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The binding of p-chloromercuribenzoate by myosin
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
93, 605-616 (1961)
The Binding of p-Chloromercuribenzoate by Myosin D A R C Y G I L M O U R ~ AN1) M A R T I N G E L L E R T -~ From the Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire Received February 21, 1961 Rabbit myosin rapidly binds 3-4 moles PCMB/10 ~ g. protein. Further binding, up to a maximum of 7-8 moles PCMB/105 g., occurs at a progressively diminishing rate. The reaction rate increases with both myosin and PCMB concentrations. The binding of 3-4 moles PCMB/10 ~ g. causes an acceleration of ATPase and inhibition of ITPase activity. These effects are more pronounced if the myosin PCMB reaction is carried out at high myosin concentration (~1 mg./ml.). Enzymic rates after such treatment are not constant in time. The changes are accelerated in the presence of substrate and are suggestive of a rearrangement of PCMB on the protein after the initial reaction. Treatment of myosin with PCMB for long times (48 hr.) avoids some of these complications. A linear relation between ITPase and PC1VIB tiger is then found. The reversal of PCMB binding by glutathione has also been studied. The results are interpreted in terms of a model of two-point attachment of substrate to the enzyme. INTRODUCTION The accelerating effect of p-chloromercuribenzoate, ~ a reagent known to bind preferentially to - - S H groups, on the A T P ase a c t i v i t y of myosin has been known for some time and has been studied b y a n u m b e r of workers (1-4). The first thorough s t u d y was t h a t of Kielley and B r a d l e y (3), who made simultaneous m e a s u r e m e n t s of the binding of P C M B and the effects on enzyme a c t i v i t y , and concluded t h a t the t i t r a t i o n of a p p r o x i m a t e l y half the accessible - - S H groups of the protein resulted in acceleration, while the binding of P C M B b e y o n d this point caused a progressively increasing 1Present address: Commonwealth Scientific and Industrial Research Organization, Canberra, Australia. 2 Present address: Section on Physical Chemistry, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland. 3 PCMB is used for p-chloromercuribenzoate throughout this paper. Other abbreviations used are ATP, ITP, and NTP for adenosine triphosphage, inosine triphosphate, and nucleoside triphosphate, respectively; ATPase, ITPase, and NTPase for their respective triphosphatases; and Tris for tris(hydroxymethyl)aminomethane.
inhibition which was complete when all the - - S H was bound. Our interest in the effect of P C M B was reawakened b y the discovery t h a t in m a n y experiments in which P C M B had been acting on the protein for only a short time (the conditions used in Kielley and B r a d l e y ' s work), the enzyme a c t i v i t y of the myosin was n o t constant with time. We therefore launched into a detailed examination of the binding of P C M B by myosin and the effects on nucleotidase a c t i v i t y , with particular emphasis on variations in these phenomena with time. Q u a l i t a t i v e l y our conclusions do n o t differ greatly from those of Kielley and Bradley, but some q u a n t i t a t i v e relationships have been revised and our d a t a show evidence of slow changes in the binding of P C M B to myosin n o t studied b y previous authors. The results have been i n t e r p r e t e d in terms of a model of the enzymic site of myosin suggested b y the kinetic studies of Blum (5). EXPERIMENTAL REAGENTS
The nucleotides were obtained as sodium salts from Sigma Chemical Company. They were dis-
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GILMOUR AND GELLERT
solved in the buffer used for the dilution of the myosin, and the p H of the solutions was a d i u s t e d to 8.0. P C M B was purified by the m e t h o d of W h i t m o r e and Woodward (6). The c o n c e n t r a t i o n of the solutions was d e t e r m i n e d from the optica' density at 232 mr,, using the molar extinction coefficient found b y Boyer (7). The optical density changes slowly with time, so solutions were kept in the cold, and for most experiments were not more t h a n 2 days old. G l u t a t h i o n e solutions were made up fresh each day. MYosiN Myosin A was prepared from r a b b i t muscle by the m e t h o d of Szent-GySrgyi (8), which involves the removal of myosin B at an ionic s t r e n g t h of a b o u t 0.15 M, followed b y the precipitation of myosin A at a b o u t 0.05 M ionic s t r e n g t h . I n some p r e p a r a t i o n s the precipitate of myosin B did not aggregate completely even in the presence of ATP, and in these cases i t was necessary to centrifuge the p r e p a r a t i o n at this stage in the Spineo prep a r a t i v e model ultracentrifuge at 50,000 X g for 40-60 rain. to remove suspended protein. After final purification, myosin A was dissolved in 0.6 M KC1 and clarified b y centrifuging at 20,000 X g for 30 min. The keeping qualities of the enzyme were i m p r o v e d b y the addition of a small a m o u n t (1% by volume) of the buffer used in the enzyme tests. Myosin A p r e p a r e d by this m e t h o d and diluted with 0.6 M KC1 showed no change in the i n t e n s i t y of scattered light, measured at 90 ° angle, on the addition of ATP. Its ATPase a c t i v i t y varied from 8 to 20 ~moles s u b s t r a t e hydrolyzed/g. protein/sec, u n d e r the conditions of our experiments. P r o t e i n c o n c e n t r a t i o n was d e t e r m i n e d b y a modified Folin m e t h o d (9). PCMB
BINDING
The time course of the c o m b i n a t i o n of P C M B with myosin was followed by measuring the change in optical density at 250 m#, the m e t h o d developed by Boyer (7). When this reaction was followed for a long time (>1000 see.), excessive increases in optical density were sometimes noted, a p p a r e n t l y due to aggregation of the myosin. Such long-time experiments were checked, therefore, b y t a k i n g aliquots from a m y o s i n - P C M B mixture, reading t h e i r optical density, t h e n adding excess glutathione and noting the rise in optical density. This yielded the a m o u n t of free P C M B r e m a i n i n g in the m y o s i n - P C M B mixture at the time, and hence, b y s u b t r a c t i o n , the a m o u n t of P C M B b o u n d to the protein. Since the readings w i t h o u t a n d w i t h g l u t a t h i o n e were t a k e n within a few seconds of
each other, slow, nonspecific changes in optical density did not interfere. I t will be n o t e d l a t e r t h a t g l u t a t h i o n e removes some P C M B from tile protein, b u t since we had found t h a t the AO.D.~0 for the c o m b i n a t i o n of P C M B with myosin was identical w i t h t h a t for the c o m b i n a t i o n with glut a t h i o n e , this did not interfere with the validity of the method. Some difficulties did arise occasionally, however, when g l u t a t h i o n e itself caused a change in the aggregation of the protein. This was overcome b y t a k i n g aliquots from. the m y o s i n - P C M B mixture, p r e c i p i t a t i n g the protein by dilution and removing it b y centrifugation, t h e n t i t r a t i n g for free P C M B in the s u p e r n a t a n t . All three methods were used to o b t a i n the results recorded below. P C M B - b i n d i n g studies were usually done at 25°C., at pH 8.0. ENZYME TESTS 5'-Nucleotidase a c t i v i t y was measured in a buffer of the following composition: Tris, 0.05 M; histidine, 0.05 M; KC1, 0.08 M; CaC12, 0.001 M; pH, 8.0. Myosin was added to a c o n c e n t r a t i o n of 0.01-0.025 mg./ml., and nucleotide, dissolved in the same buffer, at 0.001 M. E a c h test comprised a series of (usually four) m e a s u r e m e n t s of phosp h a t e evolved at successive time i n t e r v a l s at 25°C., from which a s t r a i g h t line, or curve, of subs t r a t e split against time was established. N o t more t h a n 15% of the s u b s t r a t e was split in any such experiment, and since in all experiments except t h a t concerned w i t h competitive i n h i b i t i o n between s u b s t r a t e s the initial c o n c e n t r a t i o n of s u b s t r a t e exceeded the s a t u r a t i o n level b y at least tenfold, the decrease in s u b s t r a t e c o n c e n t r a t i o n during an experiment did not affect the velocity of enzyme action. Calcium was not at a s a t u r a t i o n level, since the high c o n c e n t r a t i o n required for s a t u r a t i o n m a y precipitate nueleotide, especially I T P , at p H 8.0. I t was found, however, t h a t a fivefold increase in Ca ++ affected b o t h the unt r e a t e d and P C M B - a c t i v a t e d ATPase to the same degree, causing a b o u t a 20% increase in each. F r o m this it was concluded t h a t the level of Ca ++ in the experiments was not significant for the i n t e r p r e t a t i o n of our results. E n z y m e a c t i v i t y was stopped b y the addition of trichloroaeetie acid to a c o n c e n t r a t i o n of 30-/0. Inorganic p h o s p h a t e was measured b y the m e t h o d of Fiske a n d S u b b a R o w (10) u n d e r c o n s t a n t conditions of time and t e m p e r a t u r e for all the sequence of procedures involved, and the i n t e n s i t y of the blue color of reduced p h o s p h o m o l y b d a t e was determined b y measuring the optical density at 750 m~ in a Zeiss model PMQ I I spectrophotometer.
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B I N D I N G OF £ C M B RESULTS
BINDING OF SULFHYDRYL GROUPS
BY PCMB Figure 1 illustrates the typical time course of binding of PCMB to myosin. The three lower curves show the results obtained with three different concentrations of myosin treated with the same relative amount of PCMB (6.4 moles/10 ~ g.). It is evident that a certain number of - - S H groups, 3-4 moles/105 g. myosin, react very rapidly ( ~ 2 0 see.) in all cases, and that the remaining groups react progressively more slowly. (The three curves presumably approach a common asymptote, at very long times.) In the case of the more slowly reacting groups, it can be seen that the rate varies with the concentration of the reactants, a fact consistent with Boyer's (7) demonstration that the combination of PCMB with - - S H follows second-order kinetics. In other experiments in which smaller amounts of PCMB were applied (3-4 moles/105 g.), it was found that at the two higher myosin concentrations the reaction was complete within the time required to take the first reading of optical density (15 sec.), whereas at the lowest concentration it was complete in less than 100 sec. Most of these experi-
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F i e . 1. Time course of the reaction of myosin - - S H with P C M B . The a m o u n t of P C M B comb i n e d was m e a s u r e d b o t h b y the AO.D. at 250 m~ and b y t i t r a t i o n of free P C M B w i t h g h t a t h i o n e . Curves 1, 2, and 3, 6.4 mole P C M B added/105 g. myosin. Myosin c o n c e n t r a t i o n s : curve 1, 0.013 m g . / m l . ; curve 2, 0.14 m g . / m l . ; curve 3, 0.8 rag./ ml.; curve 4, 11.5 moles P C M B added per 105 g. myosin; myosin c o n c e n t r a t i o n 0.7 mg./ml.
ments were done with the myosin dissolved in 0.2 M KC1. It was found that, at the same pH, the reaction of PCMB with myosin - - S H was appreciably slower in the presence of the Tris-histidine buffer used in the enzyme tests. Thus, on the addition of 3.5 moles PCMB/105 g. to myosin dissolved in 0.2 M KC1 at a concentration of 0.02 mg./ml., all of the PCMB had reacted within 40 sec., whereas the same reaction performed in the Tris-histidine buffer required 200 sec. for completion. The upper curve in Fig. 1 shows the time course of PCMB combination when the reagent is added in excess of the total - - S I t content of the protein. In a number of experiments of this nature which were prolonged for several hours, the total amount of PCMB which reacted varied between 7 and 8 moles/105 g. Owing to the uncertainties introduced by the instability of the protein after long incubation in the presence of excess PCMB, it was not possible to establish whether, in fact, tile reaction approaches a true end point short of the total sulfhydryl content of the protein. Amino acid analyses (11) have established the total content of - - S H plus - - S - - S - - groups in myosin at 8.6 moles/10z g. On the time scale normally used for " - - S H titrations" (incubation times of 100-300 sec.), 5-6 moles PCMB was bound/105 g., the amount varying with the preparation and also with the age of any one preparation. In Fig. 2 the time course of the reaction of PCMB with myosin is shown in a different way. In this experiment, small amounts of PCMB were added successively to a myosin solution, and the time course was followed after each addition. Again it is evident that the first 3.5 moles reacts very rapidly, and the rest progressively more slowly. EFFECTS OF PCMB ON ENZYME ACTIVITY
Qualitatively, our results on the effect of PCMB on myosin-ATPase were in agreement with those of Kielley and Bradley (3) and other workers who have shown that the addition of PCMB up to a certain relative concentration produces activation, but that the addition of larger amounts results in inhibition. The degree of activation
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The effects on ATPase and ITPase are considered separately below.
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I00 200 300 400 500 600 700 SECONDS F ~ . 2. Time course of the reaction of myosin - - S H w i t h P C M B . P C M B added in i n c r e m e n t s of 1.2 moles/lO 6 g. myosin, at the times i n d i c a t e d by the arrows. Myosin c o n c e n t r a t i o n 0.2 mg./ml.
of the ATPase produced by PCMB varied with different myosin preparations, there being, in general, an inverse relation between the activity of the untreated myosin and the percentage activation achieved. Thus, some of the less active preparations were accelerated 3-4-fold by PCMB, whereas the increase in the most active was less than 100 %. The highest ATPase activity measured in the presence of PCMB was 36 umoles/g, myosin/
The relationship between activity and relative PC1VIB concentration for varying times of incubation, at the highest absolute concentration of both protein and reagent, is illustrated in Fig. 3. It is apparent that the addition of about 3 moles PCMB/105 g. protein results in an immediate activation of the ATPase which does not vary with the time of incubation. When about 4 moles is added, the activation measured after 25 sec. incubation decreases somewhat during the next 75 sec. With larger amounts of PCMB, only inhibition is measured, and the amount of such inhibition increases with the time of incubation. At lower myosin and PCMB concentrations, these effects are markedly slowed down, and a transitory activation becomes aparent at the higher PCMB levels (Fig. 4). This is shown more strikingly in Fig. 5, which illustrates an experiment with another myosin preparation having a lower initial activity and higher relative activation by PCMB. Figure 5 shows that at a myosin concentration of 0.024 mg./ml. a quantity of PCMB of 6.4 moles/105 g. requires approximately 20 hr. contact with L
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TIME COVRSE OF T~E EFFECT ON E~ZYME ACTIWTY The influence of the time of incubation of the myosin with PCMB on ATPase and ITPase activities was studied at three different myosin concentrations. These experiments were done by incubating the mixture of myosin and PCMB at 25°C. for varying lengths of time, at the end of which an aliquot of the mixture was added to a larger volume of buffer and substrate to start the activity run. At the lowest myosin concentration used, PCMB was added to the enzyme dissolved in the buffer at a concentration suitable for the activity test, which in this case was started by the addition of substrate.
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FIG. 3. E f f e c t of P C M B on m y o s i n ATPase. M y o s i n t r e a t e d at a c o n c e n t r a t i o n of 0.8 m g . / m l .
The times refer to i n c u b a t i o n of myosin with P C M B , before s t a r t i n g the enzyme assay.
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vidual activity runs of the same myosin preparation as that used in Figure 5, treated at a myosin concentration of 0.38 mg./ml. I t can be seen from this figure that the initial activities of samples assayed 100 and 1000 sec. after the addition of P C M B to the protein were almost identical, although the activity of the first sample had dropped to less than 60 % of its initial value after 500 sec. in the presence of substrate.
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FIG. 4. Effect of PCMB on myosin ATPase. Myosin treated at a concentration of 0.14 mg./ml. The times refer to incubation of myosin with PCMB, before starting the enzyme assay.
At high myosin concentrations, relatively small amounts of P C M B produced a marked inhibition of I T P a s e which varied little with the time of incubation (Fig. 7). Under these circumstances a revival of enzyme activity in the presence of substrate became apparent. This is seen in Fig. 8, which shows some of the individual activity runs from which the points on Fig. 7 are derived. I T P ase activity starts at a very low level but increases during the course of the experiment. At the lower myosin concentrations, inhibition of the ITPase was relatively slight for short incubation times, but increased with
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FIG. 5. Variation of myosin ATPase with time after treatment with 6.3 moles PCMB/105 g., at a myosin concentration of 0.024 mg./ml. Points record initial activities of the protein assayed after the indicated incubation times with PCMB. The inset shows the first part of the curve on an expanded time scale. the protein at 25°C. to achieve complete inhibition, although a substantial part of the activation which disappears during the first 3 hr. is apparent after only 20 sec. incubation. A striking feature of the slow decrease in activity from its maximum level is that inhibition is accelerated in the presence of substrate. This is illustrated in the series of curves in Fig. 6, which shows some indi-
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FIG. 6. Time course of myosin ATPase after treatment with 6.3 moles PCMB/105 g. at a myosin concentration of 0.38 mg./ml. The three curves, which are displaced vertically for clarity, are activity runs made after the indicated times of incubation with PCMB. Activities (v), in ~moles/ g./sec., refer to the dotted tangents.
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FIG. 7. Effect of P C M B on m y o s i n I T P a s e . M y o s i n t r e a t e d at 0.8 m g . / m l . I n c u b a t i o n t i m e s : ®, 25 see.; 0 , 1 0 0 see.; A , 4=00see. I
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runs on which Fig. 10 is based, demonstrates that the ITPase, like the ATPase, decays much more rapidly in the presence of the substrate than in its absence. Comparison of the results of the experiments on the time course of the effect of PCMB on the NTPase with the time course of the binding of PCMB as demonstrated by changes in optical density at 250 m~ reveals some anomalies. Thus, although the binding of 3-4 moles/105 g. PCMB is very rapid at all the concentrations studied, the effect of this binding at high concentrations is strong activation of ATPase and strong inhibition of ITPase, whereas at low concentrations there is little or no activation of ATPase and only weak inhibition of ITPase. This suggests that the groups which bind most rapidly at high concentration are different from those that bind most rapidly at low concentration. Moreover, at relative PCMB concentrations between 3 and 5 moles/105 g., changes in enzyme activity occur at a time when all the PCMB must have been already bound to the protein, even allowing for the slower rate of binding in the Tris-histidine buffer. These facts suggested the possibility that secondary changes were taking place in
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FIG. 8. T i m e course of m y o s i n I T P a s e after t r e a t m e n t w i t h 4.0 moles P C M B / 1 0 5 g., at a m y o sin c o n c e n t r a t i o n of 0.8 m g . / m l . T h e three curves, which are displaced v e r t i c a l l y for clarity, are a c t i v i t y r u n s m a d e after t h e i n d i c a t e d t i m e s of incubation with PCMB.
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longer incubation (Figs. 9 and 10). In these experiments ITPase activity decayed rapidly during the course of the enzyme tests. Figure 11, which shows some of the actual activity
FIG. 9. Effect of P C M B on myosin ITPase. M y o s i n treated at a concentration of 0.14 mg./m]. T h e three curves are for three different i n c u b a t i o n times, as shown.
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arms being substantially linear. The points at which the curves of inhibition of ATPase and ITPase intersect the abscissa did not coincide, a n d it was possible to obtain an enzyme preparation without any measurable ITPase activity, but still exhibiting a low activity toward ATP. (This difference may not have been significant since it was difficult to measure such low rates accurately and there was evidence that both lines showed some curvature as they approached the abscissa.) The relative PCMB concentration needed to produce complete inhibition varies with the age of the myosin preparation; for instance, for one enzyme tested the day after preparation this PCMB concentration was 7.0 moles/105 g., whereas 3 days later it had dropped to 5.8 moles.
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FIG. 10. Effect of P C M B on myosin ITPase. Myosin t r e a t e d at a concentration of 0.013 mg./ml. The three curves are for three different incubation times, as shown.
Kielley and Bradley reported that the activation, but not the inhibition, of ATPase by PCMB is reversed by ~-mercaptoethanol. We have found a similar effect of glutathione
the protein subsequent to the initial binding of PCMB. In order to determine the end point of these time-dependent changes, the effect of PCMB on enzyme activity was determined after extended incubation times, when it might be assumed that the secondary changes would have run their course. EFFECT or PCMB A~'TER LONG
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These experiments were done by treating the protein at a relatively high concentration (0.6 mg./ml.) with PCMB in amounts varying from 0 to 7 moles/105 g. The mixture was incubated for 25 see. at 25°C., then for 48 hr. at about 5°C., after which activities against ATP and ITP were assayed. Under these conditions the amount of phosphate evolved was almost always linear with time, in contrast to the experiments reported in the two previous sections, suggesting that the conditions were, in fact, saturated with respect to time of incubation. With these long incubation times, the relationship between ITPase activity and relative PCMB concentration was found to be linear, or nearly so (Fig. 12). ATPase activity showed first a rising and then a falling phase, both
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:FIG. 11. Time course of myosin ITPase after t r e a t m e n t with 6.4 moles PCMB/105 g., at a myosin concentration of 0.013 mg./ml. The three curves, which are displaced vertically for clarity, are activity runs made after the indicated times of incubation with PCMB. Activities (v), in ttmoles/g./sec., refer to the d o t t e d tangents.
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GILMOUR AND GELLERT
on both the ATPase and ITPase. The curves of activities, in relation to applied PCMB, of the enzyme treated first with PCMB and then with an excess of glutathione, each treatment being for 48 hr. in the cold, are shown in Fig. 12. It is clear that the reversal is fairly complete up to the maximum in the ATPase-PCMB curve, although both activities decline to some extent up to this point. This decline may be due to a greater tendency toward slow irreversible denaturation in the PCMBtreated enzyme, since experiments done with shorter incubation times show a more nearly complete restoration of the original activities for both substrates with amounts of PCMB up to 3 moles/10 ~ g. Beyond the maximum of the ATPase-PCMB curve, the activity curves of the glutathione-treated enzyme decline rapidly toward zero, indieating that the inhibition of the ATPase and the final part of the inhibition of the ITPase are irreversible. Although at PCMB concentrations of 5-6 moles/105 g. the effect of adding glutathione is to cause a decrease in the ATPase and an increase in the ITPase, the two activities of the glutathione-treated enzyme have a consistent relationship to one another, the ratio of ITPase activity to ATPase activity being reasonably constant throughout the entire PCMB range. An attempt was made to investigate quantitatively the removal of PCMB from the protein by glutathione, with the object of determining whether the irreversible inhibition of enzyme activity was the result of a particularly firm binding to a specific - - S H group or to a general denaturation of the enzyme due to the loading of PCMB beyond a critical level. The experiment was done by treating the myosin first with PCMB, then with a 2-3 fold excess of glutathione. Protein was precipitated by dilution with water and removed by centrifugation; then the supernatant was examined spectrophotometrically for the presence of the PCMB-glutathione compound. The ultraviolet absorption spectrum of the supernatant fell on a curve which corresponded closely with that of the authentic P C M B glutathione compound. Semiquantitative estimates of the concentration of the compound, based on the molar extinction coe~-
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FIG. 12. Effect of P C M B on myosin A T P a s e a n d I T P a s e after long i n c u b a t i o n , and after reversal by glutathione. (D----(D A T P a s e ; • ..... • ATPase after g l u t a t h i o n e t r e a t m e n t ; ~ - - - - - B I T P a s e ; • ..... • I T P a s e after g l u t a t h i o n e t r e a t ment.
cient at 250 m#, indicated that whereas all of the PCMB was removed when the myosin had been treated with 4.6 moles/105 g., less than 100 % was removed when the myosin had been treated with 7.3 moles. This suggests that the binding of PCMB to some groups on the protein was stronger than the binding between PCMB and glutathione. PROTECTION BY SUBSTRATE AGAINST
PCMB INHIBITION Further evidence that the irreversible inhibition of enzyme activity may be the result of the binding of a relativelyinaccessible - - S H group at the active site is found in the protection against inhibition afforded by the substrate. Evidence has been presented in previous sections for the acceleration of inhibition by substrate in the presence of PCMB. Protection of the enzyme by the substrate occurs at a more advanced level of inhibition. This is illustrated by Fig. 13, which shows the time course of inhibition of myosin treated at low concentration with a large
613
B I N D I N G OF P C M B
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SECONDS
FI~. 13. Effect of P C M B (122 moles/10 ~ g.) on the ATPase a c t i v i t y of myosin t r e a t e d (at 0.011 mg./ml.) in the presence and absence of substrate. P C M B added at time shown by arrow. In the upper curve A T P was present before P C M B was added; in the lower curve P C M B was present for 1600 see. before A T P was added. The d o t t e d line shows the a c t i v i t y before PCMB t r e a t m e n t . Activities (v) are in ~moles/g./see.
excess of PCMB in the presence of substrate. It was found that under these conditions the enzyme passed usually through a phase of transient activation after which the activity decayed rapidly to a low steady rate. The same preparation treated in the way in the absence of substrate was shown to be quite inactive when assayed at a time (1600 sec. after the addition of PCMB) when the enzyme treated in the presence of substrate was exhibiting a constant velocity. COMPETITIVE IXI~ImTmN OF ATPAs~ ACTIVITY BY I T P The opposing effects of PCMB on ATPase and ITPase activities could be most simply explained if the two substrates were split at separate sites, and a necessary preliminary to attempting an explanation of the foregoing results would be to determine whether or not this is so. Certain conditions of PCMB treatment establish a relationship between the activities (high ATPase, very low ITPase) suitable for the testing of competitive inhibition between the two substrates. In the experiments designed to make this test, myosin at 0.3 mg./ml, was incubated with 5.6 moles/105 g. PCMB at 25°C. for 200 sec. After such treatment the initial activities against ATP and ITP, with both substrates at a concentration of 10 -3 M, were 24.7 and 1.6 ~moles/g./sec., respectively. Myosin so treated was used to de-
termine Km's for ATPase and ITPase by the usual (l/v) vs. (1/[S]) plots. The Km's found were 2.9 X 10-5 for ATP and 9.3 X 10-~ for ITP. Then the velocities at different ATP concentrations were determined in the presence of 10-3 M ITP. The results of this experiment are shown in Fig. 14. The circles are based on actual experimental figures for the velocity at various ATP concentrations, and the triangles represent the same figures corrected for the splitting of ITP, which was calculated from the known velocities, substrate concentrations, and binding constants for the two substrates at the set of conditions represented by each point. The straight line in the figure, intersecting the ordinate at the same point at which the line for (l/v) vs. (1/[ATPD intersects, is the theoretical line for competitive inhibition, with K~ equal to the determined value of Km for ITP. Although the points show considerable scatter, due to difficulties in determining accurately the small amounts of phosphate evolved, and the tendency for v to vary with time during the runs, they show reasonably good agreement with the theoretical curve and constitute quite strong evidence that the two substrates are split at the same site. DISCUSSION
Our results show a discrepancy between the time course of the binding of PCMB to the protein and the time course of the effects on enzyme activity. This discrepancy could be due to secondary eonfigurational changes taking place in the protein or to the rearrangement of PCMB on the protein subsequent to its initial binding. Although our results do not completely exclude the first explanation, they strongly suggest that the second is true. The evidence for this is: Combination of 3-4 moles PCMB/10 5 g. protein does not produce the same effect on enzyme activity at high concentrations as at low, suggesting that the reagent may be bound to different - - S H groups under different conditions. The slow changes in enzyme activity with increasing incubation time, which are accelerated by the presence of substrate, are in such a direction as to bring the curve for activity vs. PCMB concentration nearer
614
GILMOUR AND GELLERT
the "equilibrium" position shown in Fig. 12. Where these changes are in the direction of diminishing enzyme activity it might be argued that they could be the result of either rearrangement of PCMB molecules or the destruction of the active site by secondary configurational changes; but where, as in the experiment illustrated in Fig. 8, they are in the opposite direction, then it is difficult to see how the second explanation can hold. The straight-line relationship between ITPase and PCMB concentration, show in Fig. 12, suggests that ITPase activity depends on one or more of a group of about 40 homogeneous --SH groups on the myosin molecule (assuming a molecular weight of 6.2 X 105 g. for myosin) (12). Our studies on the binding rates have shown that these - - S I t groups are far from homogeneous with respect to the kinetics of binding to PCMB, and, more importantly, it is clear that they fall into at least two sharply distinct groups with respect to equilibriurn-binding energies, since the combination of PCMB with some can be reversed by glutathione, but with others it cannot. The two groups are further distinguished by the fact that the binding of PCMB to the first causes acceleration of the ATPase, whereas binding to the second causes inhibition. It seems, therefore, that the continuity of the straight line expressing the effect on ITPase activity of the binding of PCMB to both sets of --SH groups may be fortuitous. Further, the equilibrium relationship between ATPase activity and PCMB concentration (Fig. 12) suggests that there is a sequential binding of PCMB to the two distinct sets of --SH units. Any considerable overlap of the accelerating and inhibiting effects of PCMB binding with the addition of increasing amounts of PCMB would introduce curvature into the graph of ATPase activity versus PCMB applied. In fact, this graph takes the form of two intersecting straight lines, the only curvature being immediately in the region of changeover from one straight line to the other. Thus it follows that the binding of PCMB to the same --SH unit or group of --SH units causes both acceleration of the ATPase and inhibition of the ITPase. The following picture of the titration of
l
[
1
ATP÷IO"3M/ ITP /
60
%
x 4o -I>
20 ATP
I__ 2
I 4
[~] ×
I 6
io-4
FIG. 14. Competitive inhibition by ITP of the ATP hydrolysis by PCMB-treated myosin. For explanation see text. rayosin by PCMB then emerges: The addition of PCMB in amounts up to about 27 moles/inole myosin results in a rapid random binding of the mercurial to a group of --SH units of more or less equal "accessibility", of which one or more is situated at the active center. This binding is reversible by glutathione and causes acceleration of the ATPase and inhibition (to a maximum of 70 %) of the ITPase. Addition of PCMB in amounts in excess of 27 moles/mole myosin results in a subsequent, slower, but tighter, binding to a second, less accessible group of --SH units, of which, again, one or more is situated at the active center. This binding, which is not reversible by glutathione, is complete when about 40 moles PCMB have been bound per mole of myosin. It causes inhibition of the ATPase and further inhibition of the ITPase beyond that resulting from the binding of the first group. This view of the participation of --SH units in the active center of myosin is, as stated above, qualitatively similar to that proposed by Kielley and Bradley (3). It can also be very well expressed in terms of Blum's interpretation of his data on the binding of substrate to myosin-NTPase. Blum (5) has proposed that the substituent at the 6 position of the purine or pyrimidine ring of the nucleotide substrate of myosin has
BINDING OF PCMB an important effect in determining the binding of the substrate to the enzyme. Nucleotides with an amino group on the 6 position, such as ATP, bind strongly to the enzyme, and the over-all velocity of hydrolysis of the P - - O - - P bond is low, because of the slow desorption of the products. Nucleotides with a hydroxyl on the 6 position, such as I T P , bind less strongly, and exhibit a higher hydrolysis rate. Furthermore, Blum and Felauer (13) have suggested that the acceleration of ATPase by 2,4-dinitrophenol is caused b y an interference with the binding of the amino group of the 6-amino nucleotides. From these suggestions has emerged the idea of a twopoint attachment of the nucleotides to the enzyme, one point of attachment being the polyphosphate end of the substrate, the other, the ring end. Our results suggest that an - - S H group is included in each part of the active center. The attachment of P C M B to the relatively accessible - - S H at the ringbinding site could prevent the binding of the 6-amino of A T P and thus result in an enhanced rate of hydrolysis of this substrate. The addition of larger amounts of P C M B would result in combination with the less accessible - - S H at the phosphate-binding site, thereby blocking hydrolysis of the substrate completely. I t follows from our results that the attachment of P C M B at the ring-binding site inhibits the hydrolysis of I T P as well as accelerating the over-all rate for ATP. I t is conceivable that the presence of the benzene ring of P C M B at the ring-attachment site could hinder, but not absolutely prevent, either the attachREL. PCMB CONC. MOLES/IO~
ACTIVE SITE
ATPose ITPose ACTIVITY ACTIVITY P-ATTACHMENT RING-ATTACHMENT POSITION POSITION
200
80
615
merit of the I T P molecule, or its hydrolysis, and that complete titration of this group would result in the 70 % inhibition of ITPase recorded. The same kind of hindrance would be expected to affect A T P as substrate, but in this case the inhibition is more than compensated by the acceleration resulting from the blocking of the ring binding through the 6-amino group. It seems that the kinetics of the binding of P C M B to the - - S H at the ring-binding site are such that under conditions of high concentration of both myosin and P C M B this - - S H binds the mercurial preferentially, even in comparison with others of the group of roughly 27 - - S H units on the molecule which bind P C M B less strongly than does glutathione. There is then, apparently, a subsequent migration of P C M B away from the active center, presumably to other - - S H ' s on the molecule, with the observed effect of a decrease with time of ATPase activity and an increase with time of I T P a s e activity. The demonstrated acceleration by substrate of this migration is perhaps not surprising. On the other hand, the slower binding of P C M B to the less accessible - - S H at the position of phosphate attachment is hindered by the presence of substrate on the active center. The postulated sequence of events in the titration of the protein by P C M B and the effects on ATPase and ITPase are presented schematically in Fig. 15. In another paper (14), some extensions of this model of myosin-nucleotidase are proposed, in accordance with data on the variation with temperature of the P C M B effect, but as a hypothesis to cover the data presented here the scheme shown in Fig. 15 is adequate. ACKNOWLEDGMENTS It is a pleasure to acknowledge the encouragement, suggestions, and intellectual stimulus provided by Dr. Manuel F. Morales, in whose laboratory this work was done. We are also indebted to Mr. Bruce Henry for his skilled technical assistante.
6.5
~,'aY X ~
X la.~
0
0
FIG. 15. Proposed model for effects of PCMB on myosin enzyme activity. X = position blocked by PCMB.
This work was done during tenure of a Senior Traineeship (U. S. Public Health Service Grant 2G-174) by one of us (D. G.), and was also supported by National Heart Institute Grant H-3598 on which the other of us (M. G.) was a coinvestigator.
616
GILMOUR AND GELLERT REFERENCES
1. SINGER, T. P., AND ]~ARRON, E. S. G., Proc. Soc. Exptl. Biol. Med. 56, 129 (1944). 2. PoLIS, B. D., AND MEYERI~OF, O., J. Biol. Chem. 169, 389 (1947). 3. KIELLEY, W. W., AND BRADLEY, L. B.~ J. Biol. Chem. 218, 653 (1956). 4. BLUM, J. J., Arch. Biochem. Biophys. 87, 104 (1960). 5. BLU~, J. J., Arch. Biochem. Biophys. 55, 486 (1955). 6. WI-IITMORE, 1~. C., &ND WOODWARD, G. E., Organic Syntheses Coil. Vol. I, I941, p. 159. 7. BoY~Ja, P. D., J. Am. Chem. Soc. 76, 4331 (1954).
8. SZENT-GY~RGYI, A., "Chemistry of Muscular Contraction," 2nd Ed. Academic Press, New York, 1951. 9. LOWRY, O. H., ROSBBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 10. FISKE, C. H., AND SUBBAI~OW, Y., J. Biol. Chem. 66,375 (1925). 11. KOMINZ, D. R., HOUGH, H., SYMONDS, P., AN]) LAKI, K., Arch. Biochem. 50, 148 (1954). 12. KIELLEY, W. W., AND HARRINGTON, W. F., Biochim. et Biophys. Acta 41,402 (1960). 13. BI,UM, ft. J., AND FELAUER, E., Arch. Bioehem. Biophys. 81,285 (1959). 14. GILMOUR, I)., Nature 186,295 (1960).
93, 605-616 (1961)
The Binding of p-Chloromercuribenzoate by Myosin D A R C Y G I L M O U R ~ AN1) M A R T I N G E L L E R T -~ From the Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire Received February 21, 1961 Rabbit myosin rapidly binds 3-4 moles PCMB/10 ~ g. protein. Further binding, up to a maximum of 7-8 moles PCMB/105 g., occurs at a progressively diminishing rate. The reaction rate increases with both myosin and PCMB concentrations. The binding of 3-4 moles PCMB/10 ~ g. causes an acceleration of ATPase and inhibition of ITPase activity. These effects are more pronounced if the myosin PCMB reaction is carried out at high myosin concentration (~1 mg./ml.). Enzymic rates after such treatment are not constant in time. The changes are accelerated in the presence of substrate and are suggestive of a rearrangement of PCMB on the protein after the initial reaction. Treatment of myosin with PCMB for long times (48 hr.) avoids some of these complications. A linear relation between ITPase and PC1VIB tiger is then found. The reversal of PCMB binding by glutathione has also been studied. The results are interpreted in terms of a model of two-point attachment of substrate to the enzyme. INTRODUCTION The accelerating effect of p-chloromercuribenzoate, ~ a reagent known to bind preferentially to - - S H groups, on the A T P ase a c t i v i t y of myosin has been known for some time and has been studied b y a n u m b e r of workers (1-4). The first thorough s t u d y was t h a t of Kielley and B r a d l e y (3), who made simultaneous m e a s u r e m e n t s of the binding of P C M B and the effects on enzyme a c t i v i t y , and concluded t h a t the t i t r a t i o n of a p p r o x i m a t e l y half the accessible - - S H groups of the protein resulted in acceleration, while the binding of P C M B b e y o n d this point caused a progressively increasing 1Present address: Commonwealth Scientific and Industrial Research Organization, Canberra, Australia. 2 Present address: Section on Physical Chemistry, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland. 3 PCMB is used for p-chloromercuribenzoate throughout this paper. Other abbreviations used are ATP, ITP, and NTP for adenosine triphosphage, inosine triphosphate, and nucleoside triphosphate, respectively; ATPase, ITPase, and NTPase for their respective triphosphatases; and Tris for tris(hydroxymethyl)aminomethane.
inhibition which was complete when all the - - S H was bound. Our interest in the effect of P C M B was reawakened b y the discovery t h a t in m a n y experiments in which P C M B had been acting on the protein for only a short time (the conditions used in Kielley and B r a d l e y ' s work), the enzyme a c t i v i t y of the myosin was n o t constant with time. We therefore launched into a detailed examination of the binding of P C M B by myosin and the effects on nucleotidase a c t i v i t y , with particular emphasis on variations in these phenomena with time. Q u a l i t a t i v e l y our conclusions do n o t differ greatly from those of Kielley and Bradley, but some q u a n t i t a t i v e relationships have been revised and our d a t a show evidence of slow changes in the binding of P C M B to myosin n o t studied b y previous authors. The results have been i n t e r p r e t e d in terms of a model of the enzymic site of myosin suggested b y the kinetic studies of Blum (5). EXPERIMENTAL REAGENTS
The nucleotides were obtained as sodium salts from Sigma Chemical Company. They were dis-
605
606
GILMOUR AND GELLERT
solved in the buffer used for the dilution of the myosin, and the p H of the solutions was a d i u s t e d to 8.0. P C M B was purified by the m e t h o d of W h i t m o r e and Woodward (6). The c o n c e n t r a t i o n of the solutions was d e t e r m i n e d from the optica' density at 232 mr,, using the molar extinction coefficient found b y Boyer (7). The optical density changes slowly with time, so solutions were kept in the cold, and for most experiments were not more t h a n 2 days old. G l u t a t h i o n e solutions were made up fresh each day. MYosiN Myosin A was prepared from r a b b i t muscle by the m e t h o d of Szent-GySrgyi (8), which involves the removal of myosin B at an ionic s t r e n g t h of a b o u t 0.15 M, followed b y the precipitation of myosin A at a b o u t 0.05 M ionic s t r e n g t h . I n some p r e p a r a t i o n s the precipitate of myosin B did not aggregate completely even in the presence of ATP, and in these cases i t was necessary to centrifuge the p r e p a r a t i o n at this stage in the Spineo prep a r a t i v e model ultracentrifuge at 50,000 X g for 40-60 rain. to remove suspended protein. After final purification, myosin A was dissolved in 0.6 M KC1 and clarified b y centrifuging at 20,000 X g for 30 min. The keeping qualities of the enzyme were i m p r o v e d b y the addition of a small a m o u n t (1% by volume) of the buffer used in the enzyme tests. Myosin A p r e p a r e d by this m e t h o d and diluted with 0.6 M KC1 showed no change in the i n t e n s i t y of scattered light, measured at 90 ° angle, on the addition of ATP. Its ATPase a c t i v i t y varied from 8 to 20 ~moles s u b s t r a t e hydrolyzed/g. protein/sec, u n d e r the conditions of our experiments. P r o t e i n c o n c e n t r a t i o n was d e t e r m i n e d b y a modified Folin m e t h o d (9). PCMB
BINDING
The time course of the c o m b i n a t i o n of P C M B with myosin was followed by measuring the change in optical density at 250 m#, the m e t h o d developed by Boyer (7). When this reaction was followed for a long time (>1000 see.), excessive increases in optical density were sometimes noted, a p p a r e n t l y due to aggregation of the myosin. Such long-time experiments were checked, therefore, b y t a k i n g aliquots from a m y o s i n - P C M B mixture, reading t h e i r optical density, t h e n adding excess glutathione and noting the rise in optical density. This yielded the a m o u n t of free P C M B r e m a i n i n g in the m y o s i n - P C M B mixture at the time, and hence, b y s u b t r a c t i o n , the a m o u n t of P C M B b o u n d to the protein. Since the readings w i t h o u t a n d w i t h g l u t a t h i o n e were t a k e n within a few seconds of
each other, slow, nonspecific changes in optical density did not interfere. I t will be n o t e d l a t e r t h a t g l u t a t h i o n e removes some P C M B from tile protein, b u t since we had found t h a t the AO.D.~0 for the c o m b i n a t i o n of P C M B with myosin was identical w i t h t h a t for the c o m b i n a t i o n with glut a t h i o n e , this did not interfere with the validity of the method. Some difficulties did arise occasionally, however, when g l u t a t h i o n e itself caused a change in the aggregation of the protein. This was overcome b y t a k i n g aliquots from. the m y o s i n - P C M B mixture, p r e c i p i t a t i n g the protein by dilution and removing it b y centrifugation, t h e n t i t r a t i n g for free P C M B in the s u p e r n a t a n t . All three methods were used to o b t a i n the results recorded below. P C M B - b i n d i n g studies were usually done at 25°C., at pH 8.0. ENZYME TESTS 5'-Nucleotidase a c t i v i t y was measured in a buffer of the following composition: Tris, 0.05 M; histidine, 0.05 M; KC1, 0.08 M; CaC12, 0.001 M; pH, 8.0. Myosin was added to a c o n c e n t r a t i o n of 0.01-0.025 mg./ml., and nucleotide, dissolved in the same buffer, at 0.001 M. E a c h test comprised a series of (usually four) m e a s u r e m e n t s of phosp h a t e evolved at successive time i n t e r v a l s at 25°C., from which a s t r a i g h t line, or curve, of subs t r a t e split against time was established. N o t more t h a n 15% of the s u b s t r a t e was split in any such experiment, and since in all experiments except t h a t concerned w i t h competitive i n h i b i t i o n between s u b s t r a t e s the initial c o n c e n t r a t i o n of s u b s t r a t e exceeded the s a t u r a t i o n level b y at least tenfold, the decrease in s u b s t r a t e c o n c e n t r a t i o n during an experiment did not affect the velocity of enzyme action. Calcium was not at a s a t u r a t i o n level, since the high c o n c e n t r a t i o n required for s a t u r a t i o n m a y precipitate nueleotide, especially I T P , at p H 8.0. I t was found, however, t h a t a fivefold increase in Ca ++ affected b o t h the unt r e a t e d and P C M B - a c t i v a t e d ATPase to the same degree, causing a b o u t a 20% increase in each. F r o m this it was concluded t h a t the level of Ca ++ in the experiments was not significant for the i n t e r p r e t a t i o n of our results. E n z y m e a c t i v i t y was stopped b y the addition of trichloroaeetie acid to a c o n c e n t r a t i o n of 30-/0. Inorganic p h o s p h a t e was measured b y the m e t h o d of Fiske a n d S u b b a R o w (10) u n d e r c o n s t a n t conditions of time and t e m p e r a t u r e for all the sequence of procedures involved, and the i n t e n s i t y of the blue color of reduced p h o s p h o m o l y b d a t e was determined b y measuring the optical density at 750 m~ in a Zeiss model PMQ I I spectrophotometer.
607
B I N D I N G OF £ C M B RESULTS
BINDING OF SULFHYDRYL GROUPS
BY PCMB Figure 1 illustrates the typical time course of binding of PCMB to myosin. The three lower curves show the results obtained with three different concentrations of myosin treated with the same relative amount of PCMB (6.4 moles/10 ~ g.). It is evident that a certain number of - - S H groups, 3-4 moles/105 g. myosin, react very rapidly ( ~ 2 0 see.) in all cases, and that the remaining groups react progressively more slowly. (The three curves presumably approach a common asymptote, at very long times.) In the case of the more slowly reacting groups, it can be seen that the rate varies with the concentration of the reactants, a fact consistent with Boyer's (7) demonstration that the combination of PCMB with - - S H follows second-order kinetics. In other experiments in which smaller amounts of PCMB were applied (3-4 moles/105 g.), it was found that at the two higher myosin concentrations the reaction was complete within the time required to take the first reading of optical density (15 sec.), whereas at the lowest concentration it was complete in less than 100 sec. Most of these experi-
E
$
i0100
-
2 0 iO0 TIME
30100
I 4000
L 5000
(Sec.)
F i e . 1. Time course of the reaction of myosin - - S H with P C M B . The a m o u n t of P C M B comb i n e d was m e a s u r e d b o t h b y the AO.D. at 250 m~ and b y t i t r a t i o n of free P C M B w i t h g h t a t h i o n e . Curves 1, 2, and 3, 6.4 mole P C M B added/105 g. myosin. Myosin c o n c e n t r a t i o n s : curve 1, 0.013 m g . / m l . ; curve 2, 0.14 m g . / m l . ; curve 3, 0.8 rag./ ml.; curve 4, 11.5 moles P C M B added per 105 g. myosin; myosin c o n c e n t r a t i o n 0.7 mg./ml.
ments were done with the myosin dissolved in 0.2 M KC1. It was found that, at the same pH, the reaction of PCMB with myosin - - S H was appreciably slower in the presence of the Tris-histidine buffer used in the enzyme tests. Thus, on the addition of 3.5 moles PCMB/105 g. to myosin dissolved in 0.2 M KC1 at a concentration of 0.02 mg./ml., all of the PCMB had reacted within 40 sec., whereas the same reaction performed in the Tris-histidine buffer required 200 sec. for completion. The upper curve in Fig. 1 shows the time course of PCMB combination when the reagent is added in excess of the total - - S I t content of the protein. In a number of experiments of this nature which were prolonged for several hours, the total amount of PCMB which reacted varied between 7 and 8 moles/105 g. Owing to the uncertainties introduced by the instability of the protein after long incubation in the presence of excess PCMB, it was not possible to establish whether, in fact, tile reaction approaches a true end point short of the total sulfhydryl content of the protein. Amino acid analyses (11) have established the total content of - - S H plus - - S - - S - - groups in myosin at 8.6 moles/10z g. On the time scale normally used for " - - S H titrations" (incubation times of 100-300 sec.), 5-6 moles PCMB was bound/105 g., the amount varying with the preparation and also with the age of any one preparation. In Fig. 2 the time course of the reaction of PCMB with myosin is shown in a different way. In this experiment, small amounts of PCMB were added successively to a myosin solution, and the time course was followed after each addition. Again it is evident that the first 3.5 moles reacts very rapidly, and the rest progressively more slowly. EFFECTS OF PCMB ON ENZYME ACTIVITY
Qualitatively, our results on the effect of PCMB on myosin-ATPase were in agreement with those of Kielley and Bradley (3) and other workers who have shown that the addition of PCMB up to a certain relative concentration produces activation, but that the addition of larger amounts results in inhibition. The degree of activation
608
GILMOUR AND GELLERT i
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The effects on ATPase and ITPase are considered separately below.
I
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I00 200 300 400 500 600 700 SECONDS F ~ . 2. Time course of the reaction of myosin - - S H w i t h P C M B . P C M B added in i n c r e m e n t s of 1.2 moles/lO 6 g. myosin, at the times i n d i c a t e d by the arrows. Myosin c o n c e n t r a t i o n 0.2 mg./ml.
of the ATPase produced by PCMB varied with different myosin preparations, there being, in general, an inverse relation between the activity of the untreated myosin and the percentage activation achieved. Thus, some of the less active preparations were accelerated 3-4-fold by PCMB, whereas the increase in the most active was less than 100 %. The highest ATPase activity measured in the presence of PCMB was 36 umoles/g, myosin/
The relationship between activity and relative PC1VIB concentration for varying times of incubation, at the highest absolute concentration of both protein and reagent, is illustrated in Fig. 3. It is apparent that the addition of about 3 moles PCMB/105 g. protein results in an immediate activation of the ATPase which does not vary with the time of incubation. When about 4 moles is added, the activation measured after 25 sec. incubation decreases somewhat during the next 75 sec. With larger amounts of PCMB, only inhibition is measured, and the amount of such inhibition increases with the time of incubation. At lower myosin and PCMB concentrations, these effects are markedly slowed down, and a transitory activation becomes aparent at the higher PCMB levels (Fig. 4). This is shown more strikingly in Fig. 5, which illustrates an experiment with another myosin preparation having a lower initial activity and higher relative activation by PCMB. Figure 5 shows that at a myosin concentration of 0.024 mg./ml. a quantity of PCMB of 6.4 moles/105 g. requires approximately 20 hr. contact with L
see.
TIME COVRSE OF T~E EFFECT ON E~ZYME ACTIWTY The influence of the time of incubation of the myosin with PCMB on ATPase and ITPase activities was studied at three different myosin concentrations. These experiments were done by incubating the mixture of myosin and PCMB at 25°C. for varying lengths of time, at the end of which an aliquot of the mixture was added to a larger volume of buffer and substrate to start the activity run. At the lowest myosin concentration used, PCMB was added to the enzyme dissolved in the buffer at a concentration suitable for the activity test, which in this case was started by the addition of substrate.
i I\ \ \ \
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a
400
PCMB
-, fOOX..25 Sec.
"-. ¢x
4 6 (Moles/105 g Myosin)
FIG. 3. E f f e c t of P C M B on m y o s i n ATPase. M y o s i n t r e a t e d at a c o n c e n t r a t i o n of 0.8 m g . / m l .
The times refer to i n c u b a t i o n of myosin with P C M B , before s t a r t i n g the enzyme assay.
BINDING OF PCMB I
I
1
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o3
~
==
/
/
/
-6 204 E ::L
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609
vidual activity runs of the same myosin preparation as that used in Figure 5, treated at a myosin concentration of 0.38 mg./ml. I t can be seen from this figure that the initial activities of samples assayed 100 and 1000 sec. after the addition of P C M B to the protein were almost identical, although the activity of the first sample had dropped to less than 60 % of its initial value after 500 sec. in the presence of substrate.
IO
ITPAsE
I 2
PCMB
I 4
( Moles
/105q
I 6
Myosin)
FIG. 4. Effect of PCMB on myosin ATPase. Myosin treated at a concentration of 0.14 mg./ml. The times refer to incubation of myosin with PCMB, before starting the enzyme assay.
At high myosin concentrations, relatively small amounts of P C M B produced a marked inhibition of I T P a s e which varied little with the time of incubation (Fig. 7). Under these circumstances a revival of enzyme activity in the presence of substrate became apparent. This is seen in Fig. 8, which shows some of the individual activity runs from which the points on Fig. 7 are derived. I T P ase activity starts at a very low level but increases during the course of the experiment. At the lower myosin concentrations, inhibition of the ITPase was relatively slight for short incubation times, but increased with
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FIG. 5. Variation of myosin ATPase with time after treatment with 6.3 moles PCMB/105 g., at a myosin concentration of 0.024 mg./ml. Points record initial activities of the protein assayed after the indicated incubation times with PCMB. The inset shows the first part of the curve on an expanded time scale. the protein at 25°C. to achieve complete inhibition, although a substantial part of the activation which disappears during the first 3 hr. is apparent after only 20 sec. incubation. A striking feature of the slow decrease in activity from its maximum level is that inhibition is accelerated in the presence of substrate. This is illustrated in the series of curves in Fig. 6, which shows some indi-
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FIG. 6. Time course of myosin ATPase after treatment with 6.3 moles PCMB/105 g. at a myosin concentration of 0.38 mg./ml. The three curves, which are displaced vertically for clarity, are activity runs made after the indicated times of incubation with PCMB. Activities (v), in ~moles/ g./sec., refer to the dotted tangents.
610
GILMOUI~ A N D G E L L E I ~ T I
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runs on which Fig. 10 is based, demonstrates that the ITPase, like the ATPase, decays much more rapidly in the presence of the substrate than in its absence. Comparison of the results of the experiments on the time course of the effect of PCMB on the NTPase with the time course of the binding of PCMB as demonstrated by changes in optical density at 250 m~ reveals some anomalies. Thus, although the binding of 3-4 moles/105 g. PCMB is very rapid at all the concentrations studied, the effect of this binding at high concentrations is strong activation of ATPase and strong inhibition of ITPase, whereas at low concentrations there is little or no activation of ATPase and only weak inhibition of ITPase. This suggests that the groups which bind most rapidly at high concentration are different from those that bind most rapidly at low concentration. Moreover, at relative PCMB concentrations between 3 and 5 moles/105 g., changes in enzyme activity occur at a time when all the PCMB must have been already bound to the protein, even allowing for the slower rate of binding in the Tris-histidine buffer. These facts suggested the possibility that secondary changes were taking place in
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FIG. 8. T i m e course of m y o s i n I T P a s e after t r e a t m e n t w i t h 4.0 moles P C M B / 1 0 5 g., at a m y o sin c o n c e n t r a t i o n of 0.8 m g . / m l . T h e three curves, which are displaced v e r t i c a l l y for clarity, are a c t i v i t y r u n s m a d e after t h e i n d i c a t e d t i m e s of incubation with PCMB.
ec.
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longer incubation (Figs. 9 and 10). In these experiments ITPase activity decayed rapidly during the course of the enzyme tests. Figure 11, which shows some of the actual activity
FIG. 9. Effect of P C M B on myosin ITPase. M y o s i n treated at a concentration of 0.14 mg./m]. T h e three curves are for three different i n c u b a t i o n times, as shown.
BINDING OF PCMB r
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arms being substantially linear. The points at which the curves of inhibition of ATPase and ITPase intersect the abscissa did not coincide, a n d it was possible to obtain an enzyme preparation without any measurable ITPase activity, but still exhibiting a low activity toward ATP. (This difference may not have been significant since it was difficult to measure such low rates accurately and there was evidence that both lines showed some curvature as they approached the abscissa.) The relative PCMB concentration needed to produce complete inhibition varies with the age of the myosin preparation; for instance, for one enzyme tested the day after preparation this PCMB concentration was 7.0 moles/105 g., whereas 3 days later it had dropped to 5.8 moles.
I 6
REVERSAL OF THE PCMB EFFECT WIT~ GLUTATmON~
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FIG. 10. Effect of P C M B on myosin ITPase. Myosin t r e a t e d at a concentration of 0.013 mg./ml. The three curves are for three different incubation times, as shown.
Kielley and Bradley reported that the activation, but not the inhibition, of ATPase by PCMB is reversed by ~-mercaptoethanol. We have found a similar effect of glutathione
the protein subsequent to the initial binding of PCMB. In order to determine the end point of these time-dependent changes, the effect of PCMB on enzyme activity was determined after extended incubation times, when it might be assumed that the secondary changes would have run their course. EFFECT or PCMB A~'TER LONG
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These experiments were done by treating the protein at a relatively high concentration (0.6 mg./ml.) with PCMB in amounts varying from 0 to 7 moles/105 g. The mixture was incubated for 25 see. at 25°C., then for 48 hr. at about 5°C., after which activities against ATP and ITP were assayed. Under these conditions the amount of phosphate evolved was almost always linear with time, in contrast to the experiments reported in the two previous sections, suggesting that the conditions were, in fact, saturated with respect to time of incubation. With these long incubation times, the relationship between ITPase activity and relative PCMB concentration was found to be linear, or nearly so (Fig. 12). ATPase activity showed first a rising and then a falling phase, both
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300
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:FIG. 11. Time course of myosin ITPase after t r e a t m e n t with 6.4 moles PCMB/105 g., at a myosin concentration of 0.013 mg./ml. The three curves, which are displaced vertically for clarity, are activity runs made after the indicated times of incubation with PCMB. Activities (v), in ttmoles/g./sec., refer to the d o t t e d tangents.
612
GILMOUR AND GELLERT
on both the ATPase and ITPase. The curves of activities, in relation to applied PCMB, of the enzyme treated first with PCMB and then with an excess of glutathione, each treatment being for 48 hr. in the cold, are shown in Fig. 12. It is clear that the reversal is fairly complete up to the maximum in the ATPase-PCMB curve, although both activities decline to some extent up to this point. This decline may be due to a greater tendency toward slow irreversible denaturation in the PCMBtreated enzyme, since experiments done with shorter incubation times show a more nearly complete restoration of the original activities for both substrates with amounts of PCMB up to 3 moles/10 ~ g. Beyond the maximum of the ATPase-PCMB curve, the activity curves of the glutathione-treated enzyme decline rapidly toward zero, indieating that the inhibition of the ATPase and the final part of the inhibition of the ITPase are irreversible. Although at PCMB concentrations of 5-6 moles/105 g. the effect of adding glutathione is to cause a decrease in the ATPase and an increase in the ITPase, the two activities of the glutathione-treated enzyme have a consistent relationship to one another, the ratio of ITPase activity to ATPase activity being reasonably constant throughout the entire PCMB range. An attempt was made to investigate quantitatively the removal of PCMB from the protein by glutathione, with the object of determining whether the irreversible inhibition of enzyme activity was the result of a particularly firm binding to a specific - - S H group or to a general denaturation of the enzyme due to the loading of PCMB beyond a critical level. The experiment was done by treating the myosin first with PCMB, then with a 2-3 fold excess of glutathione. Protein was precipitated by dilution with water and removed by centrifugation; then the supernatant was examined spectrophotometrically for the presence of the PCMB-glutathione compound. The ultraviolet absorption spectrum of the supernatant fell on a curve which corresponded closely with that of the authentic P C M B glutathione compound. Semiquantitative estimates of the concentration of the compound, based on the molar extinction coe~-
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FIG. 12. Effect of P C M B on myosin A T P a s e a n d I T P a s e after long i n c u b a t i o n , and after reversal by glutathione. (D----(D A T P a s e ; • ..... • ATPase after g l u t a t h i o n e t r e a t m e n t ; ~ - - - - - B I T P a s e ; • ..... • I T P a s e after g l u t a t h i o n e t r e a t ment.
cient at 250 m#, indicated that whereas all of the PCMB was removed when the myosin had been treated with 4.6 moles/105 g., less than 100 % was removed when the myosin had been treated with 7.3 moles. This suggests that the binding of PCMB to some groups on the protein was stronger than the binding between PCMB and glutathione. PROTECTION BY SUBSTRATE AGAINST
PCMB INHIBITION Further evidence that the irreversible inhibition of enzyme activity may be the result of the binding of a relativelyinaccessible - - S H group at the active site is found in the protection against inhibition afforded by the substrate. Evidence has been presented in previous sections for the acceleration of inhibition by substrate in the presence of PCMB. Protection of the enzyme by the substrate occurs at a more advanced level of inhibition. This is illustrated by Fig. 13, which shows the time course of inhibition of myosin treated at low concentration with a large
613
B I N D I N G OF P C M B
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2000
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.
2500
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FI~. 13. Effect of P C M B (122 moles/10 ~ g.) on the ATPase a c t i v i t y of myosin t r e a t e d (at 0.011 mg./ml.) in the presence and absence of substrate. P C M B added at time shown by arrow. In the upper curve A T P was present before P C M B was added; in the lower curve P C M B was present for 1600 see. before A T P was added. The d o t t e d line shows the a c t i v i t y before PCMB t r e a t m e n t . Activities (v) are in ~moles/g./see.
excess of PCMB in the presence of substrate. It was found that under these conditions the enzyme passed usually through a phase of transient activation after which the activity decayed rapidly to a low steady rate. The same preparation treated in the way in the absence of substrate was shown to be quite inactive when assayed at a time (1600 sec. after the addition of PCMB) when the enzyme treated in the presence of substrate was exhibiting a constant velocity. COMPETITIVE IXI~ImTmN OF ATPAs~ ACTIVITY BY I T P The opposing effects of PCMB on ATPase and ITPase activities could be most simply explained if the two substrates were split at separate sites, and a necessary preliminary to attempting an explanation of the foregoing results would be to determine whether or not this is so. Certain conditions of PCMB treatment establish a relationship between the activities (high ATPase, very low ITPase) suitable for the testing of competitive inhibition between the two substrates. In the experiments designed to make this test, myosin at 0.3 mg./ml, was incubated with 5.6 moles/105 g. PCMB at 25°C. for 200 sec. After such treatment the initial activities against ATP and ITP, with both substrates at a concentration of 10 -3 M, were 24.7 and 1.6 ~moles/g./sec., respectively. Myosin so treated was used to de-
termine Km's for ATPase and ITPase by the usual (l/v) vs. (1/[S]) plots. The Km's found were 2.9 X 10-5 for ATP and 9.3 X 10-~ for ITP. Then the velocities at different ATP concentrations were determined in the presence of 10-3 M ITP. The results of this experiment are shown in Fig. 14. The circles are based on actual experimental figures for the velocity at various ATP concentrations, and the triangles represent the same figures corrected for the splitting of ITP, which was calculated from the known velocities, substrate concentrations, and binding constants for the two substrates at the set of conditions represented by each point. The straight line in the figure, intersecting the ordinate at the same point at which the line for (l/v) vs. (1/[ATPD intersects, is the theoretical line for competitive inhibition, with K~ equal to the determined value of Km for ITP. Although the points show considerable scatter, due to difficulties in determining accurately the small amounts of phosphate evolved, and the tendency for v to vary with time during the runs, they show reasonably good agreement with the theoretical curve and constitute quite strong evidence that the two substrates are split at the same site. DISCUSSION
Our results show a discrepancy between the time course of the binding of PCMB to the protein and the time course of the effects on enzyme activity. This discrepancy could be due to secondary eonfigurational changes taking place in the protein or to the rearrangement of PCMB on the protein subsequent to its initial binding. Although our results do not completely exclude the first explanation, they strongly suggest that the second is true. The evidence for this is: Combination of 3-4 moles PCMB/10 5 g. protein does not produce the same effect on enzyme activity at high concentrations as at low, suggesting that the reagent may be bound to different - - S H groups under different conditions. The slow changes in enzyme activity with increasing incubation time, which are accelerated by the presence of substrate, are in such a direction as to bring the curve for activity vs. PCMB concentration nearer
614
GILMOUR AND GELLERT
the "equilibrium" position shown in Fig. 12. Where these changes are in the direction of diminishing enzyme activity it might be argued that they could be the result of either rearrangement of PCMB molecules or the destruction of the active site by secondary configurational changes; but where, as in the experiment illustrated in Fig. 8, they are in the opposite direction, then it is difficult to see how the second explanation can hold. The straight-line relationship between ITPase and PCMB concentration, show in Fig. 12, suggests that ITPase activity depends on one or more of a group of about 40 homogeneous --SH groups on the myosin molecule (assuming a molecular weight of 6.2 X 105 g. for myosin) (12). Our studies on the binding rates have shown that these - - S I t groups are far from homogeneous with respect to the kinetics of binding to PCMB, and, more importantly, it is clear that they fall into at least two sharply distinct groups with respect to equilibriurn-binding energies, since the combination of PCMB with some can be reversed by glutathione, but with others it cannot. The two groups are further distinguished by the fact that the binding of PCMB to the first causes acceleration of the ATPase, whereas binding to the second causes inhibition. It seems, therefore, that the continuity of the straight line expressing the effect on ITPase activity of the binding of PCMB to both sets of --SH groups may be fortuitous. Further, the equilibrium relationship between ATPase activity and PCMB concentration (Fig. 12) suggests that there is a sequential binding of PCMB to the two distinct sets of --SH units. Any considerable overlap of the accelerating and inhibiting effects of PCMB binding with the addition of increasing amounts of PCMB would introduce curvature into the graph of ATPase activity versus PCMB applied. In fact, this graph takes the form of two intersecting straight lines, the only curvature being immediately in the region of changeover from one straight line to the other. Thus it follows that the binding of PCMB to the same --SH unit or group of --SH units causes both acceleration of the ATPase and inhibition of the ITPase. The following picture of the titration of
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FIG. 14. Competitive inhibition by ITP of the ATP hydrolysis by PCMB-treated myosin. For explanation see text. rayosin by PCMB then emerges: The addition of PCMB in amounts up to about 27 moles/inole myosin results in a rapid random binding of the mercurial to a group of --SH units of more or less equal "accessibility", of which one or more is situated at the active center. This binding is reversible by glutathione and causes acceleration of the ATPase and inhibition (to a maximum of 70 %) of the ITPase. Addition of PCMB in amounts in excess of 27 moles/mole myosin results in a subsequent, slower, but tighter, binding to a second, less accessible group of --SH units, of which, again, one or more is situated at the active center. This binding, which is not reversible by glutathione, is complete when about 40 moles PCMB have been bound per mole of myosin. It causes inhibition of the ATPase and further inhibition of the ITPase beyond that resulting from the binding of the first group. This view of the participation of --SH units in the active center of myosin is, as stated above, qualitatively similar to that proposed by Kielley and Bradley (3). It can also be very well expressed in terms of Blum's interpretation of his data on the binding of substrate to myosin-NTPase. Blum (5) has proposed that the substituent at the 6 position of the purine or pyrimidine ring of the nucleotide substrate of myosin has
BINDING OF PCMB an important effect in determining the binding of the substrate to the enzyme. Nucleotides with an amino group on the 6 position, such as ATP, bind strongly to the enzyme, and the over-all velocity of hydrolysis of the P - - O - - P bond is low, because of the slow desorption of the products. Nucleotides with a hydroxyl on the 6 position, such as I T P , bind less strongly, and exhibit a higher hydrolysis rate. Furthermore, Blum and Felauer (13) have suggested that the acceleration of ATPase by 2,4-dinitrophenol is caused b y an interference with the binding of the amino group of the 6-amino nucleotides. From these suggestions has emerged the idea of a twopoint attachment of the nucleotides to the enzyme, one point of attachment being the polyphosphate end of the substrate, the other, the ring end. Our results suggest that an - - S H group is included in each part of the active center. The attachment of P C M B to the relatively accessible - - S H at the ringbinding site could prevent the binding of the 6-amino of A T P and thus result in an enhanced rate of hydrolysis of this substrate. The addition of larger amounts of P C M B would result in combination with the less accessible - - S H at the phosphate-binding site, thereby blocking hydrolysis of the substrate completely. I t follows from our results that the attachment of P C M B at the ring-binding site inhibits the hydrolysis of I T P as well as accelerating the over-all rate for ATP. I t is conceivable that the presence of the benzene ring of P C M B at the ring-attachment site could hinder, but not absolutely prevent, either the attachREL. PCMB CONC. MOLES/IO~
ACTIVE SITE
ATPose ITPose ACTIVITY ACTIVITY P-ATTACHMENT RING-ATTACHMENT POSITION POSITION
200
80
615
merit of the I T P molecule, or its hydrolysis, and that complete titration of this group would result in the 70 % inhibition of ITPase recorded. The same kind of hindrance would be expected to affect A T P as substrate, but in this case the inhibition is more than compensated by the acceleration resulting from the blocking of the ring binding through the 6-amino group. It seems that the kinetics of the binding of P C M B to the - - S H at the ring-binding site are such that under conditions of high concentration of both myosin and P C M B this - - S H binds the mercurial preferentially, even in comparison with others of the group of roughly 27 - - S H units on the molecule which bind P C M B less strongly than does glutathione. There is then, apparently, a subsequent migration of P C M B away from the active center, presumably to other - - S H ' s on the molecule, with the observed effect of a decrease with time of ATPase activity and an increase with time of I T P a s e activity. The demonstrated acceleration by substrate of this migration is perhaps not surprising. On the other hand, the slower binding of P C M B to the less accessible - - S H at the position of phosphate attachment is hindered by the presence of substrate on the active center. The postulated sequence of events in the titration of the protein by P C M B and the effects on ATPase and ITPase are presented schematically in Fig. 15. In another paper (14), some extensions of this model of myosin-nucleotidase are proposed, in accordance with data on the variation with temperature of the P C M B effect, but as a hypothesis to cover the data presented here the scheme shown in Fig. 15 is adequate. ACKNOWLEDGMENTS It is a pleasure to acknowledge the encouragement, suggestions, and intellectual stimulus provided by Dr. Manuel F. Morales, in whose laboratory this work was done. We are also indebted to Mr. Bruce Henry for his skilled technical assistante.
6.5
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X la.~
0
0
FIG. 15. Proposed model for effects of PCMB on myosin enzyme activity. X = position blocked by PCMB.
This work was done during tenure of a Senior Traineeship (U. S. Public Health Service Grant 2G-174) by one of us (D. G.), and was also supported by National Heart Institute Grant H-3598 on which the other of us (M. G.) was a coinvestigator.
616
GILMOUR AND GELLERT REFERENCES
1. SINGER, T. P., AND ]~ARRON, E. S. G., Proc. Soc. Exptl. Biol. Med. 56, 129 (1944). 2. PoLIS, B. D., AND MEYERI~OF, O., J. Biol. Chem. 169, 389 (1947). 3. KIELLEY, W. W., AND BRADLEY, L. B.~ J. Biol. Chem. 218, 653 (1956). 4. BLUM, J. J., Arch. Biochem. Biophys. 87, 104 (1960). 5. BLU~, J. J., Arch. Biochem. Biophys. 55, 486 (1955). 6. WI-IITMORE, 1~. C., &ND WOODWARD, G. E., Organic Syntheses Coil. Vol. I, I941, p. 159. 7. BoY~Ja, P. D., J. Am. Chem. Soc. 76, 4331 (1954).
8. SZENT-GY~RGYI, A., "Chemistry of Muscular Contraction," 2nd Ed. Academic Press, New York, 1951. 9. LOWRY, O. H., ROSBBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 10. FISKE, C. H., AND SUBBAI~OW, Y., J. Biol. Chem. 66,375 (1925). 11. KOMINZ, D. R., HOUGH, H., SYMONDS, P., AN]) LAKI, K., Arch. Biochem. 50, 148 (1954). 12. KIELLEY, W. W., AND HARRINGTON, W. F., Biochim. et Biophys. Acta 41,402 (1960). 13. BI,UM, ft. J., AND FELAUER, E., Arch. Bioehem. Biophys. 81,285 (1959). 14. GILMOUR, I)., Nature 186,295 (1960).