TIIE JOURNAL OB RIOLOGICAL CIIE~~ISTR~ Vol. 239, No. 2, February 1964

Printed in U.S.A.

The Binding of Pyrophosphate and Adenosine Triphosphate to Myosin*

A. MARTOITOSI~ AND H. MEYER

From the Department oj Muscle Research, Institute of Biological and Mrdical Xciences, Retina Foundalion, Boston i4, Massachusetts

(Reccircd for publication, May 17, 1963)

Under appropriate conditions, the myosin-catalyzed hydrolysis of B-amino nucleotides is activated by dinitrophenol (2, 3), ethylenediaminetetraacetic acid (4), and low concentrations of sulfhydryl reagents (2), while that of the 6-osynucleotides is inhibited. An explanation of this effect involving a hypothetical interaction of the B-amino group of the substrate with the en- zyme has been put forward on the basis of a kinetic analysis of 6-osy- and 6-aminonucleotide triphosphstase activity of myosin

(5). The inhibitory effect of mercurials, dinitrophenol, and EDTA

on the specific interaction of pyrophosphate with myosin (6) in concentrations known to activate myosin ATPase raised the pos- sibility that, in addition to, or instead of, the postulated effect on the interaction of the B-amino group of ATP with the enzyme, the effect of modifiers is exerted through an influence on the binding of the pyrophosphate chain of ATP molecule.

The experiments here reported represent an attempt to gain a direct insight into the events underlying activation or inhibition of myosin nucleotide triphosphatase through an analysis of the enzyme substrate interaction in the presence of modifiers.

Although no definite conclusion has been obtained regarding the exact nature of the effect of modifiers on the interaction of substrates with myosin, new information has been obtained con- cerning the existence of specialized groups within the active center involved in the interaction of pyrophosphatc and perhaps the triphosphatc chain of ATP with myosin.

EXPERIMENTAL PROCEDURE

The myosin has been prepared as described earlier (6). In a few cases the KI procedure described by Szent-Gyorgyi has been used (7). A tropomyosin-free actin preparation was obtained by the Feuer-Straub extraction method (8) followed by a recently described purification process (9).

Preparation of Creatine Phosphokinase-Creatine phosphoki- nasc has been prepared by a slightly modified method of Padieu and Mommaerts (10) and of l&by, Noda, and Lardy (11). Rabbit muscle was ground and extracted in cold with 2 volumes of 0.01 M KC1 for 15 minutes. Filtration followed through six layers of cheesecloth. The filtrate was centrifuged in an Tn-

* The work was supported by Public Health Service Research Grants B-2175-(31 and H-5949 and by grants from the National Science Foundation, the Life Insurance Medical Research Fund, and the Muscular Dystrophy Associations of America, Inc. A preliminary report was presented at the Conference on the Bio- chemistry of Muscle Contraction, Dedham, 19G2 (1).

t This work was performed during the tenure of an Established Invest,igatjorship from the American Heart Association.

ternationsl refrigerated centrifuge at 1600 X g for 15 minutes. Scetone (-18”) was added to 30% concentration, and after standing at -10” for 15 minutes, the solution was centrifuged at 1600 x g for 30 minutes at -10”. To the supernatant, cold acetone ( - 18”) was added to 60% concentra.tion, followed by 15 minutes of incubation at -10”. The precipitate was dissolved

in 0.01 M glycine buffer, pH 9, and dialyzed against the same solution. Ethanol (36%) was added, and after standing for 30 minutes at -2O”, the solution was centrifuged. The precipitate was dissolved in 0.01 M glycine buffer, pH 7.0, and dialyzed against the same solution. Ethanol was added to 50% concm- tration and the previous step was repeated. The precipitate was

dissolved in glycine buffer, 0.01 mole, pH 9, and dialyzed against the same solution. The material was stored in lyophilized state at -20”. Firefly extract was prepared according to Nanninga and Mommaerts (12).

Conditions for J[easurement of ATP Binding by Firefly Lumi- nescence Method (12, IS)-To 2.4 ml of a solution containing 0.04 M Na2HAs04, 0.02 M MgC12, and 0.3 M Na2S04, 2.5 to 3 mg of creatine phosphokinase per ml (freshly dissolved before the start of the experiment), 0.06 ml of 0.05 M creatine phosphate, and 0.2 ml of firefly enzyme were added. The steady light emission was measured in a Brice-Speiser light-scattering photometer following the addition of 0.1 ml of ATP in increasing concentrations. The emitted light intensity increased linearly with the ATP conccn- tration between 1 PM and 40 KM.

On the addition of 0.3 ml of a myosin solution or equal amounts of Na2S04 to the system, the light intensity dropped owing to dilution. Additional decrease in light intensity has been inter- preted as binding of ATP to myosin, and t.he amount of bound ATP was calculated essentially according to Nanninga and Mom- maerts (12, 13). Prior to the measurement, the myosin was precipitat’ed by dilution with 12 volumes of 1 mM Tris, pH 7, and the precipitate was dissolved in 0.3 M NazS04, pH 7.

In the experiments to be reported, the total amount of ATP per test system (2.76 and 3.06 ml, before and after, respectively, the addition of 0.3 ml of myosin or 0.3 M Na2S04) was between 7.5 and 15 m,nmoles.

Measurement of ATP Binding by Precipitation JFethod-M3;o- sin, 0.5 ml (protein concentration between 7 and 21 mg per ml), Fas precipitated with 6 ml of 1 mM Tris, pH 7.6, and the precipi- tate was washed once with the same solution. To this were added 2 ml of a solution containing 1 mM MgC12, 10 mM Tris, pH 7.6, 1 mM creatinc phosphate, 3 mg of creatine phosphokinase per ml, 2.5 to 5 pg of 14C-ATP per ml (2 to 5 PC per mg), and unlabeled ATP (if indicated in the figures), and the sample was

630

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February 1964 A. Martonosi and H. Meyer 641

incubated for 5 to 6 minutes at 2-4”. The myosin was removed by centrifugation and the radioactivity of the supernatant was measured. Three controls were used: (a) without myosin; (b) with heat-inactivated myosin; and (c) with native myosin in the presence of 5 mM nonradioactive ATP.

To test the effectiveness of the ATP-regenerating system, an aliquot was applied to Whatman No. 4 paper, and the nucleotides were separated by descendent chromatography in isobutyric acid- HzO-30% NH40H (66:33: 1) solvent. The radioactivity of the ATI’ and ADP spots were measured. The ,4TP level in the solution was maintained reasonably well by the kinase system except at the lowest ATP concentrations, where a decrease of about 25% was observed. The effectiveness of the rephosphoryl- ating system is also indicated by the fact that the control and EM-treated’ myosin preparations with widely different ATPase activities bind approximately identical amounts of ATP (Figs. 1 and 3).

!Weasurement of Pyrophosphate Binding to Myosin (6)-Pyro- phosphate binding was measured by equilibrium dialysis with 2.5 ml of protein solution in a dialysis bag of about &inch diame- ter and 5 ml of outside medium that contained radioactive pyrophosphate and other constituents described in the legends. Dialysis was performed at 4” for 36 to 48 hours. The radioac- tivity of the solution, inside and outside the dialysis bag, was measured in a Packard liquid ccintillation counter essentially according to Loftfield and Eigner (14).

Preparation of Radioactive Pyrophosphate-From I to 3 me of 32P and 100 pmoles of K2HPOd were placed in a platinum con- tainer. The pH was adjusted to about 8.5, and the solution was evaporated to dryness under an infrared lamp. The material was heated for 3 minutes in an oxygen flame, cooled in a vacuum desiccator, and dissolved in about 10 ml of HzO. Column chromatography was performed on a Dowex I-HCOI column (1 x 18 cm) under the conditions described earlier (15). The pyrophosphate containing fractions were pooled and washed Dowes 50-X8 resin was added in the H+ form (16) with stirring until no more COZ was liberated. The KHCOS-free PPi solution was lyophilized and dissolved in HSO. The concentration of PPi was determined following hydrolysis in 1 M HCl for 10

minutes at 100” by the Fiske-SubbaRow method (17). The solution was stored at -20”.

Conditions for Measurement of ATPase Activity-Calcium- activated ATPase: 0.1 M Tris (pH 7.6), 10 mM CaC12, 1 mM ATP, 0.03 1~ KCl, and 0.07 mg of myosin per ml in a total volume of 2 ml. Incubation was for 5 or 10 minutes at room temperature.

EDTA-activated ATPase: 0.1 M Tris (pH 7.6), 0.6 M KCI, 1 mM EDTA, 1 mM ATP, and 0.05 to 0.07 mg of myosin per ml. Total volume, 2 ml. Incubation was for 5 to 10 minutes at room temperature.

F-actin-activated ATPase: 0.01 M Tris (pH 7.6), 0.03 M KCl, 1 mM MgC12, 1 mM ATP, 0.05 to 0.07 mg of myosin, and 0.015 to 0.03 mg of F-actin per ml of incubation mixture. Total volume, 2 ml.

Viscosity measurements were made in an Ostwald viscometer

at 4” or at room temperat.ure. -SH groups were determined by amperometric titration with

ilgN03 according to Benesch, Lardy, and Benesch (18) or by spectrophotometric titration with CMB according to Boyer (19).

1 The abbreviations used are: EM, N-ethylmaleimide; CMB, p-chloromercuribenzoate.

Materials-All reagents used were of analytical reagent grade. Nucleoside polyphosphates were purchased from Pabst Labora- tories. EM was obtained from Schwarz BioResearch, Inc. Chelating agents were the products of Dojin Yakukagaku Ken- kyusho, 38 Kamidori-Machi, Kumamoto-Shi, Japan.

RESULTS

Effect of N-Ethylmaleimide on Interaction of PPi with Myosin-

It was first reported by Kielley and Bradley that additionof small amounts of CMB or EM to myosin increased the calcium-acti- vated and decreased the EDTA-activated ATPase activity (20). Reduction of the number of -SH groups to or below 3 or 4 moles of -SH per lo5 g of myosin, with higher concentrations of -SH reagents, gradually abolished both the calcium- and EDTA- activated ATPase and the inorganic triphosphatase activity of myosin (Fig. 1).

Myosin preparations in which Ca-activated STPase activity was inhibited by treatment with high concentration of EM were found to bind considerable amounts of pyrophosphate at both high and low free pyrophosphate concentrations, and this func- tion remained largely unaffected even when the number of -SH groups decreased to 1 to 2 moles of -SW per 1Oj g of myosin (Fig. 1).

k MOLES NEW MOLE MYOSlN

FIG. 1. The effect of EM treatment on the ATPase activity and pyrophosphate and ATP binding to myosin. Myosin (4.5 mg per ml) in 10 mM Tris, pH 7.6, was incubated with EM in concentra- tions indicated on the abscissa for 2 hours in an ice bath. Excess EM was removed by dialysis against 0.6 M KC1 and 10 mM Tris, pH 7.6. ATPase measurements were made as described in “Ex- perimental Procedure.” Inorganic triphosphate hydrolysis was measured in a solution of 10 mM CaCl2, 0.1 M Tris, pH 7.6, 1 rnM inorganic triphosphate, and 2.3 mg of myosin per ml. Incubation was for 60 minutes at room temperature. Pyrophosphate binding was determined by dialyzing 2.5 ml of EM-treated myosin against a solution which contained 0.6 M KCl, 1 mM Tris, pH 7.6, 1 rnM MgC12, and 1.5 pM or 10 PM Pa2Pi. For ATP binding, 4.95 mg of myosin, precipitated in 0.046 M KC1 and 1 mM Tris, pH 7.6, were resuspended in a solution containing 1 mM MgCln, 10 mM’ Tris, pH 7.6, 10 mM creatine phosphate, 3 mg of creatine kinase per ml, and 4.05 pM ATP-I%. Total volume, 2.4 ml. Incubation was for 5 minutes in an ice bath. Myosin was removed by centrifuga- tion, and the radioactivity of the supernatant was determined. Heat-inactivated myosin or samples containing 5 rnM unlabeled ATP served as control. The myosin preparation used in this experiment was activated by EM in the presence of Ca (10 moles of EM per mole of myosin) (see text for further explanation). e- - - -0, ATP binding; A- ---A, PPi binding at 10 PM PPi concentration; l - ---0, PPi binding at 1.5 fiM total PPi con- centration; O--O, inorganic triphosphate hydrolysis; A--A, EDTA-activated ATPase; W--W, Ca-activated ATPase; +--+, a&in-activated ATPase; O-0, ATPase without, activator.

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642 Substrate Binding to Myosin

CONCENTRATION OF FREE Pe M

FIG. 2. Pyrophosphate binding to control and EM-treated myosin Myosin was treated with 30 moles of EM per 500,000 g of protein in 0.6 M KC1 and 0.01 M Tris, pH 7.6, for 7 hours in an ice bath, followed by dialysis against 0.6 M KC1 and 0.01 M Tris, pH 7.6. The Ca-activated ATPase activity was less than 0.01 fimole of Pi per mg of protein per minute at room temperature. Equilibrium dialysis was carried out under the usual test condi- tions in 0.6 M KCl, 50 mM Tris (pH 7.2), and I mM MgC12. The free pyrophosphate concentrat,ions are indicated on the abscissa. O--O, control myosin; O--O, ERI-treated myosin.

6 5 4 3 2 1 0

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SH GROUPS/ IO 5 G MYOSIN

FIG. 3. Effect of EM on the ATPase activity, actin combina- tion, and pyrophosphate binding of myosin. Myosin (13.4 mg per ml) was treated with increasing concentrations of EM (0 to 0.4 rmole per mg of protein) in a medium containing 0.6 M KCl-10 mM Tris buffer (pH 7.6), for 2 days, followed by dialysis against 100 volumes of 0.6 M KC1 and 1 mu Tris (pH 7.6), overnight. De- termination of ATP binding by the precipitation and firefly lu- minescence method and of the Ca-activated ATPase activity were carried out as described in “Experimental Procedure.” Py- rophosphate binding was measured in the presence of 0.6 M KCl, 0.05 M Tris (pH 7.6), 1 mM MgCl2, and 4.35 PM PPi.

The combination of myosin with actin was measured with a Brice-Speiser light-scattering photometer at 90” to the incident beam (ZgO). Readings were made on actin, myosin, actomyosin, and on actomyosin following the addition of 1OW M ATP. All readings were corrected for the scattering by the protein-free solution. The results were plotted as: I, [Z90 actomyosin (AM)/ Zgo actin (A) + Zgo myosin (iM)] - 1; 2, [Zso actomyosin (AM)/Zgo actomyosin (AM) + ATP] - 1. A--A, ATP binding (precipi- tation test); +--+, ATP binding (firefly method); U--W, (A&Z/(/l + M)) - 1; O----o, (AJ!Z/(BM + ATP)) - 1; O--O, PP; binding; & - - -A, Ca-activated ATPase.

-

30 20 IO

SH GROUPS/MOLE OF MYOSIN FIG. 4. The effect of t.he firefly luminescence test medium on

the pyrophosphate binding of control and EM-treated myosin. Myosin samples (14 mg per ml) were treated with 0 to 47 moles of EM per mole of myosin in a medium containing 0.1 M Tris, pH 7.6, and 0.6 M KC1 for 5 hours in an ice bath. This was followed by dialysis against 0.6 M KC1 and 1 mM Tris buffer, pH 7.6. Py- rophosphate binding: 2.5 ml of myosin were dialyzed against 5 ml of a solution of the following composition. Solution A, 0.6 M KCl, 0.075 M Tris, pH 7.6, 2.5 PM KP3*Pi and 1.5 mM MgCl*; Solu- tion B, 0.45 M NaG?iC,, 0.06 M NazAsO+ 0.075 M Tris (pH 7.6), 2.5 PM KP3*Pi, 1.5 mM MgCl,. Dialysis was for 36 hours at 4”. De- termination of ATPase activity was carried out as described in “Experimental Procedure” before dialysis. O- - - -0, Ca-acti- vated ATPase; O---O, EDTA-activated ATPase; II--m, PPi binding in Solution A; n--n, PPi binding in Solution B.

The concentration dependence of PPi binding to control and

EM-treated myosin (Fig. 2) indicates that neither the maximal number of I’I’i binding sites nor the affinity of myosin to PPi

were affected by treatment with EM.

Not. infrequently, myosin preparations were obtained in which the Ca-activated ATPase activity was inhibited by treatment

with small concentrations of EM or CMB. In agreement with the experiments represented in Fig. 1, after the abolition of the

calcium-, EDTA-, and actin-activated ATPase activities of these

preparations by treatment with EM, the pyrophosphate binding

was only slightly reduced. The ATPase activity of myosin preparations which did not respond to EM treatment (3 to 5

moles per 5 X 1Oj g of myosin) with activation in the presence of calcium was still activated by EDTA, or actin, indicating that

the mechanism of activation in these cases is different.

Treatment of myosin with iodoacetamide results in a parallel loss of ATPase activity and of the ability to form actomyosin as

shown by Barany (21). The data in Fig. 3 extend these findings

by illustrating that, while ATPase activity and actomyosin formation are lost in a parallel fashion on treatment with high

concentrations of EM, the pyrophosphate binding is perfectly

maint,ained. Effect of EM cn Interaction of ATP and ZMyosin-The interpre-

tation of the data concerning the ATP binding to EM-treated

myosin is complicated by differences between the results obtained by two methods used for the determination of the amount of

bound ATP. As shown in Figs. 1 and 3, the amount of bound

ATP, as determined by the precipitation method, depends on the number of -SH groups in a manner similar to the pyrophos-

phate binding. On the other hand, data obtained by the firefly

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February 1964 A. Martonosi and H. Meyer 643

luminescence method indicate close parallelism between the in- hibition of ATPase activity and ATP binding.

It appeared likely that the differences between the results ob- tained by the two methods arose from differences in the composi- tion of the test system. The medium used in the firefly lumi- nescence assay contains NasHAsOl, Na2S04, and firefly extract in addition to the constituents of the medium used in the precipi- tation test.

As shown in Fig. 4, the solution used in the firefly luminescence assay, in fact, inhibits the pyrophosphate binding of myosin and the inhibition becomes more pronounced as t’he number of -SH groups decreases owing to reaction with EM. The decreased stability of the EM-treated myosin-pyrophosphate complex in the presence of Na2S04 and NazH4s04 as compared with the

Tris-KCl-Mg medium, might well account for the differences in values of ATP binding obtained by the firefly luminescence and precipitation methods.

Protective Efects of Pyrophosphate, ATP, and 2,&Dinitro- phenol on Pyrophosphate and ATP Binding to Jlyosin-Blocking of the most slowly reacting -SH groups of myosin with EM

results in a dramatic loss of both STP and pyrophosphate bind- ing. If the treatment of myosin with EM is performed in t,he presence of lop4 M PPi, 1 InM ATP, or ADP, or 1 mM dinitro-

phenol, a considerable protection of the pyrophosphate- and the

ATP-binding sites can be observed even after prolonged treat- ment with 360 to 1200 moles of EM per mole of myosin (Table I).

TABLE I

Effects of pyrophosphate, ADP, dinitrophenol, F-actin, and EDTA on inhibition of pyrophosphate binding of myosin by EIM

Conditions of EM treatment: Myosin (1.9 mg per ml) was treated with 1200 moles of EM per mole of myosin (except in the F-a&in experiment, in which 1100 moles of EM per mole of myosin were used) in the presence of 1 rnM Tris, pH 7.6, and 0.6 M KCl, for 5 to 24 hours in an ice bath with additions as indicated. At the end of incubation, the samples were precipitated by dilu- tion, washed with 0.03 M KCl, and dialyzed against 0.6 1~ KCl-1 rn>r phosphate, pH 7.6, for 48 hours. Pyrophosphate binding was measured by equilibrium dialysis in the presence of 0.6 1~ KCl, 50 my Tris, pH 7.6, and 2.67 ~31 pyrophosphate-32P. No Ca- or EDTA-activated ATPase activity was found in any of the samples used in the experiment except the one not containing EM. Addition of F-actin to the EM-treated myosin samples caused no extra increase in light scattering, and no drop in the viscosity was observed on the addition of 0.1 rnlr pyrophosphate and 1 mM MgClz to an actomyosin solution containing F-actin and EM-treated myosin. No titratable -SH groups were found with the CMB (19) and Ag titration method (18), except in the case of the control myosin, when it was 0.055 pmole per mg of protein.

Additions

PPi bound after incubation with EM

5 hr 23 hr

No addition. ( 0.16 0.00 PPi,0.2mM. _...._...._..__..._..._.... i 0.53 0.42 EDTA, 2.0 m&r. .: 0.18 ADP, 5.0 mM.. ./ DNP, 2.1 mM.. _. _. _. .I

0.78 j 8::; 0.44 0.33

F-Actin (0.3 mg per mg of myosin) ~ 1 0.00 Control (no EM). i 0.72 / 0.70 -

- CONCENTRATION OF DNP,ADPAND EDTA DURING

TREATMENT WITH NEM,M

FIG. 5. Protective effect of ADP, dinitrophenol, and EDTA on the pyrophosphate and ATP binding of myosin. Myosin (13.8 mg per ml) containing 10 mM Tris buffer, pH 7.4, and ADP, dinitro- phenol, or EDTA in concentrations indicated on the abscissu was incubated with 10 mM EM (about 360 molar excess) for 24 hours in an ice bath. Myosin was precipitated by the addition of 20 volumes of HzO. The precipitate was collected by centrifugation and washed twice with 20 volumes of 0.024 M KC1 and 1 IIM Tris, pH 7.6. The final precipitate was redissolved in 0.6 M KCl.

For measurement of the PPi binding, 2.5 ml of myosin were dialyzed against 5 ml of a solution containing 0.6 M KCI, 0.075 M Tris buffer (pH 7.6), 1.5 mM MgClz, and 3.66 pM P3’Pi for 30 to 40 hours at 4”.

ATP binding was measured by the precipitation method as de- scribed in “Experimental Procedure.” No detectable ATPase activity was found in any of these samples when assayed in the presence of Ca, EDTA, or actin as activat)ors, under the condi- tions described in “Experimental Procedure.” The combination with actin was measured by viscometry and light scattering. No -SH groups were detectable by the Ag titration method of Benesch et al. (18). O--O, PPi binding, ADP-protected; f---+: PPi binding, dinitrophenol-protected, A--A, PPi bind- ing, EDTA-protected; O--O, ATP binding, ADP-protected, q - - - -0, ATP binding, dinitrophenol-protected.

The concentration dependence of the protective effect of ADP, 2,4-dinitrophenol, or PPi on the binding of ATP or PPi is shown in Figs. 5 and 6. The similarity in the amount of ATP and pyrophosphate bound to EM-treated myosins protected by ATP, dinitrophenol, or pyrophosphate further supports the identity of the binding sites for these two compounds. It is interesting that EDTA, although its activating effect on the ATPase activity of myosin displays the same nucleotide speci- ficity as that of dinitrophenol or pyrophosphate, gives no detect- able protection against inactivation of the pyrophosphate- or ATP-binding site by EM.

The protective effect of pyrophosphate and ADP appears to be confined to the binding site of ATP and pyrophosphate. Preparations protected from the effect of EM with regard to PPi binding by ATP or pyrophosphate do not form actomyosin, as judged by the absence of the increase in viscosity on the addition of F-actin to these myosin solutions and their ATPase activity, t.ested under appropriate condibions (with Ca, actin, or EDTA as activators) is less than 1% of that of the native myosin.

Even under considerably milder conditions with 14 to 200 moles of EM per mole of myosin, 1 to 25 mM ATP or pyrophos-

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Xubstrate Binding to Myosin Vol. 239, No. 2

CONCENTRATION OF PP DURING INCUBATION WITH NEM,M

FIG. G. Protective effect of pyrophosphate against inactivation of pyrophosphate and ATP-binding sites by EM. The methods used in these experiments are essentially those described under the legend to Fig. 5 and in “Experimental Procedure.” The PPi binding was measured at a total PPi concentration of 1.3 PM. O--O, PPi binding; l - - ~ - 0, ATP binding.

B MOLES OF INHIBITOR/MOLE OF MYOSIN

FIG. 7. Action of CMB and Salyrgan on the pyrophosphate binding and ATPase activity of myosin. Myosin (7 mg per ml) was treated with CMB and Salyrgan in a medium containing 06 M KC1 and 0.02 M Tris, pH 7.6, for 1 hour in an ice bath. Equi- librium dialysis was carried out as described in “Experimental Procedure” in a medium containing 0.6 M KCl, 0.02 M Tris (pH 7.6), 1 mM MgC12, and 1.5 PM KPs2Pi at 4” for 36 hours. ATPase measuremenk were carried out as described in “Experimental Procedure.” O--O, CMB effect on PPi binding; O--O, CMB effect onCaATPase; O-0, CMB effect onEDTA-ATPase; W-a, Salyrgan effect on PPi binding; a---n, Salyrgan ef- fect on CaATPase action; APA, Salyrgan effect on EDTA- ATPase action.

phate did not protect the EDTA-activated ATPase activity

from inactivation by EM. The slow inhibition of the EDTA-

moderated ATPase caused by 0.1 M iodoacetate at pH 7 was also

unaffected by the presence of 25 mM ATP and 1 mM MgC12.

Effect of Mercurials, Proteolytic Enzymes, and Urea on ATPase

Activity and Pyrophosphate Binding of Myosin-The separation of the two functions of myosin (ATPase and PPi binding) is most easily achieved by treatment with EM. CMB and Salyr- gan cause parallel loss of ATPase activity and pyrophosphate

binding of myosin (Fig. 7) similar to the effects of prolonged digestion with trypsin or chymotrypsin or urea.

E$ect of Chelating Agents on Pyrophosphate Bindin.g and ATP- ase Activity of iMyosin-EDTA activates the ATPase activity of myosin at high ionic strength in the presence of potassium or ammonium ions but has no effect if the medium contains sodium (4). We have reported earlier that EDTA in concentrations producing the characteristic activation of myosin ATPase in- hibits the pyrophosphate binding to myosin (6).

The nest section deals with a possible correlation between the effect of EDTA on the pyrophosphate binding and ATPase ac-

FIG. 8. Effect of chelating agents on the pyrophosphate bind- ing of myosin. Myosin, 2.5 ml (5.5 mg per ml), containing 0.G M KC1 and 10 mM Tris, pH 7.6, was dialyzed for 36 to 40 hours at 4’ against 5 ml of solution cont,aining 0.6 M KCl, 0.075 M Tris (pH 7.6) and 3 PM P3’Pi. Inhibitor concentrations calculated on the bask of uniform distribution in the 7.5-ml volume are indicated on the abscissa. O--O, ethyl ether diaminetetraacetic acid; O--O, ethylene glycol bis(@-aminoethylether)-N,N’-tetraace- tic acid; O---O, 1,2-cyclohexanediaminetetraacetic acid; m---W, hydroxyethyl ethylenediaminetriacetic acid; n--a, diethylenetriaminepentaacetic acid; A--A, iminodiacetic acid; e---e, nitrilotriacetic acid.

$10

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B hO6 8 $04 d 50.2

g

fi CHELATING AGENT,

-I

M

FIG. 9. Effect of chelating agents on the myosin ATPase. Composition of the assay system: 0.6 M KCl, 0.1 M Tris (pH 7.6), 0.001 M ATP, and 0.05 mg of myosin per ml. Concentrations of chelating agents are indicated on the abscissa. Incubation was for 10 minutes at 25”. f--+, ethyl ether diaminetetraacetic acid; O--O, ethylene glycol bis(P-aminoethylether)-N,N’- tetraacetic acid; O--O, hydroxyethyl ethylenediaminetriacetic acid; .&--a, 1,2-cyclohexanediaminetetraacetic acid; A--A, diethylenetriaminepentaacetic acid; O--U, iminodiacetic acid; m---m, nitrilotriacetic acid; e---3, dihydroxyethylglycine; X--X, EDTA.

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February 1964 A. Martonosi and H. Meyer 645

tivity of myosin. (a) One group of chelating agents, EDTA, ethyl ether diaminetetraacetic acid; 1,2-cyclohesanediamine-

tetraacetic acid, hydroxyethyl ethylenediaminetriacetic acid,

and diethylenetriaminepentaacetic acid, inhibits at low concen- trations the pyrophosphate binding of myosin (Fig. 8) and

activates it’s ATPase activity (Fig. 9). The concentration de-

pendence of the inhibition of pyrophosphate binding and of the activation of myosin ATPase is rather similar. (b) A second

EDTA, M

FIG. 10. Inhibition of the pyrophosphate binding of myosin by EDTA in the presence of potassium and sodium. Pyrophos- phate binding was determined by equilibrium dialysis in a medium containing 0.6 M KC1 or NaCl, 0.01 M potassium or sodium phos- phate buffer, pH 7, at a total pyrophosphate concentration of 1.04 X 1OW M. In the case of the Na experiments, myosin was dialyzed before the experiment against 0.6 M NaCl containing 1O-2 M sodium phosphate, pH 7. Protein concentration: 9.2 mg per ml in the K and 9.7 mg per ml in the Na experiments. 0, sodium; 0, potassium.

z 0.9 65

g 0.0

g 0.7

? 0.6

g a 05 . z 3 0.4

g a 0.3 cl. g 0.2

2 0.1

EDTA, M

FIG. 11. The inhibitory effect of EDTA on the pyrophosphate binding of myosin at various pyrophosphate concentrations. Py- rophosphate binding was determined by dialyzing 2.5 ml of myosin (5.55 mg per ml) against 5 ml of 0.6 M KCI-0.075 M Tris, pH 7.6, for 36 to 40 hours at 4”. The concentrations of the KP3’Pi in the four sets of experiments were as follows: Experiment 1, O--O, 0.2 MM; Experiment 2, O--O, 1.3 PM; Experiment 3, O-0, 5.3 MM; Experiment 4, A--@,, 13.3 pM. The myosin preparation used in this experiment was about 20 days old, which explains the somewhat low value of pyrophosphate binding in the absence of EDTA. The concentrations of EDTA are in- dicated on the abscissa, and were calculated on the basis of uni- form distribution in 7.5 ml of volume.

* The measurements were made by Dr. J. Seidel. 1 The numbers in parentheses represent the number of experi-

ments .

group of compounds, viz., ethyleneglycol bis(P-aminoethyl- ether)-llr , Wtetraacetic acid, iminodiacetic acid, and nitrilotri-

acetic acid, did not inhibit the pyrophosphate binding of myosin

(Fig. 8) and did not activate the myosin ATPase (Fig. 9) at concentrations less than 0.5 mM. Even at, much higher concen- trations, only slight effects were observed. Similar effects on

the ATPase activity were earlier described by Ebashi et al. (22). The inhibitory effect, of EDTA on the pyrophosphate binding of

IO-4

ADP. M

FIG. 12. sin, 2.5 ml

Inhibition of pyrophosphate binding by ADP. Myo- (5.5 mg per ml) was dialyzed against 5 ml of a solution

containing 0.6 M KC1 and 1.5 mM Tris, pH 7.6. The concentra- tions of pyrophosphate calculated for a total volume of 7.5 ml were as follows: O--O, 0.2 PM; O-0, 1.2 pM; O----O, 5.3 FM; A--A, 13.3 pM. The concentration of ADP calculated for a volume of 7.5 ml is indicated on the abscissa.

TABLE II

Effect of CMB on pyrophosphate binding, ATPase activity, and calcium content on myosin

Treatment of myosin with CMB was carried out as described by Kitagawa et al. (23). ATPase activity was assayed in the presence of 10 rn$r CaCl2,lO rn*r Tris (pH 7.6), and 1 ma ATP; myo- sin concentration was 0.095 mg per ml. Pyrophosphate binding was measured by equilibrium dialysis in the presence of 0.6 M KCl, 0.075 M Tris, pH 7.6, and 7.8 PM PZ2Pi. The calcium content has been measured by EDTA titration following wet ashing with calcein as an indicator.*

Additions PP i binding

Control. . CMB, 20 moles per mole

of myosin.. CMB, 25 moles per mole

of myosin.

mole PP< bound/ mole myosilz

0.40 (3)t

0.18 (3)

0.09 (2)

ATPase

plnole Pi/?&g protein/min

0.57 (2)

0.25 (2)

0.18 (2)

Sound Ca++ + Mgff content

pm&s/g

5.0 (3)

3.3 (3)

0.15 (3)

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G4G Substrate Binding to Myosin Vol. 239, No. 2

myosin can be fully reversed by dialysis against 0.6 BI KC1 and 0.01 ar Tris buffer, pH 7.6.

In contrast to the monovalent cation specificity of the activat- ing effect of EDTA on the myosin ATPase, its effect on the pyrophosphate binding is identical in potassium- and sodium- containing media (Fig. 10). The inhibitory effect of EDTA on the PPi binding of myosin is not influenced by changes in the pyrophosphate concentration from lo-’ to 10h5 31 (Fig. ll), while the inhibitory effect of XDP is strongly reduced at the higher pyrophosphate concentrations (Fig. 12).

Kitagawa, Yoshimura, and Tonomura (23) reported that the treatment of myosin with CMB followed by the addition of cysteine results in a complete and irreversible loss of the firmly bound calcium and magnesium from the myosin, accompanied by minor changes in the ATPase activity and actin-complesing ability. We have been able to confirm the removal of the bound cations by CMB treatment under the conditions described by Kitagawa et al. (23) (Table II). However, in our case, the re- moval of the bound cations is accompanied by a parallel loss of the ATPase activity and pyrophosphate binding suggesting the involvement of the bound cations in these t,lvo functions.

DISCUSSION

It has been shown that treatment of myosin with moderate concentrations of EM causes the inhibition of the calcium, EDT&, and act.in-activated ATPase activity, while the inter- action of pyrophosphate and ATP with myosin remains largely unaffected. The separation of hydrolytic activity from the sub- strate binding is obtained when the number of unreacted -SH groups is between 2 and 4 per 100,000 g of myosin. Further blocking of myosin -SH groups with a large excess of EM leads to a gradual inhibition of the pyrophosphate and ATP binding as the number of unreacted -SH groups decreases below I. The presence of pyrophosphate, ATP, or dinitrophenol protects the pyrophosphate- and ATP-binding site against inactivation by high concentrations of EM. n-0 protection of the ATPase activity was found.

These experiments appear to indicate the existence of distinct functional groups which are connected: (a) with the hydrolysis of ATP; (6) with the interaction of PPi and ATP wit.h myosin.

EM appears to be exceptionally suitable for the selective modi- fication of groups in the first class. Other inhibitors of the dTPase activity (mercurials, urea, and proteolytic enzymes) produced a para.llel inhibition of the pyrophosphate binding.

It seems likely for a number of reasons that the effects of EM are due to its reaction with -SH groups. EM, under our esperi- mental conditions, is preferentially reactive with -SH groups. The ATPase activity, PPi and ATP bindings of myosin arc also inhibited by other -SH reagents such as CMB or Salyrgan. Therefore, the effects of EM on both ATPase activity and PPr bindings will be interpreted as the consequence of its reaction with -SH groups. One has to keep in mind, however, the possibility that EM reacts with other groups of functional signi- ficance (24) even if the modification of a few amino or imidazole residues might escape detection.

Whether the -SH groups in question are directly participating in the active center or their reaction with EM interferes Ivith the ATPase and binding functions only by virtue of their proximity to the active site is not decided. Close spatial relationship between the -SH (or other) groups implicated in the PPi bind- ing and the PPi-binding site appears likely from experiments

demonstrating the protective effect of ATP, PPr, or dinitro- phenol against inactivation of the substrate binding site by EM. The earlier finding by Morales (25) that pyrophosphate protects a small number of -SH groups against reaction with CMB is of interest in this respect, although the inhibitory effect of CM.13 on the binding of PPi by myosin is not counteracted by 0.1 11111 PPi.

The binding of ATP and pyrophosphate to EM-treated myosin appears to depend pronouncedly on the composition of the test medium, a dependence which cannot be observed with the native myosin. The increased sensitivity of the binding of ATP and pyrophosphate by EM-treated myosin to arsenate and tiulfate indicates that although both the affinity and the number of pyrophosphate or ATP-binding sites of myosin remain unaffected by treatment with moderate concentrations of EM when tested in 0.6 M KC1 solution, certain changes in the structure of the binding site take place during EM treatment. These changes are brought into focus under the combined action of arsenate and sulfate. This serves as an explanation for the discrepancy between the ATP-binding data obtained by the precipitation and firefly luminescence methods. From the correlation between the disappearance of ATPase activity and AT1 binding as de- termined by the firefly luminescence method, it appears probable that a common factor is involved in the loss of these two func- tions.

The participation of Mg in the pyrophosphate binding of myosin is indicated by the inhibitory effect of chelating agents on the pyrophosphate binding. The relative effectiveness of various chelating agents increases parallel with the increase of the stabil- ity constant of their Mg complex. An interesting case is that of ethylensglycol bis(P-aminoethylether)N, N’-tetraacetic acid, which is much less effective than EDTA in inhibiting the binding of pyrophosphate to myosin; it forms a weak chelate with Mg, whereas the stability constant of its calcium chelate is equal to that of the EDTA-calcium complex. The close correlation between the effectiveness of various chelating agents in displacing pyrophosphate from the binding site and the stability of their magnesium chelates makes the role of Ca in the pyrophosphate binding somewhat unlikely. The results might be interpreted in terms of the removal of Mg by EDTA from the binding site or as formation of a complex of EDTA with bound Mg at or near the pyrophosphate binding site without displacement of the bound Mg. The former explanation seems more likely since EDTA is not bound to myosin (26).

The effectiveness of various chelating agents in inhibiting the PPr binding to myosin and activating the myosin ATPase is re- markably similar. This similarity suggests but not necessarily proves that there is a common feature in ATPase acivity and PPr binding which is affected by EDTA.

The inhibitory effect of EDTS on the pyrophosphate binding appears to be identical in sodium- and potassium-containing media, while its activating effect on the myosin ATPase depends on the presence of high concentrations of potassium. The cation specificity of the activating effect of EDTA on the myosin ATP- ase led to the proposal that the effect of EDTA might be exerted through its potassium complex (27). This explanation is not likely to hold for the EDTA effect on the PPi binding which is not influenced by PIY’a and K.

On account of its effects on actomyosin systems, pyrophos- phate has long been regarded as a simple analogue of ATP. This view has gained further support from studies which showed that there is only one binding site for pyrophosphate (Gergely et al.

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February 1964 A. Martonosi and H. Meyer 647

(6); see however Tonomura and Morita (28)) and ATT (12, 13)

in 4 to 5 X lo5 g of myosin. Since the pyrophosphate binding is inhibited by ADI’ or IDP as well as by actin, and the inhibi- tion in the case of AD1 appears to be competitive, it seems likely that the binding site for pyrophosphate is identical with a portion of the ATPase center of myosin, presumably the site of mterac- tion n-it,h the phosphate chain of ATP.

The activation of ATPnse and inhibition of ITPase activity by EDTA occurs at EDTA concentrations which inhibit the PPi binding to myosin. With EM, on the other hand, the pyrophos- phate binding is completely unaffected at EM concentrations which activate the myosin ATI’ase. This difference between the two classes of modifiers might reflect some differences in their mechanism of action on the ATPaae activity. On the assump- tion that the pyrophosphate binding occurs at a site where the triphoyphate chain of ATP is partly attached, it seems probable that EDTA exerts its effect on the ATPase mainly through modi- fying the interaction of the triphosphate chain with the active site, while the ATPase-activating effect of EM would be exerted primarily at the site of interaction of the enzyme with the 6-NH2 group of the nucleotide ring as proposed originally by I31um (5). This interpretation of the ATPase-activating effect of EDTA and EM involving two different sites of action is supported also by the fact that EDTA activates the ATPase activity of myosin preparations which are not activated by small concentrations of EM.

SU&IMJIART

1. i\Iyosin preparations have been obtained, by partial blocking of the sulfhydryl groups with N-ethylmaleimide, that do not have an adenosinetriphosphatase activity but still combine with adenosine 5’.triphosphate and inorganic pyrophosphate.

2. The pyrophosphate-binding site is protected from the in- activating effect of high concentrations of N-et,hylmaleimide when inorganic pyrophosphate, adenosine 5’-diphosphate, or 2,4- dinitrophenol are present.

3. No protection by inorganic pyrophosphate, adenosine 5’. diphosphate, or 2,4-dinitrophenol is observed under identical conditions against inhibition of the adenosinetriphosphatase ac- tivity and actin-binding ability of myosin by N-ethylmaleimide.

4. Chelating agents which activate the myosin adenosinetri- phosphatase inhibit the binding of pyrophosphate to myosin.

5. The inhibition of pyrophosphate binding by ethylenedi- aminetetraacetic acid is identical in Sa- or K-containing media.

Acknowledgments-Our thanks are due to Dr. John Seidel for carrying out the Ca and Mg determinations and to Dr. John Gergely for the numerous corrections in the manuscript.

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A. Martonosi and H. MeyerThe Binding of Pyrophosphate and Adenosine Triphosphate to Myosin

1964, 239:640-647.J. Biol. Chem. 

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