end of the preparation of the liver enzyme to inhibit the attack of proteases. Practically, the procedures evolved above for all three enzymes, with either Na+ or Tris+ gradients, yield sharper chromatograms and permit their applications to relatively large amounts of protein (ca. 25 pmol of enzyme).

III. RESULTS

A. Physical Studies Sedimentation velocity studies at about

pH 8 and 20°C on the crystalline calf muscle type and calf liver type revealed single sedimenting components. Both enzymes displayed the usual concentration dependencies [see Fig. 3(S)] and yielded extrapolated values of s$,,, W equal to 2.1,~ and 2.3, S for the calf muscle type and calf liver type, respectively.

Sedimentation equilibrium studies in the presence or absence of 4 M guanidin- ium chloride-O. 1 M p-mercaptoethanol indicated the absence of a subunit structure

and the presence of a single polypeptide chain in both calf muscle and liver isoenzymes. Estimates of the molecular weights of the native structure for the calf isoenzymes were conducted by the procedure of Yphantis (38) [see Fig. 4(S), A and Bl and yielded iI& = 21,200 f 200 for the calf muscle myokinase and 25,600 + 200 for the calf liver adenylate kinase.

Dodecyl sulfate polyacrylamide gel electrophoresis (35) revealed the presence of single bands for the calf muscle and calf liver isoenzymes, as well as for the rabbit muscle myokinase, but showed two bands (a heavy chain and a light chain) for the rabbit anti-calf muscle myokinase globulin. Estimates of their molecular sizes by this technique (7% gels) are given in Fig. 5(S) and are as follows: calf muscle myokinase, 21,000; rabbit muscle myokinase, 23,500; calf liver adenylate kinase, 25,500; light chain and heavy chain, 25,000 and 42,000, respectively. On 3.5% stacking gels at pH 8.9 (39) the antibody appears to migrate as a single band with a mobility in the neighborhood of ca. 130,000 to 160,000. Therefore, there are two light chains and two heavy chains in the antibody with a molecular weight of ca. 134,000 by this technique.

Electrophoresis studies by liquid boundary electrophoresis at protein concentrations of about 5-8 mg/ml (cf. Ref. 1) yielded variable results in the pH 6.5 to 8.5 range for the rabbit muscle enzyme, apparently due to a strongly nonideal system, either as a result of unusual charge interactions with the ionic components of the buffers or due to changes in its frictional coeffl- cient as a result of preferential binding of solvent components. On the alkaline side, the enzyme proved to be too unstable to approach its isoelectric point (ca. pH g-10) calculated from its amino composition, neglecting charge effects (43). Attempts to employ isoelectric focusing (32) led to the surprising observation that the apparent isoelectric point varied from ca. 8.3 (with 2% Ampholine, pH 3-10, plus L-lysine) at a load of ca. 13.5 mg on the llO-ml Ampho- line column to ca. pH 9.4 at a load of ca. 4 mg (with otherwise identical conditions and with either 2% pH 3-10 Ampholine plus L-lysine or 2% pH 7-10 Ampholine


plus L-lysine) to ca. pH 10.2 at a load of 1 mg of myokinase (with 3% pH 7-10 Am- pholine plus L-lysine and L-arginine). A plot of apparent fl vs load yields an extrapolated isoelectric point of ca. 10.6. Therefore, at relatively high protein concentrations, the rabbit muscle enzyme apparently acts in a nonideal fashion in several electrophoretic systems. Electro- phoresis on cellulose acetate was then studied, a technique which permits evaluation of the electrophoretic behavior at microgram to nanogram levels of protein. As pointed out in Ref. 40, effects due to electroendosmosis seemed to be absent on Sepraphore III (Gelman) cellulose acetate strips as checked by the use of the un- charged blue dextran (Pharmacia) macro- molecule. However, there are slight differences in degrees of adsorption or retention between the several lots of Sepraphore III studied, making it important for compar- ative studies to employ the same lot. Elec- trophoresis on cellulose acetate was conducted at an average temperature of ca. 8°C on Sepraphore III strips with use of a Gelman No. 51101 chamber (refrigerated) and a Spinco Duostat power supply as described (40, 41). Detection of enzyme activity on the strips after the runs was made either with a coupled-enzyme-dye reaction suitable for adenylate kinase activity (see Refs. 40 and 41) or by staining for protein, clearing, and analyzing densi- tometrically (40). In Fig. 3(P) (A-C), the results of a large number of electrophoretic studies of the rabbit muscle myokinase on cellulose acetate are described. These data encompassed several buffer species, ionic strengths (0.025 to 0.2), and pH values (2.5-12). A plot of the p&, values obtained at each fixed ionic strength vs (r/2jU2 yielded a linear plot which permitted an extrapolation to a pl, = 1O.60 [Fig. 3(P)]. This value is now in good agreement with the estimate made from its amino acid composition (43) and points to the efficacy of this procedure. Similarly, in Fig. 6(S), the data are presented for the calf isoenzymes at a single (I/2) = 0.05. The apparent pI values differ only slightly for the two calf isoenzymes (viz., 9.6, for the liver adenylate kinase and 10.0, for the muscle myokinase), and they would not be readily

separated by this technique. Anti-enzyme interactions are described

in Fig. 4(P), where it may be seen from Fig. 4A(P) that there is essentially no inhibition of the calf liver enzyme by the anti-calf muscle myokinase (Ab). Not shown here are the data at relatively large ratios of Ab to calf muscle enzyme (Ag; initially at 9.9 x 1OWB M), where close to 100% inhibition is achieved. On the basis of mass action, for the case of (a) a stoichi- ometryof2Agto1Ab,i.e.,forEZ*Ie2 E + I, &’ = [(E)2(I)I/(E,I), where I = Ab, E = Ag, and i = 1 - (uJIJ,,) = fraction inhibition, the following equation may be derived:

It (X = (2 - 4’ Kd) + E

(4 (2 - 2i)’ E, t’

where subscript t implies total concentration. A plot of the left-hand side vs [(2 - i)/(2 - 2i)12 should be linear with Et = the ordinate intercept and the slope = J&‘/E,. For (b) a stoichiometry of 1 Ag to 1 Ab, i.e., for E.1 * E + I, Kd’ = [(E)(I)I/EI, the equation

results, and a plot of 1,/i vs [l/(1 - i)l should be linear with a Y-intercept = Et and a slope = Kd ’ . As may be seen [Fig. 4C(P)l, case (a> appears to hold and a Kd’ = 2.7 x lo-l4 M* may be calculated at 37.5”C for the stoichiometry of 2 Ag to 1 Ab. As a measure of self-consistency, Et is estimated to be 8.7 x lo-* M and close to the 9.9 x 10V8 M calf muscle myokinase added initially, but there may have been some inactive enzyme in the sample. The inhibition reaction of Ag and Ab appears to follow a second-order reaction which takes into account the stoichiometry of 2:1, i.e.,

h I = 2.303 b (a - 2x) t t(a - 2b)

log - ~ 0 a (b - x) ’

and yields a value of K ’ = 5.0 x 106 M-’ * min-’ at 30°C. An Arrhenius plot is linear [Fig. 4B(P)] and an energy of activation of 6.3 kcal/mol is obtained for the reaction of rabbit anti-enzyme and calf muscle myokinase. Not shown here is the

44 KUBY ET AL.

6. APPARENT ISOELECTRIC POINTS FROM CELLULOSE ACETATE ELECTROPHORESIS OF RABBIT MUSCLE MYOKINASE

GLYCINE-NaOH ,Y,-,\

A. CELLULOSE ACETATE STRIP FI FCTQWWY!FSIS t6

OF RABBIT MUSCL

,KCl,

I -.

,, L-C”.,.“, ..-. -- - O.OSN BUFFER .E MYOKINASE

r : +4 10m3M P-MERCAPTOETHANOL 7 i

-0.025 -0.050 -O.,OO 1

0.051 BUFFER m ,ON,C STRENGTH ADJUSTED WITH KCI

p t2

0 r co-

- 0.025~ - 0.025M BUFFER &Jo 0 - -

t30-

O-

-IO- 0.

-20 4

c I I I 4

pHkEASURE0 0151 IO 12 10.0 11.0

ptt htEASUREOot5') ISOELECTRIC POINT E,XTRAPOLATED TO ZERO

C. IONIC STRENGTH FOR RABBIT MUSCLE MYOKINASE

/ pl, = 10.60

PI IO.? OPP

10.00:7 0.6

If i

FIG. 3(P). Cellulose acetate strip electrophoresis of crystalline rabbit muscle adenylate kinase. (A) Electrophoresis on cellulose acetate (Sepraphore III, 2.5 x 17-cm strips from Gel- man) as a function of pH measured at 5°C at several fixed ionic strengths; 150 V (7 to 10 mA) for 60 min in a Gelman chamber 51101 (refrigerated) and a Spinco Duostat power supply. O-O, ionic strength (I/2) = 0.025; 0-Q ionic strength (I/2) = 0.050; A-A, ionic strength (I/2) = 0.100. 0, 0, and A, 0.05 M buffer (with ionic strength adjusted with KCl); all with 0.1 mM EDTA and 1 mM P-mercaptoethanol [see also B and Fig. 6(S) insert for list of buffers]. Izi---izI, ionic strength (I/2) = 0.025, 0.025 M buffer at 0.025 (I/2). I = imidazole and T = Tris at same pH and (I/2). 0 and A, cacodylate buffer. Relative electrophoretic mobility is expressed in millimeters per hour. (B) Electrophoresis in the neighborhood of its apparent isoelectric point (pH 10-11) at several fixed ionic strengths in 0.05 M glycine-NaOH (KC11 or 0.05 M /3-alanine-NaOH (KC11 buffer containing 0.1 mM EDTA and 1 mM /3-mercaptoethanol. Estimated values for pl.,, (apparent isoelectric points) are given for each (I/Z). (0 A plot of pl,,, (from B) vs (I/2)L’z to yield pZO = 10.6,.

observation9 that the anti-enzyme will also tion of the calf muscle myokinase is com- inhibit the human muscle-type enzyme as pared with that derived from the structure well, pointing to a similarity in structures. of the rabbit muscle myokinase’O (see Ref.

B . Chemical Properties 42), which in turn is in good agreement with that estimated earlier (43). The ho-

In Table IV(P), the amino acid composi- lo Kuby et al., unpublished work (and see Ref. y Tsai and Kuby, unpublished work. 42) and manuscript in preparation, 1978.


0 L -m

FIG. 4(P). Titration of calf muscle myokinase and calf liver adenylate kinase by purified rabbit anti-calf muscle myokinase. (A) Residual activity (percentage) vs anti-calf muscle myokinase at pH 7.7, 0.2 (I/2) buffer (50 mM Tris, 37 mM HCl, 0.163 M NaCl, 1 mM EDTA, 1 mM DTE, 1 mg/ml of albumin) at 37°C for 6 h. O-O, calf muscle myokinase, initially 9.9 x 1O-8 M; A-A, calf liver adenylate kinase. (B) Arrhenius energy of activation for reaction of calf muscle myokinase (Ag) and purified rabbit anti-calf muscle myokinase (Ab). (C) Kinetic determination of the dissociation constant for the reaction of calf muscle myokinase with anti-calf muscle myokinase.

mology is surprisingly high and they differ in only 10 out of 193 residues with but a few notable differences, e.g., the two histidines in the calf myokinase versus the three histidines in the rabbit myokinase.

C. Kinetic Properties

A more complete description of the kinetic parameters and their evaluation for the calf isoenzymes and for the rabbit muscle myokinase will be presented elsewhere (see Footnote 6). For the present, an interesting comparison is shown of the potent inhibition by p1,p5-di(adenosine- 5’)pentaphosphate or ApsA (44) of rabbit and calf muscle myokinase compared to the case of the calf liver adenylate kinase [see Figs. 7(S)-9(S)]. Thus, for the forward reaction (i.e., MgATP*- + AMP*- +I, ApsA acts as a competitive inhibitor with

respect to either substrate, with Ki = Cl- 0.5) x 10e8 M for both muscle enzymes, but, surprisingly, a 30-80 times larger value is obtained for the liver-type enzyme [or (3-8) x lo+ MI [Table V(P)]. Thus, while 60-70% inhibition may be obtained at 3 x 10e8 M ApsA for either muscle-type enzyme, 2 x 10” M ApsA is required to yield 70-80% inhibition of the liver type, pointing either to a more rigid structure for the liver type or to a different mecha- nism of inhibition.

Of interest is the observation that ApsA acts as a noncompetitive inhibitor of the muscle types for either substrate in the reverse reaction (i.e., with respect to either ADP3- or MgADP-), with a Ki = (2- 0.6) x lo-* M for the muscle enzymes, but with a Ki of only (3-4) x lo* M for the liver enzyme. Therefore, although the “multisubstrate inhibitor” (44) ApRA may

46

TABLE IV(P)

KUBY

AMINO ACID COMPOSITIONS OF CALF MUSCLE AND RABBIT MUSCLE ATP-AMP TRANSPHOSPHORYLABE

Amino acid Calf mus- Rabbit cle myoki- muscle

nase myoki-

number of PolY- residues peptide per poly- chain of peptide molecular chain of

molecular weight 21,400

weight from 21,400) structure10

Aspartic acid 130 13’ Threonine 14 12 Serine 10 10 Glutamic acid 26b 26” Proline 7 6 Glycine 18 18 Alanine 10 12 Valine 15 16 Methionine 6 5 Isoleucine 8 9 Leucine 18 18 Tyrosine 7 7 Phenylalanine 5 5 Lysine 20 20 Histidine 2 3 Arginine 12 11 Half-cystine (as cysteine) 2 2 Tryptophan 0 0

Total residues per chain 193 193

a Includes asparagine. b Includes glutamine.

act, in part, as a transition state analog, additional explanations are required in view of the peculiar and mixed nature of its inhibition pattern of the muscle types and the less effective inhibitory nature of the ApsA with respect to the liver type.

IV. DISCUSSION

For the isolation of the cytoplasmic muscle type on a relatively large scale it is convenient to employ, for the early steps, the acid denaturation step from the 10 mM KC1 extract, followed by the zinc precipitation step, similar to that first described in Ref. 1 for the rabbit muscle myokinase. However, for the enzyme from liver, which is largely mitrochondrial, ho- mogenization in Na,HPO, was found nec-

ET AL.

essary to extract the enzyme, and removal of the cell debris could be readily accom- plished by simply adjusting the pH to 4.6. Before the acid denaturation step could be employed on the relatively dilute enzyme, a zinc fractionation followed by further concentration with (NH&SO, precipitation proved necessary. The liver’extracts were found to contain relatively high protease activities toward the adenylate kinase. Thus, whenever possible, phenylmethanesulfonyl fluoride was employed for partial inhibition of these activities. But, nevertheless, toward the end of the isolation scheme, some protease activities were apparently concentrated together with the enzyme and were not inhibited. The use of 5’-AMP was effective in stabi- lizing the enzyme against their attack. The low-capacity phosphocellulose cbro- matography at pH 7.1 with Na+ gradients proved to be a very effective cbromato- graphic tool for preparing almost 70-80% pure muscle-type myokinase. Curiously, under similar conditions, the liver type acts as if it were more basic to the low- capacity phosphocellulose column, peak- ing at ca. 8 mmho [Fig. 2B(S)] vs ca. 4.5 mmho for the calf muscle enzyme [Fig. lA(S)]. Yet their apparent isoelectric points at 0.05 (r/2) as measured by cellulose acetate electrophoresis are ca. 9.6 for the liver enzyme vs ca. 10.1 for the muscle enzyme [Fig. 6(S)]. Incidentally, should both isoenzymes be present in significant amounts in the same cell, rather than attempting their separation by electropho- retie methods, low-capacity phosphocellulose chromatography would be the method of choice.

For the muscle type, it is convenient to employ a Sephadex G-100 gel filtration step conducted at high ionic strengths [viz., 1.0 M (NH,),SO,I to remove any traces of denatured protein, followed by crystallization and recrystallization to ho- mogeneity. For the liver type, after Frac- tion IV [see Table III(P)], the following order of chromatographies proved most effective: a DEAE-cellulose filtration step at first to remove greenish-colored proteins, followed successively by first a high- capacity cellulose phosphate chromatogra-

CRYSTALLINE ADENYLATE KINASE ISOENZYMES

TABLE V(P)

47

INHIBITION OF ATP-AMP TRANSPHOSPHORYLASES BY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

Enzyme source

Forward reaction

Ki (with varied MgATP*-)

K,

Rabbit muscle Calf muscle

Comp. u Comp.

4.6, (ko.20) X lo-’ M 5.2, (20.12) x 1O-9 M

Calf liver

Comp.

2.9, (20.23) X lo-’ M

(with varied AMP2-) 9.2, (20.84) x 1O-9 M 1.1, (kO.10) x 10-e M 8.49 (k0.35) X lo-’ M

Reverse reaction Non-Comp. (1 Non-Comp. Non-Comp.

K, (with varied MgADPm) 5.6, (k0.44) X 1o-9 M 1.83 (~0.57) x 1O-8 M 2.5, (20.55) x 1O-6 M

K, (with varied ADP3-) 1.9, (20.59) X lo-’ M 2.0, (e0.12) X lo-’ M 3.9, (20.68) X lo@ M

a Comp., competitive inhibition; Non-Comp., noncompetitive inhibition.

phy step (because of the relatively high protein load at this stage) over a steep Na+ gradient and then a low-capacity phosphocellulose step with a narrow Na+ gradient to remove essentially all of the reddish-colored proteins and to provide almost a 60-fold purification. This was followed by a carboxymethyl cellulose chromatography with Tris+ gradients, a Seph- adex G-100 gel filtration as with the muscle-type enzyme, and one crystallization to obtain homogeneous protein after ca. a 700-fold purification and at a sufficient scale to permit sequence studies. Both calf isoenzymes, as well as the improved preparation of the rabbit muscle myokinase, seemed to be homogeneous by sedimentation velocity, by dodecyl sulfate-polyacrylamide gel electrophoresis, and by cellulose acetate electrophoresis under a variety of conditions.

Calculations based on the amino acid composition, neglecting charge effects (43) of the rabbit muscle myokinase, which was originally assigned an isoelectric point of 6.1 by liquid boundary electrophoresis in 0.1 (I/2> phosphate or Veronal- NaCl buffers at 0.8% protein concentration, demonstrated that the isoelectric point would have to be greater than 9. This anomalous electrophoretic behavior has since been found in the porcine myokinase which was sequenced (48), in the human muscle enzyme (61, in the adenylate kinase from human erythrocytes (141, and also now in the calf muscle my-

okinase (42; see above). Explanations are now offered here which invoke a type of nonideality in electrophoretic measurements resulting from the relatively high protein concentrations employed. Thus, at microgram or nanogram levels, the protein migrates uniformly on cellulose acetate and yields a calculated plo, extrapolated to zero ionic strength, of ca. 10.6 [see Fig. 3(P)], which is now in good agreement with the theoretical calculations (43). Similarly, the calf muscle isoenzyme gives a papp = 10.1 at 0.05 r/2) [Ref. 42; see Fig. 6(S)l. Although the calf isoenzymes are similar in some properties [cf. p&, = 9.6 for liver type, Fig. 6(S)], they differ significantly in their molecular weights (26,000 vs 21,400 for the liver type and muscle type, respectively) and especially in their immunological behavior. Interestingly, the homologous muscle types (calf and rabbit, which differ in their amino acid composition in only 10 out of 193 residues) did not prevent the calf muscle enzyme from acting as a good antigen in the rabbit. The rabbit anti-muscle protein proved to be amenable to purification from the antiserum and yielded a typical globulin structure with apparently two heavy chains and two light chains with an overall molecular weight of 134,000 [Fig. 5(S)l. This globulin acted as a powerful inhibitor only toward the muscle type, being largely unreactive toward the liver type, and its intrinsic dissociation constant could be evaluated kinetically

48 KUBY ET AL.

[Fig. 4(P)] for a 2:l stoichiometry (of 2 Ag:l Ab) of 3 x lo-l4 Mu. Moreover, the anti-calf muscle globulin also acts as an anti-enzyme toward the human muscle myokinase, pointing to a very high degree of homology between their structures (see Ref. 20).

But most interesting is the finding presented here that Ap,A (44) does not act similarly as an inhibitor toward both muscle type and liver type and requires almost two orders of magnitude higher concentrations to cause the same percentage inhibition in the liver type as in the muscle type [Table V(P)]. Moreover, for both the muscle-type and liver-type enzyme, the nature of the inhibition changes qualitatively from competitive inhibiton with respect to either substrate in the forward reaction (MgATP*- or AMP2-) to noncompetitive in the reverse reaction with either substrate (MgADP- or ADP3-) [see Figs. 7@)-9(S)]. Thus, in agreement with Ref. 44, it is too trivial an explanation for the “multisubstrate inhibitor” Ap5A to act as a transition state analog, and a more so- phisticated explanation must be sought. One notes that ApsA would likely bear a net charge of 5- under the pH 7.4 conditions of the kinetic studies employed here, and, at lO+ to lOpa M initial concentrations of ApjA in the presence of 1 mM uncom- plexed Mg2+, it would likely be converted quantitatively to a complex bearing a net charge of l-, i.e., to Mg2(ApsA)-. The explanation for its action as a noncom- plexed inhibitor with respect to ADP3-, for example, or its competitive nature with respect to MgATP2- could then lie in the structure of the magnesium chelate itself, which may or may not contain within it a structure analogous to that of the particu- lar substrate being varied. The explanation for the huge differences inKi between muscle type and liver type may lie in a more rigid structure for the liver type. Thus, as indicated in Ref. 42, from substrate binding studies on fragments of the rabbit muscle myokinase, Residues l-44 of the rabbit muscle myokinase may contain the binding sites for MgATP2- and MgADP-, and the terminal sequence of Residues 171-193 may contain the binding

sites for AMP2- and ADP2-. Therefore, for an interaction between the binding sites to occur, a head-to-tail bending of the molecule must take place. Such a large conformational change in a more rigid structure would then make the multisubstrate inhibitor Ap,A a much less effective inhibitor. Therefore, it will be of interest to compare eventually the structure of the liver-type and muscle-type isoenzymes.

We gratefully acknowledge the assistance of the following individuals: Mr. Stephen Hofmeister, Mr. P. Parham, Mr. R. Tainter, and Ms. Linda Millsaps.

1. NODA, L., AND KUBY, S. A. (195’7) J. Biol. Chem. 226, 541449.

2. NODA, L, AND KDY, S. A. (1963) in Methods in Enzymology (Colowick, S., and Kaplan, N. O., eds.), Vol. 6, p. 223, Academic Press, New York.

3. NODA, L., AND KUBY, S. A. (1957) J. Biol. Chem. 226, 551-558.

4. KUBY, S. A., PALMIERI, R. H., OKABE, K., YUE, R., JACOBS, H. K., FRISCHAT, A., AND CRESS, M. C. (1978) Submitted for publication.

5. SCHIRMER, I., SCHIRMER, R. H., SCHULZ, G. E., AND THUMA, E. (1970) FEBS. Lett. 10, 333- 338.

5a. NODA, L., SCHULZ, G. E., AND VON ZABERN, I. (1975) Eur. J. Biochem. 51, 229-235.

6. THUMA, E., S~HIRMER, R. H., AND SCHIRMER, I. (1972) Biochim. Biophys. Actu 268, 81-91.

7. MARKLAND, F. S., AND WADKINS, C. L. (1966) J. Biol. Chem. 241, 4124-4135.

8. MARKLAND, F. S., AND WADKINS, C. L. (1966) J. Biol. Chem. 241, 4136-4145.

9. CRISS, W. E., SAPICO, V., AND LITWACK, G. (1970) J. Biol. Chem. 245, 6346-6351.

10. SAPICO, V., LITWACK, G., AND CRISS, W. E. (1972) B&him. Biophys. Acta 258,436-445.

11. CHIGA, M., AND PLAUT, G. W. E. (1960) J. Biol. Chem. 235, 3260-3265.

12. CRISS, W. E., PRADHAN, T. J., AND MORRIS, H. P. (1974) Cancer Res. 34, 3062-3065.

13. PRADHAN, T. J., AND CRISS, W. E. (1976) Enzyme 21, 327-331.

14. TSUBOI, K. K., AND CHERVENKA, C. H. (1975) J. Biol. Chem. 250, 132-140.

15. CHIN, C.-S., Su, S., AND RUSSELL, P. J. (1967) B&him. Biophys. Actu 132, 361-369.

16. FILDES, R. A., AND HARRIS, H. (1966) Nature (London) 209, 261-263.

17. GIBLETT. E. R. (1969) Genetic Markers in Hu- ,

ACKNOWLEDGMENTS

REFERENCES


man Blood, pp. 512-519, F. A. Davis, Phila- 35. WEBER, K., AND OSBORN, M. (1969) J. Biol. delphia. Chem. 244, 4406-4412.

18. BROWNSON, C., AND SPENCER, N. (1972) Bio- 36. PALMIERI, R. H., YUE, R. H., JACOBS, H. K., them. J. 130, 797-804. MALAND, L., WV, L., AND KUBY, S. A. (1973)

19. BROWNSON, C., AND SPENCER, N. (1972) Bio- J. Biol. Chem. 248, 4486-4499. them. J. 130, 805-811. 37. KRESS, L. F., BONO, V. H., AND NODA, L. (1966)

20. VON ZABERN, I., WITTMANN-LIEBOLD, B., UN- J. Biol. Chem. 241, 2293-2300. TUCHT-GRAU, R., SCHIRMER, R. H., AND PAI, 38. YPHANTIS, D. A. (1964) Biochemistry 3, 207. E. F. (1976) Eur. J. Biochem. 68, 281-290. 39. DAVIS, B. J. (1964) Ann. N.Y. Acad. Sci. 121,

21. KLETHI, J., AND MANDEL, P. (1968)Nature (Lon- 404. don) 218, 467-468. 40. YUE, R. H., JACOBS, H. K., OKABE, K., KEUTEL,

22. RUSSELL, P. J., HORENSTEIN, J. M., GOINS, L., H. J., AND KUBY, S. A. (1968) Biochemistry 7, JONES, D., AND LAVER, M. (1974) J. Biol. 4291-4298. Chem. 249, 1874-1879. 41. KEUTEL, H. J., OKABE, K., JACOBS, H. K.,

23. KEUTEL, H. J., JACOBS, H. K., OKABE, K., YUE, ZITER, F., MALAND, L., AND KUBY, S. A. R. H., AND KUBY, S. K. (1968) Biochemistry 7, 4283-4290.

(1972) Arch. Biochem. Biophys. 150, 648-678.

24. MAHOWALD, T. A., NOLTMANN, E. A., AND 42. KIJBY, S. A., HAMADA, M., PALMIERI, R. H.,

KUBY, S. A. (1962) J. Biol. C&m. 237, 1535. TSAI, W. C., JACOBS, H. K., MALAND, L.,

25. OLSON, 0. E., AND KUBY, S. A. (1964) J. Biol. Wu, L., AND FISCHER, A. (1976) Fed. Proc.

Chem. 239, 460. Abstr. (ASBC Abstr.) 35, 1629.

26. KUBY, S. A. (1967) Nat. Acad. Sci. Nat. Res. 43. MAHOWALD, T. A., NOLTMANN, E. A., AND

Count. Publ. 1344, 247. KUBY, S. A. (1962) J. Biol. Chem. 237, 1138-

27. OLIVER, I. T. (1955) Biochem. J. 61, 116. 1145.

28. GORNALL, A. G., BARDAWILL, C. J., AND DAVID, 44. LIENHARD, G. E., AND SECEMSKI, I. I. (1973) J.

M. M. (1949) J. Biol. Chem. 177, 751. Biol. Chem. 248, 1121-1123.

29. ARNON, R., AND SHAPIRA, E. (1968) Biochemistry 45. JACOBS, H. K., AND KUBY, S. A. (1970) J. Biol.

7, 4196. Chem. 245, 3305-3314.

30. SANDERS, M. M., WALSH, K. A., AND ARNON, R. 46. KUBY, S. A., AND NOLTMANN, E. A. (1962) in

(1970) Biochemistry 9, 2356. The Enzymes (Bayer, P. D., Lardy, H., and

31. SACHS, D. H., AND PAINTER, E. (1972) Science Myrback, K., eds.), 2nd ed., Vol. 6, p. 515,

175, 781-782. Academic Press, New York.

32. LKB Instruction Manual (1968) No. l-8100-EOl, 47. KUBY, S. A., MAHOWALD, T. A., AND NOLT-

LKB, Stockholm, Sweden. MANN, E. A. (1962) Biochemistry 1, 748-762.

33. NOLTMANN, E. A., GUBLER, C. J., AND KUBY, S. 48. HEIL, A., M~~LLER, G., NODA, L., PINDER, T., A. (1961) J. Biol. Chem. 236, 1225-1230. SCHIRMER, H., SCHIRMER, I., AND VON ZA-

34. RATLIFF, R. L., WEAVER, R. H., LARDY, H. A., BERN, I. (1974) Eur. J. Biochem. 43, 131-141. AND KUBY, S. A. (1964) J. Biol. Chem. 239, 49. ANSARI, A. A., AND SALAHUDDI~, A. (1973) Bio- 301. them. J. 135. 705-711.

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Studies on adenosine triphosphate transphosphorylases: Isolation and several properties of the crystalline calf ATM-AMP transphosphorylases (adenylate kinases) from muscle and liver