Role of adenosine triphosphate in the relaxation of skeletal muscle myofibrils

ARCHIVES

Role

OF

BIOCHEMISTRY

AND

of Adenosine

BIOPHYSICS

568-576

110,

Triphosphate Muscle

in the Myofi

E. EISENBERG2 Department

of Biophysics,

State

(1965)

University

AND

Relaxation

of Skeletal

brils’ c.

Aloos

of New

York,

Bu$alo,

New

York

Received January 7, 1965 Recent studies of relaxation have shown that, in a calcium-depleted system, the ATPase of actomyosin is inhibited and syneresis is delayed. The hydrolysis of ATP during such a delay must somehow cause the onset of syneresis, and it probably does so by lowering the ATP concentration, although the liberated ADP and inorganic phosphate have also been reported to play a role. We have re-examined the roles of ATP and ADP by directly measuring the ATP concentration remaining at the moment when myofibrils flocculate. Our results indicate that the delay in flocculation represents primarily the time required for the concentration of ATP to fall below a critical “threshold” level which is required for relaxation, although added ADP appears to increase this threshold level. We have also found that, when “relaxation” is promoted by an increase in the concentration of EGTA, relaxing-factor granules, KCI, or MgC12, the threshold ATP level is correspondingly reduced. We offer an interpretation of these results based on the postulate that the actin-myosin interaction is inhibited by the binding of ATP to a “relaxing” site on the myosin, distinct from the ATPase site.

It has long been recognized that ATP has two effects on actomyosin systems in vit7-0. Not only does its hydrolysis serve as the primary source of energy for contraction, but in addition, its presence appears to be necessary for relaxation, This latter function is manifested as the “plasticizing effect” of ATP on glycerinated muscle fibers and its inhibitory effect on the ATPase and syneresis of actomyosin (14). In more recent studies of relaxation, major attention has been given to the central role of calcium ion rather than to the relaxing effect of ATP. These studies have shown that in the presenceof the natural relaxing factor, or of a synthetic calcium-chelator like 1 This investigation was supported by P.H.S. research grants GM-07255 and GM-10249 from the National Institute of General Medical Sciences, U.S. Public Health Service. 2 For this work, Evan Eisenberg was awarded a Dr. Richard S. Brookings and Dr. Robert Carter Medical School Prize by Washington University School of Medicine, St. Louis, Missouri, in 1964.

EGTA,3 the ATPase activity of actomyosin systems is inhibited and syneresis is delayed (5-7). The hydrolysis of ATP during such a delay must in some manner cause the onset of syneresis, and it probably does so by lowering the ATP concentration until the actomyosin no longer responds to the relaxing agent (8). It has also been reported, however, that the liberated ADP and Pi play a role in inducing syneresis (4). In the present study, we have re-examined the roles of the changing ATP and ADP concentrations by directly measuring the ATP concentration remaining at the moment when myofibrils flocculate. Our results indicate that the delay in flocculation in a calcium-depleted system represents primarily the time required for the concentration of ATP to fall below a critical “threshold” level which is required to maintain the relaxed state (3, 9, lo), although the 3 Abbreviations used : EGTA, bis(aminoethylether)tetraacetic ganic orthophosphate.

568

ethyleneglycolacid; P;, inor-

ATP

AND

RELAXATION

ADP concenkation appears to modify the value of this threshold level. Furthermore, we have examined the dependence of the threshold ATP level on the concentrations of EGTA, relaxing factor granules, KCl, and MgC12, and have found that when ‘kelaxation” is promoted by an increase in the concent’ration of any of these agents, the threshold ATP level is always rorrespondingly reduced. MATERIALS

AND

METHODS

iUyo$brils. The method for the preparation of myofibrils was essentially that of Perry (11). Rabbit muscle was homogenized in a solution containing 0.1 M KCl, 0.01 M imidazole, 4 mM EDTA, and 4 mM MgCl,, adjusted to pH 7.0 with NaOH. After centrifugation, the sediment was homogenized once more in this solution and centrifuged, and the myofibrils were then washed three times in 0.1 ,V KC1 containing 10 mM imidazole-HCl buffer at pH 7.0 (“KC1-buffer” solut,ion), collecting only the upper myofibril layer of the sediment each time. The final sediment was diluted slightly with “KC1-buffer” solut,ion and then mixed with an equal volume of cold glycerol and stored at -20”. For use in experiments, the myofibrils were washed 5 times in “KC1-buffer” solut,ion and finally resuspended at a concentration of 10-30 mg per milliliter and stored at 0” for not more than a week. The protein concentration of this stock suspension was determined by microKjeldahl nit,rogen assay, or by the Lowry method

(12). Relaxing factor granules. For the preparation of granules, the rabbit muscle was homogenized in 3 volumes of 0.15!11 KCl-1 mM NaHC03 (5), and the suspension was centrifuged for 1 hour at 14,000g. The supernatant liquid was filtered through glass wool and then recentrifuged as before. From this second supernatant, the granules were sedimented by centrifugat,ion for 90 minutes at lOO,OOOg, suspended in “KC1-buffer” solution, sedimented again in the same way, and finally resuspended in the “KCl-buffer.” Coarse aggregates were removed by brief centrifugation at 30009, and the suspension was stored for no more than a week at 0”. The protein concentrat,ion was determined by the same methods used for the myofibrils. Flocculation time. The determination of flocculation time (5) was carried out in a total volume of 2.5 ml. All components of the mixture except the myofibrils were combined in a test tube and brought to 25” in a water bath, and then, at a given time, the myofibrils were rapidly mixed in. The tubes were observed thereafter, with gentle agita-

OF

MYOFIBRILS

569

tion, until flocculation of the suspension occurred, this end point being abrupt) and easily observed with suitable illumination (Fig. 1). In samples observed simultaneously, differences of less than 1 minute in t,he time of flocculation could be clearly det,ermined. ildenosine triphosphate determination. At. the time of flocculation, 2.0 ml of cold 1O7o HCIOa were rapidly added, and the suspension was chilled in ice and then centrifuged briefly. From t,he supernatant, exactly 0.2 ml was withdrawn and mixed with 5.0 ml of 0.05 il4 KHCOI. The KCIOa precipit,ate was allowed to settle, and the solution, after suitable dilution, was analysed for ATP by the firefly method of St,rehler and Totter (13). The light emitted upon the addition of a crude firefly-tail extract was recorded by means of a photomultiplier, and the resulting record of light intensit,y was extrapolated back to the moment of addition of t,he firefly extract to obtain a value which was proportional to the ATP concentration and essentially unaffected by ADP or other sample components. Standard mixtures were prepared which were identical to the reaction mixture except for the omission of the myofibrils and the addition of various known concentrations of ATP in the range expected for the samples. These were treated exactly like the reaction mixture samples, and the light intensities for these standards were used for the calculation of the ATP concentrations in the reaction mixtures. This procedure was found to be quite reproducible, giving accuracies of roughly 107; or better. Adenosine triphosphatase measurements. The ATPase activity of the myofibrils was measured in a mixture identical to that used for the flocculation time determination. At selected time intervals after the addition of myofibrils, small aliquots were removed from the suspension and mixed with lo-20 volumes of cold 5% trichloroacetic acid. The protein precipitates were centrifuged down, and samples of the supernatants were analyzed for Pi by a modification of the isobutanol extract’ion procedure of Berenblum and Chain (14). Throughout the delay preceeding flocculation, the time course of Pi production was nearly linear, and its slope was used as the ATPase rate. Chemicals. The ADP and ATP were purchased from commercial suppliers, and stock solutions were adjusted to pH 7.0 with NaOH and stored frozen. The concentration of these stocks was determined spectrophotometrically (15). The desiccated firefly tails were also purchased commercially, and the EGTA was kindly supplied by Geigy Industrial Chemicals, Ardsley, New York, to whom we express our gratitude. All other chemicals were reagent grade commercial products.

RESULTS

Threshold adenosine triphosphate level. When the syneresis of myofibrils is delayed by a relaxing agent such as EGTA, the onset of the macroscopically observable flocculation is quite sharp (Fig. 1). If flocculation is induced by the fall in the ATP concentration which occurs during this delay, then it follows that the physical state of the myofibrils must change abruptly over a narrow range of ATP concentration, which we refer to as the “threshold ATP level.” The experiment in Table I was designed to test the validity of this approach. The ATP level remaining at the t’ime of syneresis was measured under conditions where flocculat)ion was delayed about 15 minutes. Another sample was then prepared containing just this concentration of ATP together wit’h the amounts of ADP and Pi calculated to have been present at syneresis, and in this case flocculat,ion occurred almost instantly. However, when the

4

5

6

ATP, ADP, and Pi concent,rations were changed slightly to resemble the conditions just before syneresis, a short but definite delay in flocculation was observed, which confirms that the transition from relaxation to syneresis is abrupt and occurs only at the threshold ATP level. Role of adenosine diphosphate and Pi. The last line of Table I shows that at the previously measured threshold concentration of ATP, a reduction in the ADP and Pi levels (with the MgClz maintained equivalent to the total nucleotide) led to a brief but definite delay in flocculation, suggesting that the liberated ADP and Pi contribute to the initiation of syneresis. This effect may be regarded as an influence of ADP and Pi on the threshold ATP level, and indeed when the effect of added ADP is examined, as shown in Fig. 2, it is evident that the presence of extra ADP, alt’hough it does not affect the ATPase rate, does increase t’he

7

a

9

min.

FIG. 1. Flocculation of myofibrils. All samples contained 2 rnM ATP, 2 m&I MgClz, 1 mM EGTA, 20 mM KCl, 12 mM imidazole buffer, pH 7, and 2.5 mg/ml myofibrils. The number under each tube indicates the time in minutes which had elapsed after addition of myofibrils. The lower photograph shows the same set of tubes as the upper photograph but 1 minute later. The checked tube flocculated in this int>erval.

ATP TABLE EFFECTS

OF ATP, ADP, OF MYOFIBXILLAR Initial

;\TP

cont. ADP&PiC

END

A?jD

l
OF

571

iLlYOFIBHILS

I Pi ON THE INIIUTION

30 -

FLOCCUL.ITION~~

(I&M) M&I?

Floccula-

Threshold

tio&::,lay

“%,;,’

oA ) i5 -

4.0 0.8 1.2 0.8

3 2 2.8 0.2

4 .o 4.0 4.0 1 .Ob

‘I All samples contained 1 imidazole buffer at pH 7.0, and 5.3 fibril protein? in addition to indicated concentrations of ATP, ADP, Pi (as Ka-salts, pH 7), and MgC12. Total volume = 2.5 ml. * 15 mM KC1 added to maintain ionic strength, c ADP and Pi were added at equal concentration.

r\TP concentration remaining at the moment of flocculation. Similarly, as shown in Fig. 3, a higher initial ATP concentration, which res&s in a higher concentration of ADP at the time of flocculation, also leads to a higher threshold ATP level. It should be noted, however, that even in the absence of ADP, a low ATP concentration can induce syneresis, whereas, as shown by the initial conditions in Fig. 2, at an ATP concentration well above the threshold level, a considerable rise in aDI’ does not induce immediate syneresis. Therefore, alt,hough ADP increases the threshold ATP level, the physical state of the myofibrils appears to be considerably nlore sensit’ive to changes in the ATP concent ration t’han in the ADP concentration. It) then follows that, t’he role of ATP-gencrating transphosphorylase enzymes in pronloting relaxation in z&o (5) may be primarily a consequence of their effect on the concentration of ATP rat’her t,han ADP. Likewise, the increase in flocculation delay with an increase in added ADP in Fig. 2 may be due to the conversion of some of the ADP to dTI’ by the myokinase activity in the system, so that a somewhat longer time is required for t’he ATP concentration to fall to its threshold level. It should be noted that the system used in these experiments would not be affected by calcium contanklation in the ATP and ADP preparatjionc, calcium chelat’ion by nucleotides, or changes in the calcium-binding

-I

;

o’

Initial

ADP

a-- 0 3

2

CON.,

mM

FIG. 2. Effect of ADP on t,hreshold ATP concentration, ATPase activity, and flocculation delay, in presence of EGTA. All samples contained 2 mM ATP, 1 mM EGTA, 10 mM imidazole buffer, pH 7, and 2.16 mg/ml myofibrils. Magnesium chloride cone. maintained equal to tot,al nucleotide. Potassium chloride was added to maintain ionic strength at about 0.08.

15 .z 3 6 -g 10 2 $

-x-x-x

A TRuse

4 \ -s-,-1-,-

’ _~

f

3

2

Initial

ATP

Cont.,

4

mM

FIG. 3. Effect of initial ATP concentration on threshold ATP concentration, ATPase activit.y, and flocculation delay, in presence of EGTA. All samples contained 1 mM EGTA, 10 mill’ imidazole bufl’er, pH 7, and 3.0 mg/ml myofibrils. Magnesium chloride cont. equaled initial ATP cont. Potassium chloride was added to maintain ionic strength at about o.o(~.

EISENBERG

oI +0'

A

8 EGTA

16

12 Cont.,

.?O

,&vi

FIG. 4. Effect of EGTA concentration on flocculation delay and threshold ATP. All samples contained 4 mM ATP, 4 mM MgC12, 10 mM imidazole buffer, pH 7, 80 mM KCl, and 2.2 mg/ml myofibrils .

activit,y of any relaxing factor granules which might occur in the myofibrils, because EGTA

AND

MOOS

EGTA than with the highest concentration of granules, whereas the threshold ATP concentration was not as low with EGTA as with granules. However, this apparent contradiction can probably be explained on two grounds. First, the flocculation delay with granules may have been shortened by the ATPase activity of the granules themselves (16, 17). Second, the free magnesium concentration at the time of flocculaton may have been higher with the granules than with EGTA, and, as will be shown below (Fig. S), this would lead to a lowered threshold ATP concentration. Such an increase in the free magnesium concentration could result from the conversion of ADP to IMP by the myokinase activity of the granules in combination with the ATPase and deaminase activities in the system, since IMP binds magnesium much less strongly than does ADP. In support of this explanation, we have observed by paper chromatography and spectrophotometry that, at high concentrations of granules, the principal nucleotide product formed in the system is indeed IMP. It might be noted that we have found the flocculation delay to be unaffect,ed by added AMP or IMP.

1.

was added in considerable excess over any contaminant calcium which could be present. Efects of EGTA and relaxing factor granules. The action of other agents which regulate the transition from “relaxation” to “contraction” in vitro may also be considered in terms of alterations in the threshold ATP level. Both EGTA and relaxing factor granules are now thought to inhibit the syneresis of myofibrils by lowering the free calcium concentration in the suspension (6). The effects of these agents on the threshold ATP level are shown in Figs. 4 and 5. As the concentration of each was increased, leading to an increase in the flocculation delay, the threshold ATP level fell, suggesting that a decreasein the free calcium concentration in 0.52 0.78 0.16 the suspension lowers the threshold ATP Added Granule Cone, mg/ml level at which contraction occurs. FIG. 5. Effect of concentration of muscle granFrom a comparison of Figs. 4 and 5, it ules on threshold ATP concentration and flocculaappears that EGTA and relaxing-factor tion delay. All samples contained 4 m&l ATP, granules might act by different mechanisms, 4 mM MgCL, 10 mM imidazole buffer, pH 7,80 mM since the flocculation delay was longer with KCl, and 2.2 mg/ml myofibrils.

--

AT!?

AND

RELAXATION

Effects of ionic strength and magnesium. Alt,hough a removal of free calcium is thought to be the physiologic mechanism of relaxation in vivo (6, IS), other agents which do not’ affect the free calcium concentrat.ion, such as an increase in the ionic strength or magne-

OF

573

MYOFIBRILS .

40 -

1.0

Threshold

ATP

.; 30 -.30 .s I 2

r

0.2

P .4 ; L 5 t-1-1

.2

0

i0

IO

20

KC.1 Cont.,

30

mM

FIG. 6. Effect, of ionic strength on t,hreshold ATP concentration and flocculation delay in the presence of EGTA. All samples contained 3 mM ATP, 3 mM MgC12,lO mM imidazole buffer, pH 7, 1 ml\{ EGTA, and 2.68 mg/ml myofibrils.

I *-,----lI-I-,I--

0

'0

10

20 KCI

30 Concentration,

40

50

60

m I.4

FIG. 7. Effect of ionic strength on threshold ATP concentration and flocculation delay in the presence of granules. All samples contained 4 mM ATP, 4 m&f MgC12, 10 mM imidazole buffer, pH 7, 1.04 mg/ml granules, and 2.2 mg/ml myofibrils.

0

1

4

3

MgCI,

Cont.,

--

0

-I-,-

3

5 mM

FIG. 8. Effect of magnesium concentration on ATP threshold, ATPase activity, and flocculation delay, in the presence of EGTA. All samples contained 3 mM ATP, 1 mM EGTA, 10 mM imidazole buffer, pH 7, and 2.1 mg/ml myofibrils. Potassium chloride was added to maintain ionic strength at about 0.05.

sium concentration, also promote “relaxation” in vitro (4, 10). Figures 6 and 7 show the effect of increasing the ionic strength in the presence of EGTA or relaxing granules. In both cases, as the delay in syneresis was prolonged by an increase in the ionic strength, the threshold ATP level was reduced. When the magnesium concentration was varied in t’he presence of EGTA (Fig. 8), a similar result was obtained-the greater the inhibition of the myofibrillar ATPase or the delay in syneresis, the lower the threshold ATP level. It therefore appears that not only agents which lower the free calcium concentration, but also other agents which delay the syneresis of myofibrils, may do so by lowering the threshold ATP concentration at which syneresis occurs, as well as by inhibiting the ATPase and thus increasing the time required t*o reach the threshold ATP level. DISCUSSION

We have shown that when the ATP-induced syneresis of isolated myofibrils is delayed under the influence of a relaxing agent,

574

EISENBEIIG

the principal parameter which triggers the abrupt transit’ion to bhe flocculated state is the fall in the ATP concent’rat’ion below a critical t,hreshold level which is required to maintain relaxation (Table I). The response of myofibrils to various agents may be examined by invest’igating changes in this threshold ATP level, and we have made such a study here by directly measuring the ATP concentration remaining at the moment of myofibrillar flocculation under different COIIditions. During a delay in flocculation, not only is ATP destroyed, but ADP and Pi accumulate, and these agents have been reported to affect the t’hreshold ATP level (4). A direct test’ of t,he influence of AD!? (Figs. 1 and 2) showed t’hat the addition of ADP does increase the threshold ATP, but no significant acceleration of the myofibrillar ATPase was observed. lLIaruyarna and Gergely (4), on the other hand, reported that added ADP raised both the ATP threshold and the ATPase activity of actomyosin gel. The explanation for this difference in results is not clear, although it may be due to differences in iVIgClz concentration and ionic strength, or possibly to the use of actomyosin rather than myofibrils. In our study of the effects of various “relaxing” agents, we have found that in every case, an increased delay in flocculation is accompanied by a lowered ATP threshold. Addition of EGTA (Fig. 4) or relaxing factor granules (Fig. 5) clearly lowered the threshold ATP level, as did an increase in ionic strength (Figs. 6 and 7) or MgClz concentration (Fig. 8) in a calcium-depleted system. It is of interest to speculate on why Dhe relaxing ability of Obese varied agent’s is paralleled by their effects on the threshold ATP concentration. An impressive body of evidence indicates that a key element in the control of muscle cont,raction is the regulation of the amount of calcium bound bo the contractile protein system, relaxation being caused by a loss of bound calcium (8, 18, 19). In vitro chelating agents like EGTA, and relaxing-factor granules, lower the free calcium concentration in the medium and thereby remove bound calcium from the myofibrils and inhibit contraction. An increase in the ionic st’rength or

AN11

MOOS

the free magnesium concentration may decreasethe affinity of the protein for calcium, leading again t’o a loss of bound calcium (8). The control exerted by the ATP caoncent,ration, on the other hand, does not appear to operat,e in this way. Actomyosin will superprecipitate at’ a low concentration of ATP even when most of its bound calcium has been removed by EGTA (8), and similarly, myofibrillar ATPase in the presenceof added free calcium ion is inhibit,ed by a high ATP concentrat’ion (10 mM), witshout, a loss of bound calcium (19). It seems,t,herefore, that the transition of myofibrils from the relaxed to the contracted state in vitro may be governed by both the bound calcium level and the ATP concentration. Since the relaxing effect of ilT1’ is not, exerted through an influence on calcium binding, the ATP must act directly on the contract,ile protein, perhaps by binding at a second site on the myosin, distinct from the hydrolytic ATPase site. Such a possibility has been noted by Weber and Portzehl (2) and is supported by the finding of Hasselbath (20) t’hat t’he relative effectiveness of various nucleotides is different for relaxat’ion than for contraction and hydrolysis. This second site might therefore be called the “relaxing binding site” as opposed to the hydrolytic binding site for ATP. We may then postulate that t’he binding of ATP (possibly as Mg-ATP) to this “relaxing site” interferes with the binding of actin to myosin and thereby inhibits contraction in myofibrils and “clears” (dissociates) a&omyosin. According to this model, the minimum concentration of ATP required to prevent the actin-myosin interaction, i.e., the ATP-threshold, is a measure of the affinity of the actin for its binding site relative t.o that of ATP for the “relaxing-binding site.” We have assumedthat the actin-binding sit,e and the “relaxing-binding site” for ATP are separate, because if they were identical it would be diflicult to explain why ADP raises the t*hreshold ATP level; on the contrary, one might then expect the binding of ADP, like that of ATP, to inhibit the act’in-myosin interaction. By postulating that the two sites are at least partly separate, however, we can explain the observed effect of ADP as a cornpetition between ADP and :1T1’ for the

ATP

AN11

RELi\NATION

“relaxing site,” with only the binding of ATP inhibiting the interact.ion of myosin with act,in. The fact that the removal of calcium decreases the ATl’ threshold (Figs. 4 and .ri) indicates that the presence of calcium bound to t#he protein system favors the binding of artin. To understand this action of calcium we must consider the role of ‘
OF

,575

MYOFIBRILS

(2.5). In either case, like a reduction in the free calcium concentration, these agents must decrease the affinity of act’in for myosin, and therefore it is reasonable t’hat they should also lower t,he ATl’ threshold level. In summary, t-hen, we have interpreted tbc relationship between the relaxing effects of various agents and their effect’s on the threshold ATl’ concent’ration with two assumpt,ions: first, that the syneresis of myofibrils represents an abrupt’ increase in the affinity of a&n for myosin; and second, that this affinity is governed by a balance between the effect, of various relaxation-promoting agenk, and the effect, of the falling ,4TI’ caoncentration, with the latter effect being due t’o the binding of ATP to a ‘(relaxing” sii e 011the myosin. ACKNOWLEl)(;MENT

Mr. this

We are grateful David Garrison project.

to Mr. Frederick for technical

Barlow assistance

and in

REFERENCES 1. H.\SSELB.\CH, W., ANDWEBER, H.H.,Hiochim. Biophys. Ada 11, IGO (1953). 2. WEBER, H. H., POR,TZEHL, H., Progr. Bioph?ys. Biophys. C’hem. 4, GO (1954). 3. %~Iu-~.IMA, Ii., .IKU GERGELY, J., J. Bid. Chem 237, 1095 (1962). 4. MARUYAM.\, K., AND (~EKGELY, J., J. Hial. Chem. 237, 1100 (1962). as a 5. LORAND, L., AND MOI,NAR, J., in “Muscle Tissue” (K. Rodahl and S. M. Horvath, eds.), p. 97. McGraw Hill, New York (19G2). G. WEBEIL, A., HEHZ, It., ASD REISS, I., J. Gen. Phg.sioZ. 46, 679 (1963). 7. EBASHI, P.,J. Riochenl. 50, 230 (1961). 8. WEBEH, A., .\xD HERZ, R., J. Biol. Chem. 238, 599 (1963). K., AND ~HIKAIv.L Y., J. Rio9. b~.\RUYAM.\, them. 65, 110 (1964). Y., .\ND YOSHIMUHA, J., ., I?. J., J. Biol. Chcm. 193, 265 (1951). 13. STKEHLER, B. L., AND TOWEH, J. It., Arch. Biochem. Biophys. 40, 28 (1952). I., .ANU CH.\IS, E., Biochem. J. 11. BEHENBLUM, 32, 295 (1938).

576

EISENBERG

15. BOCK, R. M., LING, N.-S., Morell, S. A., LIPTON, S. H., Arch. Riochem. Biophys. 253 (1956). 16. MARTONOSI, A., AND FERETOS, R., J. Chem. 239, 648 (1964). 17. MARTONOSI, A., AND FERETOS, R., J. Chem. 239, 659 (1964). 18. Symposium on “The Relaxing Factor of cle,” Federation Proc. 23, 885 (1964). 19. SEIDEL, J. C., AND GERGELY, J., J. Chem. 238, 3648 (1963).

AND AND 62,

Biol. Biol. MusBiol.

MOOS

2Q. HASSELBACH, W., Biochim. Biophys. Acta 20, 355 (1956). 21. EBASHI, S., AND EBASHI, F., J. Biochem. 66, 604 (1964). 22. LAKI, K., MARUYAMA, K., AND KOMINZ, D. R., Arch. Biochem. Biophys. 98, 323 (1962). 23. MARTONOSI, A., J. Biol. Chem. 237, 2795 (1962). 24. KATZ, A. M., J. Biol. Chem. 239,3304 (1964). 25. MARUYAMA, K., ISHIKAWA, Y., AND EBASHI, S., J. Biochem. 66, 581 (1964).