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Observations on the role of sulfhydryl groups in the functioning of actomyosin
ARCHIVES
OF BIOCHEMISTRY
AND
Observations
BIOPHYSICS
(1962)
97,321-328
on the Role of Sulfhydryl Functioning
Groups
in the
of Actomyosin
.J. J. BLUM The Gerontology Branch, National Heart Institute, National Institute of Health, Public an,d Welfare, Bethesda, Health Service, United States Department of Health, Education, Maryland, and the Baltimore City Hospitals, Baltimore, MarylarLd
Received October 17, 1961 The reaction of N-ethylamaleimide (NEM) with actomyosin causes the same pattern of activation and inhibition of ATPase activity at low KC1 in the presence of Mg” as at high KC1 in the presence of Ca”. The reaction of enough NEM to cause maximal ATPase activation does not interfere with superprecipitation or with the ability of relaxing factor grana to prevent superprecipitation, but does decrease the tendency of actomyosin to dissolve at high ATP concentrations. Further reaction with NEM reverses the activation of ATPase activity and prevents superprecipitation. Superprecipitation can be completely inhibited before there is any net inhibition of ATPase activity. It is suggested that two -SH groups are involved in the functioning of myosin, and their roles in ATPase activity and in mechanochemical coupling arc discussed. INTRODUCTIOX
The interaction between myosin and actin certainly involves the -SH groups of myosin (l-3) but probably does not involve the -SH groups of actin (3, 4). The exact role of the --SH groups on myosin in t.his interaction, is, however, not yet cIear. Bailey and Perry (3) reported that several reagents which reacted with the --SH groups of myosin destroyed both the adenosinetriphosphatase (ATPase) activity and the affinity of the myosin for actin. Weber and his colleagues (2, 5) have examined the effects of a variety of -SH reagents on the ATPase activity and contractility of actomyosin (AM) systems, and have accumulated evidence that when groups of myosin are blocked the -SH there is a concomitant loss of ATPase activity and of contractility. This view was challenged by Turba and Kuschinsky (6), who reported that oxarsan treatment of myosin A could inhibit superprecipitation without causing any appreciable change in ATPase activity. Their results were, how-
ever, challenged by Mugikura (7, 8) who reported that oxarsan caused a parallel loss of ATPase and superprecipitability. More recently, there has been a growing realiaation that the -SH groups of myosin are heterogeneous both in respect to ATPase activity (9, 10) and with respect to the interaction between myosin and actin. Thus, BBr6ny and B&rBny (11) demonstrated that although certain -SH reagents inhibited both the ATPase activity and the actin-combining ability of myosin A, these reagents would inhibit the ATPase activity of myosin B but would not dissociate it into actin and myosin A. B&dny and BBrbny (11) also found that high concentrations of iodoacetamide could completely inhibit the ATPase activity of myosin A without interfering with its actin combining ability, thus suggesting t’hat the -SH groups of myosin are involved to different extents in the ATPase and in the contractile funct,ions of myosin. We have recently studied patterns of ATPase activation and inhibition caused by reaction of several -SH reagents with 321
322
BLUM
myosin at high KC1 concentrations in the presence of Ca+ + (12,13). It was suggested that two -SH groups are involved in the hydrolytic functioning of the active site, and some of the properties of the individual -SH groups were partially analyzed. We have now found that activation of ATPase by low concentrations of N-ethylmaleimide p-chloromercuribenzoate and (NEW (PCMB) can be observed at 0.1 M KC1 in the presence of Mg++. This observation (which has also been made by W. W. Kielley and T. Sekine, personal communication) has permitted us to compare the effects of these -SH reagents on both the contractile and the enzymic properties of myosin, and thus further analyze the role of the -SH groups of myosin in these processes. A preliminary account of this work has already appeared (14). EXPERIMENTAL NEM and the sodium salts of adenosine triphosphate (ATP) and PCMB were obtained from the Sigma Chemical Company. PCMB was purified by the methods of Whitmore and Woodward (15), and its concentration was determined from absorbance measurements at 232 rnp, using the extinction coefficient of Boyer (16). NEM concentration was determined from absorbancy measurements at 300 mp, using the extinction coefficient of Alexander (17). Actomyosin was prepared from 24-hr. extract of rabbit muscle, using the KCl-PO,EDTA extraction fluid described by Mommaerts (18). In addition to three reprecipitation cycles for. purification, the myosin B was partially freed of low molecular weight aggregates of myosin A by discarding the supernatant of a 45-min. centrifugation step at 18,000 x g in 0.28 M KCl. Protein concentration was determined by the modified Folin ‘method of Lowry et al. (19). The actomyosin was reacted with NEM in two ways. In the first method, identical amounts of NEM were added to aliquots of actomyosin in 0.6 M KCl, 0.02 M Tris, pH. 7.50 and were permitted to react for known times at 25.5”C. The reaction was terminated by adding an excess of cysteine. A control was prepared by mixing the NEM and cysteine first, and then adding the mixture to an aliquot of actomyosin. The tubes were kept at 25.5”C. until the longest reaction was complete and were then put in an ice bath until the contents of each tube was transferred to a dialysis sac. The dialysis tubing was pretreated with disodium ethylenediamine tetraacetate (EDTA) and cysteine
and washed with glass-distilled water before use. The dialysis sacs were individually dialyzed for at least 24 hr. at 4°C. against a solution of 0.1 iM KCI, pH 7.0, containing either 0.05 M Tris or 0.01 M histidine and 0.005 M potassium oxalate, as specified below. In the second method, increasing amounts of NEM were allowed to react with the actomyosin at 0°C. for 24 hr. in 0.6 M KCI, 0.02 M Tris, pH 7.5. Each sample was then dialyzed against one of the 0.1 M KCl, pH 7.0 buffers described above. Essentially the same procedure was used with PCMB, except that the reaction at 0” was allowed to proceed for 70 hr. before dialysis was begun. After dialysis was completed, the actomyosin samples were kept at 0°C. until assayed for ATPase activity and superprecipitability. Grana containing the relaxing factor system were prepared by the procedure of Nagai et al. (20) and were used within 24 hr. of preparation. Superprecipitation was measured at room temperature (about 25°C.). Except when the effects of grana were to be tested, the dialyzed actomyosin was added to a small beaker containing the appropriate amount of ATP and Mg++ and the mixture was transferred to a Wintrobe hematocrit tube. When the effects of grana were to be tested, the grana were incubated with the ATP for about 1 min. prior to the addition of the actomyosin, and the mixture was then transferred into a Wintrobe tube. The hematocrit tubes were spun for exactly 2 min. in a clinical centrifuge at 1780 X g. The precipitate height was measured and normalized so that it refers to a column of liquid 10 cm. long. ATPase activity was measured by the FiskeSubbaRow technique as described earlier (12). In experiments where high ATP concentration was used, the samples to be assayed were diluted in a sufficient volume of trichloroacetic acid (final concentration, 3.3%) to insure that the unhydrolyzed ATP would not interfere with the development of the blue color (21). The ATP and actomyosin were incubated at the temperature of the assay (25.5”C. unless otherwise specified) for about 60-70 sec., at which time the first of two aliquots was taken. The second aliquot was taken between 100 and 500 sec. later, depending on the expected ATPase activity. When the effect of relaxing factor grana was to be measured, the grana were first mixed with ATP, and aliquots of this mixture were put into the 25.5” bath and immediately mixed with the actomyosin. Samples for orthophosphate analysis were drawn after the 60sec. incubation period. In addition, samples of grana were mixed with buffer instead of actomyosin, and the ATPase activity due to grana alone was measured at several time points up to the end of the 5-min. incubation period. The ATP-
ROLE
OF -SH
GROUPS
ase activity of the actomyosin could then be obtained by subtracting the activity due to the grana. The units of ATPase activity in all the figures and tables throughout this paper are pmoles P/sec./g. RESULTS
117henthe ATPase activity of actomyosin that has reacted with NEM for various times is assayed at low salt in the presence of Mg++, it is found (Fig. 1) that the pattern of activation and inhibition resembles the pattern found for NEM and PCMB (10) at high KC1 in the presence of Ca+ + or Mg+ +. In the experiment shown in Fig. 1, the reaction was terminated before any net inhibition of ATPase activity occurred. With longer times of exposure (or higher concentrations of NEM), complete inhibition is obtained, although this is not shown in Fig. 1. It can be seen that the re-
TIME
IN
ACTOMYOSIN
323
action of NEM with the group responsible for activating ATPase does not inhibit superprecipitation, while the reaction of NEM with the group responsible for the loss of this activation causes a loss of superprecipitability. It should be noted that SUperprecipitation is completely inhibited befor there is any net inhibition of ATPase (Fig. 1). In the presence of relaxing factor grana, there is an inhibition of the ATPase activity of untreated actomyosin and a tendency for the actin and myosin to dissociat,e and thus form a solution rather than a suspension (22). In our experience the amount of ATPase inhibition caused by the grana has been variable but is not very sensitive to pretreatment of the actomyosin with NEM. The effect of relaxing factor grana on actomyosin (AM) not treated with NEM or
Imin.1
FIG. 1. Effect of NEM treatment on ATPase, superprecipitation, and response to relaxing factor of actomyosin. Actomyosin (6.9 mg./ml.) was reacted with NEM (5.5 moles/lo5 g.) in 0.6 M KCI, 0.02 M Tris, pH 7.5, at 25.5”C. for the times shown on the abscissa and then dialyzed against 0.1 M KCl, 5 X lo-’ M potassium oxalate, 0.01 M histidine, pH 7.0. BTPase activity (0, 0, left-hand ordinate) and superprecipitation (Cl, W, right-hand ordinate) were measured in the histidine buffer with 4 X lOA 1M ATP, 2 X 10.’ M Mg”, 2.1 mg. AM/ml. Solid symbols, grana present (0.7 mg. grana/mg. AM); open symbols, no grana. The two plus (+) symbols in the upper left indicate that in the presence of grana, untreated AM and AM treated for 1 min. with NEM, dissolved upon the addition of ATP. In this figure and in Figs. 3 and 4 the height of the control AM column after centrifugation in the presence of Mg”, but not ATP, is shown by an arrow which points to a vertical bar that extends one standard deviation above and below the average value.
324
BLUM
treated with NEM for only 1 min. was to dissolve it, as indicated in Fig. 1. With increasing time of exposure to NEM, the AM became less soluble in the ATP-grana mixture, and even before maximum ATPase activation was achieved it was clear that NEM treatment did not prevent the relaxing action of the grana-ATP system. Thus, although brief exposure to NEM affects the solubility of AM in ATP-grana, no concentration of NEM tested appeared to prevent the relaxing action of grana, provided, of course, that superprecipitation was still observable in the absence of grana. The experiment shown in Fig. 1 demonstrated an effect of NEM treatment on the solubility properties of AM in high concentration of ATP-grana. Since the ATP-grana system caused relaxation in the NEMtreated AM, it was of interest to inquire
whether NEM changed the solubility properties of AM with respect to high concentrations of ATP alone. When myosin is reacted with high concentrations of NEM (Fig. 2)) it is found that even in the absence of ATP the height of the centrifuged AM column decreases with increasing exposure to the NEM. In the presence of low ATP (1O-3 M, 3 x 10V3 M), superprecipitation is complete until the ATPase activity begins decreasing from its maximum (cf. Fig. 1). Superprecipitation is inhibited before there is any net loss of ATPase activity at these two concentrations of ATP. At higher ATP (10m2 M) , the AM that has not been treated with NEM or has been treated for only 1 min. dissolves (Fig. 2)) but AM that is treated with NEM for more than 1 min. no longer dissolves. It is clear that some -SH groups of AM are
l
I 5
. 10
15 TIME
. 20
25
30
(min. ]
FIG. 2. Effect of increasing ATP concentration on ATPase activity and superprecipitation of NEM-treated AM. Actomyosin (3.1 mg./ml.) was reacted with NEM (10 moles/lO’ g.) for the times shown on the abscissa in 0.6 M KCl, 0.02 M Tris, pH. 7.5 at 25.5”C., and then dialyzed against 0.1 M KCl, 0.05 M Tris, pH. 7.5 for 36 hr. ATPase (solid lines, solid symbols, left-hand ordinate) and superprecipitation (dashed lines, open symbols, right-hand ordinate) were measured in 0.11 M KCl, 2 X lo-* M Mg++, 0.05 M Tris, pH 7.5, with final concentrations of ATP of 1 (0, O), 3 (W, Cl), and 10 mM (A, A). The curve with the inverted triangles (V) shows the height of t.he AM column after centrifuging in the absence of ATP. The plus (+) symbols in the upper left of the graph indicate that at 0 and 1 min. of NEM treatment, 10 mM ATP dissolved the AM. The ATP used in this experiment was converted to the potassium form by equilibration with an excess of well-washed Dowex 50 in the K’ form.
ROLE
OF -SH
GROUPS
IN
325
ACTOMYOSIN
sentially independent of the -SH groups of actomyosin, alt,hough these groups can modulate the sensitivity of the actomyosin to the high ATP. When NEM activates the ATPase activity of actomyosin, it also increases the sensitivity of the AM to superoptimal inhibition. The increased sensitivity to superoptimal inhibition is removed as increasing NEM reverses the activation of ATPase activity. The data shown in Figs. 1 and 2 were obtained from timed exposures of AM to excess NEM. The question arises whether similar results may be obtained by long exposures of actomyosin to small amounts of NEM. When this was done, it was found that a similar patt’ern was obtained (Fig. 3). There was no inhibition of superprecipitation until enough NEM was added to begin decreasing the ilTPase activity from its maximum. In this experiment the reaction patt,ern of the -SH groups with NEM is such that there was about a 30% loss of ATPase activity when the superprecipitation was completely inhibited.
involved in determining the solubility of AM in high concentrations of ATP. Qualitatively similar result’s have been obtained when the ionic strength was held constant as the ATP concentration increased. The data of Fig. 2 also bear on the role of -SH groups in the superoptimal inhibition of the ATPase activitv of AM (23, 24). If we set the ATPase activity measured in 1O-3 M as loo%, then increasing the ATP to 3 x lo-” M caused a 20% inhibition of the activity of untreated AM. This increased to a 45% inhibition with increasing time of treatment, up to about 4 min. and then decreased to about 20% inhibition again with increasing exposure to NEM. At higher ATP (lo-’ J1) the activity of untreated AM is inhibited by 88% as compared to the activity in low3 M ATP. Brief NEM treatment increases the superoptimal inhibition to 94%, while further NEM treatmerit again removes this extra sensitivity and returns the inhibition to about 82%. The ability of high ATP to cause superoptimal inhibit,ion of ATPase is thus es-
-8
.6
-4
-2
,’ / ._,_*-*-O---------o-----9------,3
2
MOLES NEM/IO'
I(
L
I
-0
gms. MYOSIN
FIG. 3. Effect of low concentrations of NEM on ATPase activity and superprecipitation of BM. Actomyosin (4.23 mg./ml.) was reacted with NEM in the amounts shown on the abscissa for 24 hr. at 0°C. in 0.6 M KCI, 0.02 M Tris, pH 7.5 and then dialyzed against 0.1 M KCl, 0.005 M potassium oxalate, 0.01 M histidine at pH 7.0 for 23 hr. STPase activity (O-O, left-h an d or d’ma t e ) and superprecipitation (O-0, right-hand ordinate) were measured in the histidine buffer with 4 X 10e3 M ATP and 2 X 10.’ M Mg”. Also see legend for Fig. 1.
BLUM
326
Although the reaction of NEM with -SH groups forms strong covalent bonds, this is not true for the reaction of PCMB with -SH groups. Nevertheless, the bonds formed with PCMB are sufficiently stable so that dialysis against buffered salt solutions will not remove all the bound PCMB. When actomyosin was reacted with very low concentrations of PCMB, it was found (Fig. 4) that the relations between ATPase activity and superprecipitation were similar to the relations found using NEM. In the presence of Mg++ at low KCl, the ATPase was activated by low concentrations of PCMB and superprecipitation was not inhibited. Higher concentrations of PCMB decreased the ATPase activity towards the control value and prevented superprecipitation (Fig. 4). In this experiment the maximum activation occurred after treatment with about 0.06 mole PCMB/105 g., or 0.38 mole/mole myosin A (25). Even if the actin content of the AM were as much as SO%, this would mean that less than 1 mole PCMB had reacted with the myosin to
cause maximal activation. This suggests the possibility that PCMB catalyzed the oxidation of some -SH groups and prevents one from using the data in Fig. 4 for computing the stoichiometry of the reaction between PCMB and actomyosin. The data so far presented show that the -SH group involved in activating the ATPase activity does not prevent superprecipitation when it reacts with NEM or PCMB. It has been suggested (12) that cysteine ethyl ester (CEE) or X-aminoethylisothiouronium bromide (AET) reacts only with this -SH group on myosin A. It was therefore of interest to establish whether reaction of AM with AET would inhibit superprecipitation. The data presented in Table I show that superprecipitation was not inhibited, thus further indicating that the -SH group involved in the activation of ATPase is not involved in superprecipitation. DISCUSSION
From
the results
of a study
of the inter-
-6
6’
>_,l_AYoOO
d . 2
4
(MOLES
. 6
. 8
PCMB/lO’ gnu. MYOSIN)
10
0
x10’
FIG. 4. Effect of PCMB on superprecipitation and ATPase activity of AM. Actomyosin (4.39 mg./ml.) was reacted with PCMB in the amounts shown on the abscissa for 70 hr. at 0°C. in 0.6 M KCI, 0.02 M Tris, pH 7.5 buffer. It was then dialyzed for 22 hr. against 0.1 M KCl, 5 X lo-’ M potassium oxalate, 0.01 M histidine buffer at pH 7.0, and assayed for ATPase activity (O-O, left-hand ordinate), and superprecipitation (O-Q, right-hand ordinate) using 2 X 1Om8 M Mg++ and 4 X lo-* M ATP. Also see legend to Fig. 1.
ROLE
OF --SH
GROUPS
action between myosin A and PCMB (in high KC1 with Ca+ +) , it was suggested that two -SH groups were involved in the functioning of the active hydrolytic site (13). The reaction of -SH1 with PCMB activated the ATPase activity, while the reaction of -SH, with PCMB reversed this activation and then inhibited hydrolysis. The present data (Figs. 1,3, and 4) show that at low KC1 in the presence of Mg++ the same pattern of activation, reversal of activation, and inhibition are obtained when eit.her NEM or PCMB interacts with actomyosin. The reaction pattern of these -SH groups will depend on the concentrations of AM and of -SH reagent, on the temperature, and on the ionic conditions. Under conditions such as were used in these experiments the rcact’ions seem to be fairly well separated. The data demonstrate clearly that the reaction of -SH1 with PCMB or NEM does not prevent superprecipitation. When more -SH reagent reacts it is possible to completely inhibit superprecipitat,ion before there is any net loss of ATPase activity. This could be interpreted in two ways: (a) One could assume that there are three -SH groups involved in t,he functioning of actomyosin. Reaction of -SH1 with e.g., NEM, enhances the ATPase activity. Reaction of -SH, reverses t,his activation and prevents TABLE
I
EFFECT OF AET ON ATPASE ACTIVITY SUPERPRECIPITATION OF ACTOMYOSIN
AND
Activity Supegxi&tate Mg++ pm&s
Control AET
1.77 1.37
Mg” + Ca++
IN
ACTOMTOSIN
327
superprecipitation. At higher NEM concentrations -SHB reacts and causes loss of hydrolytic activity. (b) If, as seems more likely, there are only two -SH groups per active site, then reaction of -SHI with NEM would activate ATPase activity and reaction of -SH, would reverse this activation and prevent superprecipit’ation.l In the absence of actin or ATP a secondary small conformation change involving -SH2 would occur, leading to t’he loss of hydrolytic activity. In t.he presence of act,in or ATP, however, this secondary change would be impeded and one would observe inhibition of superprecipitation without any net loss of ATPase activity. To the extent that superprecipitation is a satisfactory model of the contractile process, these results indicate that the transduction of chemical free energy of ATP into mechanical energy does not depend crucially on either the binding of ATP t’o the site (26) or on the over-all rate of splitting of the ATP (27). What does seem to be required is that the hydrolysis occurs via a pat,hway involving a particular sulfhydryl group which we have tentatively designated as -SH2. If this group reacts with NEM or PCMB, hydrolysis may still proceed but it will no longer be coupled to the contractile machinery even in the presence of Mg++. Other types of groups, of course, may also be required for mechanochemical coupling (11,28). Physiologically, the relaxing factor system found in grana appears to control the mechanochemical coupling process (20)) but it is not known via what groups of the act’in-myosin system t.his control is ex-
P/sec./g.
0.31 0.51
;lt3 0.3
Actomyosin (6.65 mg./ml.) was reacted with 5 X 10-S M AET for 8 hr. at 0°C in 0.6 M KCl, 0.02 M Tris, pH 7.5, and then dialyzed for 18 hr. against 0.1 M KCl, 0.05 M Tris, pH 7.5 buffer. Superprecipitation was measured using 10-a M ATP, 2 X 10e3 M Mg++, and 3.32 mg./ml. AM. The precipitate height in the absence of ATP was about 8 cm. for the control and for the AET treated protein. ATPase was also measured in the low KC1 buffer, with 10-s M ATP and either 2 x lo-3 M Mg++ or 2 X 10-Z M blg++ plus 5 X 10-s M Ca++.
‘When -SH, of the myosin is reacting with YEM, it is also possible that -SH groups on the actin are reacting with NEM and that these, rather than --SH? , cause t,he loss of superprecipitability. Aside from evidence suggesting that the --SH groups of ac*tin are not involved in the interaction betwr,en myosin and actin (3> 4), the present experiments have been conducted over a wide range of NEM to AM ratios and for several different, AM preparat.ions. In all cases the loss of superprecipitabilitv coincided with the decrease of ATPase actktp from its peak, a result which would not be expected if the reaction of the -SH groups of actin with NEM caused the loss of superprecipitability.
328
BLUM
erted. The data presented in Fig. 1, however, show that -SH1 is not required for the relaxing factor to be active. The role of the -SH groups of myosin in the superoptimal inhibition caused by high ATP is not clear. The data of Fig. 2 show that although reaction of -SH1 and -SH2 with NEM can modify the details of the superoptimal inhibition, the superoptima1 inhibition is not greatly affected by the NEM treatment. Tonomura et al. (29), however, found that treatment of myosin A with PCMB followed by removal of the PCMB with cysteine abolished the superoptimal inhibition ordinarily observed in the presence of actin and high ATP. A loss of superoptimal inhibition was also observed when small concentrations of PCMB reacted with actomyosin (30). Since the major difference in assay conditions was that Tonomura et al. (29, 30) had more Mg++ in their system than ATP, it may be that superoptimal inhibition depends on the complex form of the substrate molecule as well as on the state of the -SH groups of the myosin. Although reaction of actomyosin with NIX does not prevent superoptimal inhibition under our conditions, it is evident from the data in Figs. 1 and 2 that the solubility of AM in high ATP is decreased by react’ion of the -SH groups with NEM. Because of the high NEM concentrations used, one cannot decide whether the group involved is -SH1 , since it is probable that other -SH groups on either the myosin or the actin (or both) also react with NEM. ACKNOWLEDGMENT The author wishes to thank Mr. P. J. Buchanan for his highly skilled technical assistance. REFERENCES 1. GODEAUX, J., Bull. sot. roy. sci. LiBge 00, 21627 (1944). 2. WEBER, H. H., AND PORTZEHL, H., Advances in Protein Chem. 7, 161 (1952). 3. BAILEY, K., AND PERRY, S. V., Biochim. et Biophys. Acta 1, 506 (1947). 4. KUSCHINSKY, G., AND TURBA, F., Biochim. et Biophys. Acta 6,426 (1951).
5. WEBER, H. H., AND PORTZEHL, H., Progr. in Biophys. and Biophys. Chem. 4,60 (1954). 6. TURBA, F., AND KUSCHINSKY, G., Biochim. et Biophys. Acta 8, 76 (1952). 7. MUCIKURA, H., Sapporo Med. J. (Japan) 5, 175 (1954). 8. MUGIKURA, H., Sapporo Med. J. (Japan) 6, 97 (1955). 9. GILMOUR,D., AND GELLERT,M., Arch. Bioche,q. Biophys. 93,605 (1961). 10. KIELLEY, W. W., AND BRADLEY, L. B., J. Biol. Chem. 218,653 (1956). K., Biochim. et Bic11. B~RIINY, M., ANDB~~Y, phys. Acta 35,293 (1959). Biophys. 87, 104 12. BLUM, J. J., Arch. Biochem. (1960). Biophys. 97, 309 13. BLUM, J. J., Arch. Biochem. (1962). 14. BLUM, J. J., J. Gen. Physiol. 45, 5938 (1962). 15. WHITMORE,F. C., ANDWOODWARD,G. E. in “Organic Syntheses Collected,” Vol. I, p. 159. Wiley and Sons, New York, 1941. 16. BOYER, P. D., J. Am. Chem. Sot. 76, 4331 (1954). 17. ALEXANDER, N. M., Anal. Chem. 30, 1292 (1958). W. F. H. M., Methods in Med. Re‘.8. MOMMAERTS, search 7, 1 (1958). !9. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L., AND RAXDALL, R. J., J. Biol. Chem. 193, 265 (1951). NAGAI, T., MAKINOSE, M., ANDHASSELBACH,W., Biochim. et Biophys. Acta 43,223 (1960). 2i, BLUM, J. J., ANDCHAMBERS,R. W., Biochim. et Biophys. Acta 18,601 (1955). 2:, I 4 MUELLER, H., Biochim. et Biophys. Acta 39, 93 (1960). 23. HASSELBACH,W., AND WEBER, H. H., Biochim. et Biophys. Acta 11, 160 (1953). 24 PIZHHY~‘S.V., AND GREY, T. C.. Biochem. J. 64, I’
OF BIOCHEMISTRY
AND
Observations
BIOPHYSICS
(1962)
97,321-328
on the Role of Sulfhydryl Functioning
Groups
in the
of Actomyosin
.J. J. BLUM The Gerontology Branch, National Heart Institute, National Institute of Health, Public an,d Welfare, Bethesda, Health Service, United States Department of Health, Education, Maryland, and the Baltimore City Hospitals, Baltimore, MarylarLd
Received October 17, 1961 The reaction of N-ethylamaleimide (NEM) with actomyosin causes the same pattern of activation and inhibition of ATPase activity at low KC1 in the presence of Mg” as at high KC1 in the presence of Ca”. The reaction of enough NEM to cause maximal ATPase activation does not interfere with superprecipitation or with the ability of relaxing factor grana to prevent superprecipitation, but does decrease the tendency of actomyosin to dissolve at high ATP concentrations. Further reaction with NEM reverses the activation of ATPase activity and prevents superprecipitation. Superprecipitation can be completely inhibited before there is any net inhibition of ATPase activity. It is suggested that two -SH groups are involved in the functioning of myosin, and their roles in ATPase activity and in mechanochemical coupling arc discussed. INTRODUCTIOX
The interaction between myosin and actin certainly involves the -SH groups of myosin (l-3) but probably does not involve the -SH groups of actin (3, 4). The exact role of the --SH groups on myosin in t.his interaction, is, however, not yet cIear. Bailey and Perry (3) reported that several reagents which reacted with the --SH groups of myosin destroyed both the adenosinetriphosphatase (ATPase) activity and the affinity of the myosin for actin. Weber and his colleagues (2, 5) have examined the effects of a variety of -SH reagents on the ATPase activity and contractility of actomyosin (AM) systems, and have accumulated evidence that when groups of myosin are blocked the -SH there is a concomitant loss of ATPase activity and of contractility. This view was challenged by Turba and Kuschinsky (6), who reported that oxarsan treatment of myosin A could inhibit superprecipitation without causing any appreciable change in ATPase activity. Their results were, how-
ever, challenged by Mugikura (7, 8) who reported that oxarsan caused a parallel loss of ATPase and superprecipitability. More recently, there has been a growing realiaation that the -SH groups of myosin are heterogeneous both in respect to ATPase activity (9, 10) and with respect to the interaction between myosin and actin. Thus, BBr6ny and B&rBny (11) demonstrated that although certain -SH reagents inhibited both the ATPase activity and the actin-combining ability of myosin A, these reagents would inhibit the ATPase activity of myosin B but would not dissociate it into actin and myosin A. B&dny and BBrbny (11) also found that high concentrations of iodoacetamide could completely inhibit the ATPase activity of myosin A without interfering with its actin combining ability, thus suggesting t’hat the -SH groups of myosin are involved to different extents in the ATPase and in the contractile funct,ions of myosin. We have recently studied patterns of ATPase activation and inhibition caused by reaction of several -SH reagents with 321
322
BLUM
myosin at high KC1 concentrations in the presence of Ca+ + (12,13). It was suggested that two -SH groups are involved in the hydrolytic functioning of the active site, and some of the properties of the individual -SH groups were partially analyzed. We have now found that activation of ATPase by low concentrations of N-ethylmaleimide p-chloromercuribenzoate and (NEW (PCMB) can be observed at 0.1 M KC1 in the presence of Mg++. This observation (which has also been made by W. W. Kielley and T. Sekine, personal communication) has permitted us to compare the effects of these -SH reagents on both the contractile and the enzymic properties of myosin, and thus further analyze the role of the -SH groups of myosin in these processes. A preliminary account of this work has already appeared (14). EXPERIMENTAL NEM and the sodium salts of adenosine triphosphate (ATP) and PCMB were obtained from the Sigma Chemical Company. PCMB was purified by the methods of Whitmore and Woodward (15), and its concentration was determined from absorbance measurements at 232 rnp, using the extinction coefficient of Boyer (16). NEM concentration was determined from absorbancy measurements at 300 mp, using the extinction coefficient of Alexander (17). Actomyosin was prepared from 24-hr. extract of rabbit muscle, using the KCl-PO,EDTA extraction fluid described by Mommaerts (18). In addition to three reprecipitation cycles for. purification, the myosin B was partially freed of low molecular weight aggregates of myosin A by discarding the supernatant of a 45-min. centrifugation step at 18,000 x g in 0.28 M KCl. Protein concentration was determined by the modified Folin ‘method of Lowry et al. (19). The actomyosin was reacted with NEM in two ways. In the first method, identical amounts of NEM were added to aliquots of actomyosin in 0.6 M KCl, 0.02 M Tris, pH. 7.50 and were permitted to react for known times at 25.5”C. The reaction was terminated by adding an excess of cysteine. A control was prepared by mixing the NEM and cysteine first, and then adding the mixture to an aliquot of actomyosin. The tubes were kept at 25.5”C. until the longest reaction was complete and were then put in an ice bath until the contents of each tube was transferred to a dialysis sac. The dialysis tubing was pretreated with disodium ethylenediamine tetraacetate (EDTA) and cysteine
and washed with glass-distilled water before use. The dialysis sacs were individually dialyzed for at least 24 hr. at 4°C. against a solution of 0.1 iM KCI, pH 7.0, containing either 0.05 M Tris or 0.01 M histidine and 0.005 M potassium oxalate, as specified below. In the second method, increasing amounts of NEM were allowed to react with the actomyosin at 0°C. for 24 hr. in 0.6 M KCI, 0.02 M Tris, pH 7.5. Each sample was then dialyzed against one of the 0.1 M KCl, pH 7.0 buffers described above. Essentially the same procedure was used with PCMB, except that the reaction at 0” was allowed to proceed for 70 hr. before dialysis was begun. After dialysis was completed, the actomyosin samples were kept at 0°C. until assayed for ATPase activity and superprecipitability. Grana containing the relaxing factor system were prepared by the procedure of Nagai et al. (20) and were used within 24 hr. of preparation. Superprecipitation was measured at room temperature (about 25°C.). Except when the effects of grana were to be tested, the dialyzed actomyosin was added to a small beaker containing the appropriate amount of ATP and Mg++ and the mixture was transferred to a Wintrobe hematocrit tube. When the effects of grana were to be tested, the grana were incubated with the ATP for about 1 min. prior to the addition of the actomyosin, and the mixture was then transferred into a Wintrobe tube. The hematocrit tubes were spun for exactly 2 min. in a clinical centrifuge at 1780 X g. The precipitate height was measured and normalized so that it refers to a column of liquid 10 cm. long. ATPase activity was measured by the FiskeSubbaRow technique as described earlier (12). In experiments where high ATP concentration was used, the samples to be assayed were diluted in a sufficient volume of trichloroacetic acid (final concentration, 3.3%) to insure that the unhydrolyzed ATP would not interfere with the development of the blue color (21). The ATP and actomyosin were incubated at the temperature of the assay (25.5”C. unless otherwise specified) for about 60-70 sec., at which time the first of two aliquots was taken. The second aliquot was taken between 100 and 500 sec. later, depending on the expected ATPase activity. When the effect of relaxing factor grana was to be measured, the grana were first mixed with ATP, and aliquots of this mixture were put into the 25.5” bath and immediately mixed with the actomyosin. Samples for orthophosphate analysis were drawn after the 60sec. incubation period. In addition, samples of grana were mixed with buffer instead of actomyosin, and the ATPase activity due to grana alone was measured at several time points up to the end of the 5-min. incubation period. The ATP-
ROLE
OF -SH
GROUPS
ase activity of the actomyosin could then be obtained by subtracting the activity due to the grana. The units of ATPase activity in all the figures and tables throughout this paper are pmoles P/sec./g. RESULTS
117henthe ATPase activity of actomyosin that has reacted with NEM for various times is assayed at low salt in the presence of Mg++, it is found (Fig. 1) that the pattern of activation and inhibition resembles the pattern found for NEM and PCMB (10) at high KC1 in the presence of Ca+ + or Mg+ +. In the experiment shown in Fig. 1, the reaction was terminated before any net inhibition of ATPase activity occurred. With longer times of exposure (or higher concentrations of NEM), complete inhibition is obtained, although this is not shown in Fig. 1. It can be seen that the re-
TIME
IN
ACTOMYOSIN
323
action of NEM with the group responsible for activating ATPase does not inhibit superprecipitation, while the reaction of NEM with the group responsible for the loss of this activation causes a loss of superprecipitability. It should be noted that SUperprecipitation is completely inhibited befor there is any net inhibition of ATPase (Fig. 1). In the presence of relaxing factor grana, there is an inhibition of the ATPase activity of untreated actomyosin and a tendency for the actin and myosin to dissociat,e and thus form a solution rather than a suspension (22). In our experience the amount of ATPase inhibition caused by the grana has been variable but is not very sensitive to pretreatment of the actomyosin with NEM. The effect of relaxing factor grana on actomyosin (AM) not treated with NEM or
Imin.1
FIG. 1. Effect of NEM treatment on ATPase, superprecipitation, and response to relaxing factor of actomyosin. Actomyosin (6.9 mg./ml.) was reacted with NEM (5.5 moles/lo5 g.) in 0.6 M KCI, 0.02 M Tris, pH 7.5, at 25.5”C. for the times shown on the abscissa and then dialyzed against 0.1 M KCl, 5 X lo-’ M potassium oxalate, 0.01 M histidine, pH 7.0. BTPase activity (0, 0, left-hand ordinate) and superprecipitation (Cl, W, right-hand ordinate) were measured in the histidine buffer with 4 X lOA 1M ATP, 2 X 10.’ M Mg”, 2.1 mg. AM/ml. Solid symbols, grana present (0.7 mg. grana/mg. AM); open symbols, no grana. The two plus (+) symbols in the upper left indicate that in the presence of grana, untreated AM and AM treated for 1 min. with NEM, dissolved upon the addition of ATP. In this figure and in Figs. 3 and 4 the height of the control AM column after centrifugation in the presence of Mg”, but not ATP, is shown by an arrow which points to a vertical bar that extends one standard deviation above and below the average value.
324
BLUM
treated with NEM for only 1 min. was to dissolve it, as indicated in Fig. 1. With increasing time of exposure to NEM, the AM became less soluble in the ATP-grana mixture, and even before maximum ATPase activation was achieved it was clear that NEM treatment did not prevent the relaxing action of the grana-ATP system. Thus, although brief exposure to NEM affects the solubility of AM in ATP-grana, no concentration of NEM tested appeared to prevent the relaxing action of grana, provided, of course, that superprecipitation was still observable in the absence of grana. The experiment shown in Fig. 1 demonstrated an effect of NEM treatment on the solubility properties of AM in high concentration of ATP-grana. Since the ATP-grana system caused relaxation in the NEMtreated AM, it was of interest to inquire
whether NEM changed the solubility properties of AM with respect to high concentrations of ATP alone. When myosin is reacted with high concentrations of NEM (Fig. 2)) it is found that even in the absence of ATP the height of the centrifuged AM column decreases with increasing exposure to the NEM. In the presence of low ATP (1O-3 M, 3 x 10V3 M), superprecipitation is complete until the ATPase activity begins decreasing from its maximum (cf. Fig. 1). Superprecipitation is inhibited before there is any net loss of ATPase activity at these two concentrations of ATP. At higher ATP (10m2 M) , the AM that has not been treated with NEM or has been treated for only 1 min. dissolves (Fig. 2)) but AM that is treated with NEM for more than 1 min. no longer dissolves. It is clear that some -SH groups of AM are
l
I 5
. 10
15 TIME
. 20
25
30
(min. ]
FIG. 2. Effect of increasing ATP concentration on ATPase activity and superprecipitation of NEM-treated AM. Actomyosin (3.1 mg./ml.) was reacted with NEM (10 moles/lO’ g.) for the times shown on the abscissa in 0.6 M KCl, 0.02 M Tris, pH. 7.5 at 25.5”C., and then dialyzed against 0.1 M KCl, 0.05 M Tris, pH. 7.5 for 36 hr. ATPase (solid lines, solid symbols, left-hand ordinate) and superprecipitation (dashed lines, open symbols, right-hand ordinate) were measured in 0.11 M KCl, 2 X lo-* M Mg++, 0.05 M Tris, pH 7.5, with final concentrations of ATP of 1 (0, O), 3 (W, Cl), and 10 mM (A, A). The curve with the inverted triangles (V) shows the height of t.he AM column after centrifuging in the absence of ATP. The plus (+) symbols in the upper left of the graph indicate that at 0 and 1 min. of NEM treatment, 10 mM ATP dissolved the AM. The ATP used in this experiment was converted to the potassium form by equilibration with an excess of well-washed Dowex 50 in the K’ form.
ROLE
OF -SH
GROUPS
IN
325
ACTOMYOSIN
sentially independent of the -SH groups of actomyosin, alt,hough these groups can modulate the sensitivity of the actomyosin to the high ATP. When NEM activates the ATPase activity of actomyosin, it also increases the sensitivity of the AM to superoptimal inhibition. The increased sensitivity to superoptimal inhibition is removed as increasing NEM reverses the activation of ATPase activity. The data shown in Figs. 1 and 2 were obtained from timed exposures of AM to excess NEM. The question arises whether similar results may be obtained by long exposures of actomyosin to small amounts of NEM. When this was done, it was found that a similar patt’ern was obtained (Fig. 3). There was no inhibition of superprecipitation until enough NEM was added to begin decreasing the ilTPase activity from its maximum. In this experiment the reaction patt,ern of the -SH groups with NEM is such that there was about a 30% loss of ATPase activity when the superprecipitation was completely inhibited.
involved in determining the solubility of AM in high concentrations of ATP. Qualitatively similar result’s have been obtained when the ionic strength was held constant as the ATP concentration increased. The data of Fig. 2 also bear on the role of -SH groups in the superoptimal inhibition of the ATPase activitv of AM (23, 24). If we set the ATPase activity measured in 1O-3 M as loo%, then increasing the ATP to 3 x lo-” M caused a 20% inhibition of the activity of untreated AM. This increased to a 45% inhibition with increasing time of treatment, up to about 4 min. and then decreased to about 20% inhibition again with increasing exposure to NEM. At higher ATP (lo-’ J1) the activity of untreated AM is inhibited by 88% as compared to the activity in low3 M ATP. Brief NEM treatment increases the superoptimal inhibition to 94%, while further NEM treatmerit again removes this extra sensitivity and returns the inhibition to about 82%. The ability of high ATP to cause superoptimal inhibit,ion of ATPase is thus es-
-8
.6
-4
-2
,’ / ._,_*-*-O---------o-----9------,3
2
MOLES NEM/IO'
I(
L
I
-0
gms. MYOSIN
FIG. 3. Effect of low concentrations of NEM on ATPase activity and superprecipitation of BM. Actomyosin (4.23 mg./ml.) was reacted with NEM in the amounts shown on the abscissa for 24 hr. at 0°C. in 0.6 M KCI, 0.02 M Tris, pH 7.5 and then dialyzed against 0.1 M KCl, 0.005 M potassium oxalate, 0.01 M histidine at pH 7.0 for 23 hr. STPase activity (O-O, left-h an d or d’ma t e ) and superprecipitation (O-0, right-hand ordinate) were measured in the histidine buffer with 4 X 10e3 M ATP and 2 X 10.’ M Mg”. Also see legend for Fig. 1.
BLUM
326
Although the reaction of NEM with -SH groups forms strong covalent bonds, this is not true for the reaction of PCMB with -SH groups. Nevertheless, the bonds formed with PCMB are sufficiently stable so that dialysis against buffered salt solutions will not remove all the bound PCMB. When actomyosin was reacted with very low concentrations of PCMB, it was found (Fig. 4) that the relations between ATPase activity and superprecipitation were similar to the relations found using NEM. In the presence of Mg++ at low KCl, the ATPase was activated by low concentrations of PCMB and superprecipitation was not inhibited. Higher concentrations of PCMB decreased the ATPase activity towards the control value and prevented superprecipitation (Fig. 4). In this experiment the maximum activation occurred after treatment with about 0.06 mole PCMB/105 g., or 0.38 mole/mole myosin A (25). Even if the actin content of the AM were as much as SO%, this would mean that less than 1 mole PCMB had reacted with the myosin to
cause maximal activation. This suggests the possibility that PCMB catalyzed the oxidation of some -SH groups and prevents one from using the data in Fig. 4 for computing the stoichiometry of the reaction between PCMB and actomyosin. The data so far presented show that the -SH group involved in activating the ATPase activity does not prevent superprecipitation when it reacts with NEM or PCMB. It has been suggested (12) that cysteine ethyl ester (CEE) or X-aminoethylisothiouronium bromide (AET) reacts only with this -SH group on myosin A. It was therefore of interest to establish whether reaction of AM with AET would inhibit superprecipitation. The data presented in Table I show that superprecipitation was not inhibited, thus further indicating that the -SH group involved in the activation of ATPase is not involved in superprecipitation. DISCUSSION
From
the results
of a study
of the inter-
-6
6’
>_,l_AYoOO
d . 2
4
(MOLES
. 6
. 8
PCMB/lO’ gnu. MYOSIN)
10
0
x10’
FIG. 4. Effect of PCMB on superprecipitation and ATPase activity of AM. Actomyosin (4.39 mg./ml.) was reacted with PCMB in the amounts shown on the abscissa for 70 hr. at 0°C. in 0.6 M KCI, 0.02 M Tris, pH 7.5 buffer. It was then dialyzed for 22 hr. against 0.1 M KCl, 5 X lo-’ M potassium oxalate, 0.01 M histidine buffer at pH 7.0, and assayed for ATPase activity (O-O, left-hand ordinate), and superprecipitation (O-Q, right-hand ordinate) using 2 X 1Om8 M Mg++ and 4 X lo-* M ATP. Also see legend to Fig. 1.
ROLE
OF --SH
GROUPS
action between myosin A and PCMB (in high KC1 with Ca+ +) , it was suggested that two -SH groups were involved in the functioning of the active hydrolytic site (13). The reaction of -SH1 with PCMB activated the ATPase activity, while the reaction of -SH, with PCMB reversed this activation and then inhibited hydrolysis. The present data (Figs. 1,3, and 4) show that at low KC1 in the presence of Mg++ the same pattern of activation, reversal of activation, and inhibition are obtained when eit.her NEM or PCMB interacts with actomyosin. The reaction pattern of these -SH groups will depend on the concentrations of AM and of -SH reagent, on the temperature, and on the ionic conditions. Under conditions such as were used in these experiments the rcact’ions seem to be fairly well separated. The data demonstrate clearly that the reaction of -SH1 with PCMB or NEM does not prevent superprecipitation. When more -SH reagent reacts it is possible to completely inhibit superprecipitat,ion before there is any net loss of ATPase activity. This could be interpreted in two ways: (a) One could assume that there are three -SH groups involved in t,he functioning of actomyosin. Reaction of -SH1 with e.g., NEM, enhances the ATPase activity. Reaction of -SH, reverses t,his activation and prevents TABLE
I
EFFECT OF AET ON ATPASE ACTIVITY SUPERPRECIPITATION OF ACTOMYOSIN
AND
Activity Supegxi&tate Mg++ pm&s
Control AET
1.77 1.37
Mg” + Ca++
IN
ACTOMTOSIN
327
superprecipitation. At higher NEM concentrations -SHB reacts and causes loss of hydrolytic activity. (b) If, as seems more likely, there are only two -SH groups per active site, then reaction of -SHI with NEM would activate ATPase activity and reaction of -SH, would reverse this activation and prevent superprecipit’ation.l In the absence of actin or ATP a secondary small conformation change involving -SH2 would occur, leading to t’he loss of hydrolytic activity. In t.he presence of act,in or ATP, however, this secondary change would be impeded and one would observe inhibition of superprecipitation without any net loss of ATPase activity. To the extent that superprecipitation is a satisfactory model of the contractile process, these results indicate that the transduction of chemical free energy of ATP into mechanical energy does not depend crucially on either the binding of ATP t’o the site (26) or on the over-all rate of splitting of the ATP (27). What does seem to be required is that the hydrolysis occurs via a pat,hway involving a particular sulfhydryl group which we have tentatively designated as -SH2. If this group reacts with NEM or PCMB, hydrolysis may still proceed but it will no longer be coupled to the contractile machinery even in the presence of Mg++. Other types of groups, of course, may also be required for mechanochemical coupling (11,28). Physiologically, the relaxing factor system found in grana appears to control the mechanochemical coupling process (20)) but it is not known via what groups of the act’in-myosin system t.his control is ex-
P/sec./g.
0.31 0.51
;lt3 0.3
Actomyosin (6.65 mg./ml.) was reacted with 5 X 10-S M AET for 8 hr. at 0°C in 0.6 M KCl, 0.02 M Tris, pH 7.5, and then dialyzed for 18 hr. against 0.1 M KCl, 0.05 M Tris, pH 7.5 buffer. Superprecipitation was measured using 10-a M ATP, 2 X 10e3 M Mg++, and 3.32 mg./ml. AM. The precipitate height in the absence of ATP was about 8 cm. for the control and for the AET treated protein. ATPase was also measured in the low KC1 buffer, with 10-s M ATP and either 2 x lo-3 M Mg++ or 2 X 10-Z M blg++ plus 5 X 10-s M Ca++.
‘When -SH, of the myosin is reacting with YEM, it is also possible that -SH groups on the actin are reacting with NEM and that these, rather than --SH? , cause t,he loss of superprecipitability. Aside from evidence suggesting that the --SH groups of ac*tin are not involved in the interaction betwr,en myosin and actin (3> 4), the present experiments have been conducted over a wide range of NEM to AM ratios and for several different, AM preparat.ions. In all cases the loss of superprecipitabilitv coincided with the decrease of ATPase actktp from its peak, a result which would not be expected if the reaction of the -SH groups of actin with NEM caused the loss of superprecipitability.
328
BLUM
erted. The data presented in Fig. 1, however, show that -SH1 is not required for the relaxing factor to be active. The role of the -SH groups of myosin in the superoptimal inhibition caused by high ATP is not clear. The data of Fig. 2 show that although reaction of -SH1 and -SH2 with NEM can modify the details of the superoptimal inhibition, the superoptima1 inhibition is not greatly affected by the NEM treatment. Tonomura et al. (29), however, found that treatment of myosin A with PCMB followed by removal of the PCMB with cysteine abolished the superoptimal inhibition ordinarily observed in the presence of actin and high ATP. A loss of superoptimal inhibition was also observed when small concentrations of PCMB reacted with actomyosin (30). Since the major difference in assay conditions was that Tonomura et al. (29, 30) had more Mg++ in their system than ATP, it may be that superoptimal inhibition depends on the complex form of the substrate molecule as well as on the state of the -SH groups of the myosin. Although reaction of actomyosin with NIX does not prevent superoptimal inhibition under our conditions, it is evident from the data in Figs. 1 and 2 that the solubility of AM in high ATP is decreased by react’ion of the -SH groups with NEM. Because of the high NEM concentrations used, one cannot decide whether the group involved is -SH1 , since it is probable that other -SH groups on either the myosin or the actin (or both) also react with NEM. ACKNOWLEDGMENT The author wishes to thank Mr. P. J. Buchanan for his highly skilled technical assistance. REFERENCES 1. GODEAUX, J., Bull. sot. roy. sci. LiBge 00, 21627 (1944). 2. WEBER, H. H., AND PORTZEHL, H., Advances in Protein Chem. 7, 161 (1952). 3. BAILEY, K., AND PERRY, S. V., Biochim. et Biophys. Acta 1, 506 (1947). 4. KUSCHINSKY, G., AND TURBA, F., Biochim. et Biophys. Acta 6,426 (1951).
5. WEBER, H. H., AND PORTZEHL, H., Progr. in Biophys. and Biophys. Chem. 4,60 (1954). 6. TURBA, F., AND KUSCHINSKY, G., Biochim. et Biophys. Acta 8, 76 (1952). 7. MUCIKURA, H., Sapporo Med. J. (Japan) 5, 175 (1954). 8. MUGIKURA, H., Sapporo Med. J. (Japan) 6, 97 (1955). 9. GILMOUR,D., AND GELLERT,M., Arch. Bioche,q. Biophys. 93,605 (1961). 10. KIELLEY, W. W., AND BRADLEY, L. B., J. Biol. Chem. 218,653 (1956). K., Biochim. et Bic11. B~RIINY, M., ANDB~~Y, phys. Acta 35,293 (1959). Biophys. 87, 104 12. BLUM, J. J., Arch. Biochem. (1960). Biophys. 97, 309 13. BLUM, J. J., Arch. Biochem. (1962). 14. BLUM, J. J., J. Gen. Physiol. 45, 5938 (1962). 15. WHITMORE,F. C., ANDWOODWARD,G. E. in “Organic Syntheses Collected,” Vol. I, p. 159. Wiley and Sons, New York, 1941. 16. BOYER, P. D., J. Am. Chem. Sot. 76, 4331 (1954). 17. ALEXANDER, N. M., Anal. Chem. 30, 1292 (1958). W. F. H. M., Methods in Med. Re‘.8. MOMMAERTS, search 7, 1 (1958). !9. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L., AND RAXDALL, R. J., J. Biol. Chem. 193, 265 (1951). NAGAI, T., MAKINOSE, M., ANDHASSELBACH,W., Biochim. et Biophys. Acta 43,223 (1960). 2i, BLUM, J. J., ANDCHAMBERS,R. W., Biochim. et Biophys. Acta 18,601 (1955). 2:, I 4 MUELLER, H., Biochim. et Biophys. Acta 39, 93 (1960). 23. HASSELBACH,W., AND WEBER, H. H., Biochim. et Biophys. Acta 11, 160 (1953). 24 PIZHHY~‘S.V., AND GREY, T. C.. Biochem. J. 64, I’