Cyclic AMP-dependent and independent protein kinase phosphorylation of canine cardiac myosin light chains

oToumalof MolecularandCellularCardiology(1977) 9, 501-513

Cyclic AMP-dependent and Independent Protein Kinase Phosphorylation of Canine Cardiac Myosin Light Chains Y. SURENDRANATH R.EDDY,* BARRY J. R. PITTS Am) ARNOLD SCHWARTZt Department of Cell Biophysics, Baylor Collegeof Medicine, Houston, Texas 77030, U.S.A.

(Received 3 May 1976, acceptedin revisedform 8 oTuly1976) Y. S. REDDY,B.J.R. Prrrs ANDA. SCHWARTZ.CyclicAMP-dependent and Independent Protein Kinase Phosphorylation of Canine Cardiac Myosin Light Chains. oToumalof Molecular and Cellular Cardiolo~ (1977) 9, 501-513. Myosin was isolated from purified cardiac myofibrils and characterized for K+-ATPase and Ca~+-ATPase activities. The light chain (LG) fraction was obtained by exposing myosin to pH 11.5 followed by (NH4)2SO4 fractionation. Sodium dodecyl sulphate (SDS) gel electrophoresis revealed two components with molecular weights of 30 000 (LC1) and 20 000 (LC2) daltons. No detectable cyclic AMP-dependentprotein ldnase activitywas found. Cyclic AMP markedly stimulated phosphorylation of the myosin light chain fraction in the presence of added protein kinase. Alkaline phosphatase dephosphorylated the phosphorylated light chains. LC2 was phosphorylated by cyclic AMP-dependent protein kinase. K~Y WORDS:Myosin light chains; Protein kinase; Phosphorylase b kinase; Cyclic AMP; Phosphorylation.

I. Introduction Stimulation of protein phosphorylation in animal cells by cyclic AMP involves the binding of cyclic AMP to the regulatory subunit of protein kinase and the release of the catalytic subunit of the enzyme [17]. Possible physiological substrates which are known to be involved in glycogenolysis [5] and muscle contraction have been identified [3, 28, 31, 32, 37]. Perrie a al. [27] reported that under certain conditions the skeletal muscle myosin light chains migrated as four bands (designated as ML1, MLs, MLs, ML,), MLs (the phosphorylated form) could be converted to MLs (the dephosphorylated form) by alkaline phosphatase. The ML3 fraction of Petrie et al. [27] corresponded to the DTNB [5,5Ldithiobis-(2-nitrobenzoic acid)] light chain of Weeds and Lowey [4/]. Cardiac muscle myosin from rabbit, in contrast, appears to contain only two light chains with molecular weights of 20 000 and 30 000 daltons [11-13]. Perrie et aL [27] have reported lower values of 19 000 and 24 000 and also observed a minor component with a molecular weight of 28 000 daltons. A small incorporation of asp into this latter component was observed when cardiac myosin light * Present address and reprint requests to: Y. S. Reddy, Ph.D., Senior Investigator, Cardiovascular Research Laboratories, Texas Heart Institute, P.O. Box 20269, Houston, Texas 77030, U.S.A. t Present address: The University of Cincinnati College of Medicine, Department of Pharmacology and Cell Biophysics,231 Bethesda Avenue, Cincinnati, Ohio 45267, U.S.A.

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Y . s . REDDY, B. J. R. Pia't$ AND A. SCHV~ARTZ

chains were incubated with phosphorylase b kinase and [y.asp] ATP but the authors suggested that this was due to a proteolytic fragment of TN-T (a component of troponin). The authors suggested that the lack of phosphorylation of the 18 500 myosin component in rabbit cardiac muscle could be due to high endogenous phosphorylation that persists through the isolation procedure. McPherson et al. [24] isolated phosphorylated heavy and fight cardiac myosin subunits 2 days after injecting dogs with asp. The amounts of bound phosphate in each of the two fight chains and the heavy chain in moles/mole purified subunits were 0.17, 0.73 and 4.5, respectively. The present report deals with the isolation of canine cardiac myosin fight chain fractions prepared from purified myofibrils and the cyclic AMP-dependent protein kinase-catalyzed phosphorylation of these fight chains.

2. M a t e r i a l s a n d M e t h o d s

Preparation of myofibrils Canine cardiac myofibrils were isolated as described by Solaro et al. [307.

EGTA, azide, and quinidine-sensitive A TPase activities of myofibrils Protein fractions (0.2 to 0.5 rag) were incubated in a volume of 1 ml containing (in final concentration) 2 m_MATP, 2 m_MMgCls, 50 m_~ KC1 and 20 m~ imidazole -HC1 (pH 7.0) at 37~ for 5 rain in the presence or absence of 1.6 mu EGTA [ethyleneglycol bis ([~-aminoethyl ether)-N,N'-tetraacetic acid], 10 mM sodium azide or 1 rn~ quinidine. The reaction was stopped by the addition of 10% cold trichloroacetie acid and the inorganic phosphate was determined by the method of Fiske and SubbaRow [8]. Quinidine interferes with colorimetric determinations of inorganic phosphate and it was, therefore, necessary to remove the drug by extraction with choloroform [20]. (Na+,K+)-ATPase activities were determined by incubation at 37~ with 2.5 rnM ATP, 5 m_MMgCI~, 1 m_MEDTA (ethylenediamine tetraacetate), 100 mu NaC1, 10 mu KCI and 30 rnM histidine-HC1 (pH 7.4) in the presence or absence of 0.5 mu ouabain. Cardiac myosin was prepared from purified myofibrils as described by Huszar and Elzinga [13]. Protein concentrations were determined by the method of Lowry et al. [19].

SDS-polyacrylamide gel electrophoresis Electrophroesis was performed by the technique of Weber and Osborn [39] ruing 10% polyacrylamide gels at a constant current of 8 mA/tube for 4 h. Molecular

P H O S P H O R Y L A T I O N O F C A R D I A C MYOSIN L I G H T C H A I N S

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weights were determined by the use of a calibration curve obtained by plotting the electrophoretie mobilities of various purified proteins against the logarithms of their known molecular weights.

Isolation of cardiac myosin light chains The light chains were isolated from purified myosin (0.5 to 2.0 mg/ml) by exposure to pH 11.5 at 0~ for 1 h, followed by (NH4)~SO4 fraetionation. Heavy chains were collected between 0 to 40% saturation of (NH4)~SO4, while the light chains were collected between 40 to 70% saturation. This procedure is similar to that described by Frederiksen and Holtzer [9].

Phosphorylation of light chains Phosphorylation was carried out by the procedure of Brostrom et al. [4]. The protein-bound 8~p was assayed by the method ofMiyamoto et al. [26]. In some experiments, phosphorylation was carried out as follows. The enzyme phosphorylase b kinase (0.04 to 0.2 mg/ml, Sigma) was incubated at 30~ with myosin light chain fractions (0.8 to 1.5 mg/ml) in 20 n ~ 2-(Ar-morpholino) ethanesulfonic acid buffer (MES) (pH 8.0) containing 3 rnM magnesium acetate, 0.5 mM CaC19, 25 mM NaF, 1 mM dithiothreitol and 2.5 mM [y32P]ATP of known specific activity. Samples were withdrawn and the reaction was stopped with 0.5 vol. of 15% TCA containing 2.5 rnM ATP, centrifuged at 2000 rev/min at 5~ and washed 3 times with ice-cold 5% TCA; 8~p was determined by the Cerenkov method. Phosphorylation of myosin light chain fraction using protein kinase was carried out as follows. (Protein kinase purchased from Sigma.) The enzyme (0.05 mg/ml) was incubated at 30~ with myosin light chain fraction (0.8 to 1.5 mg/ml) in 20 mM MES (pH 7.0), 3 rnM magnesium acetate, 25 mM NaF, 1.0 mat dithiothreitol and 2.0 mM EGTA. The reaction was stopped with 0.5 vol. of I5% cold TCA and the precipitate was diluted with an equal volume of 5% TCA containing 2.5 rnM ATP. The precipitate was washed 3 times with 5% TCA at 5~ Protein bound 3~p was measured by the Cerenkov method. The controls contained all the reactants except protein, which was added after the addition of TCA.

Determination of phosphate Phosphorous covalendy bound to the myosin light chain fraction was determined in two ways. (1) Phosphorylated protein was dissolved in 0.2 ml of 0.5 N NaOH and diluted with 10 ml of distilled water. The 3up radioactivity was determined by the Cerenkov method. The amount of phosphate incorporated into the protein was

504

Y . S . REDDY~ B. J. R. PITTS AND A. SCHWARTZ

calculated from the specific radioactivity of the [T82P] ATP. This procedure was used for determination of the time course of incorporation of ~2p into the myosin light chain fraction. (2) Direct determination of total phosphate. Protein was precipitated, washed and digested in 2.5 N NaOH at 100~ for 20 rain [40]. After neutralization, the phosphate was measured with ammonium molybdate malachite green [14].

Removal of endogenousphosphatefrom myosin light chainfraction Myosin light chain fraction (12.0 mg), dissolved in 200 mM KCI, 100 n ~ TrisHCI and 10 m_~ ~-mercaptoethanol (~ME), was incubated with 16 units ofE. r alkaline phosphatase (480 ~g) (enzyme to substrate ratio 1:25). The mixture was incubated at 30~ for 60 min. The reaction was stopped by boiling at 92~ for 20 rain. The solution was dialyzed against 1 liter of 10 mM ~ME. After dialysis, the amount of phosphate bound to the protein was estimated by the method of Itaya and Ui [14]. Rephosphorylation of dephosphorylated myosin light chain fraction was carried out as described above.

3. Results Triton X-100 treatment of cardiac myofibrils completely removed azide and quinidine-sensitive ATPase activities and eliminated the ouabain-sensitive (Na +, K+)-ATPase (Figure 1). The preparation was, therefore, free of contamination by active mitochondrial fragments [36, 38], sarcoplasmic reticulum [10, 23] and (Na +, K+)-ATPase [20]. Recent studies have shown that the specific activity of purified sarcolemmal (Na +, K+)-ATPase is in excess of 2.7 ~maol Pi mg protein-t rain -t [30]. From this value, we calculate that the contamination of the Triton X-100 treated-myofibrils by this membrane enzyme was less than 0.08%. Eighty percent of the total myofibrillar ATPase (0.2 btmol mg-t min-1) was inhibited by EGTA. SDS polyacrylamide gel electrophoresis of cardiac myosin isolated from highly purified myofibrils revealed that the heavy chains remained at the top of the gel and two light chains, LCt and LC~, migrated in very distinct bands towards the bottom of the gel (Plate 1). Occasionally, however, we observed faint bands just below the heavy chains. SDS gels of the cardiac myosin light chains revealed only two bands with molecular weights of 30 000 (LC1) and 20 000 (LC2). Examination of the effects of Ca 2+ and K + on the ATPase activities of cardiac myosin preparation confirmed the results previously reported by Fenner et al. [7] and indicated that treatment of the myofibrils with Triton X-100 did not alter the properties of the myosin subsequently isolated from them. The time-dependent incorporation of 82p from [y-3~P]ATP into the light chain fraction revealed that maximum incorporation (0.01 mol/mol protein), was reached after 20 rain (data not shown). At pH 5.0 there was no cyclic AMP-dependent incorporation of

PHOSPHORYLATION

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505

phosphate, but as the pH was increased, the incorporation of 3sp into the light chain fraction was dependent on cyclic AMP. Added protein kinase (2 to 5 ttg) increased the pH-dependent phosphorylation to some degree in the absence of cyclic AMP (Figure 2). This may be due to autophosphorylation. There was a decrease in cyclic AMP-dependent phospborylation at pH 8.0 or higher, although in one experiment no decrease in phosphorylation at pH 9.0 was observed. In the preceding studies, bovine heart protein kinase was employed. Protein kinase from bovine brain, isolated as described by Miyamoto et al. [26"] also catalyzed phosphorylation of cardiac myosin light chain. However, the maximum stimulation of phosphorylation in the presence of cyclic AMP was 50% rather than 100%. In all the above experiments, the incorporation of 3sp into the light chain fraction was very low, i.e. 0.01 mol]mol protein. Endogenous phosphate was not removed from these preparations. This low incorporation of asp may be due to high in vivo phosphory|ation of myosin light chain fractions, a phenomenon reported in skeletal muscle myosin fight chain (DTNB) by Perry and Cole [28] and suggested for cardiac muscle myosin by Petrie et al. [27]. Therefore, we estimated the total endogenous phosphate in cardiac myosin light chain fraction. Endogenous phosphate present in the cardiac myosin light chain fraction was removed by using E. coli alkaline phosphatase (Table 1). In seven preparations, three contained no detectable bound phosphate and the remainder contained 20% of bound phosphate present, even after 60 min of treatment with E. coli alkaline phosphatase. We have used these preparations for rephosphorylation studies using cyclic AMP-dependent protein kinase and phosphorylase b kinase. The endogenous phosphate level after E. coli alkaline phosphatase treatment was 0.055 mol]mol protein. In the presence of cyclic AMP and protein klnase, 0.17 mol/mol protein 0.25 UNWASHED IC

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FIGURE 1. EGTA,azide,quinidineand ouabain-sensidveATPaseactivitiesof unwashed,washed and Triton X-100 treatedmyofibrils.The activitiesshownare differencesbetweenthose obtainedin the presenceand absenceofthe inhibitor.Assayswerecarriedout as indicatedin Methods.

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FIGURE 2. Effect of pH on the phosphorylation of myosin light chain fraction at various pH values, as indicated above. The reaction medium contained 0.16 mg protein in 0.2 ml. Other conditions were as described in Methods. Light chains alone ([[]---[[]), light chains + 10-~M cyclic AMP ( H ) , light chaim + protein kinase ( 0 ~ 0 ) . light chains + protein kinase + 10-6Mcyclic AMP ((]k--~). TABLE I. Endogenous bound phosphate present in the myosin light chain fraction was removed by E. coli alkaline phosphatase. The conditions for the removal o f phosphate are described in Methods Preparation number 1 2 3 4 5 6 7

mol bound phosphate/mol protein 0.25 0.20 0.35 0.60 0.60 0.36 0.22 S.E.M. 0.37 4- 0.064

tool of bound phosphate present after E. roll alkaline phosphatase treatment 0.045 0.045 0.062 0.055 Not detectable Not detectable Not detectable S.E.M. 0.03 -4- 0.01

P H O S P H O R Y L A T I O N OF C A R D I A C MYOSIN L I G H T CHAINS

507

were i n c o r p o r a t e d after 120 rain, w h e r e a s t h e i n c o r p o r a t i o n of 3~p b y p h o s p h o r y l a s e b kinase was a b o u t 0.1 tool/tool p r o t e i n ( F i g u r e 3). T h e p h o s p h o r y l a t e d light c h a i n fraction was i n c u b a t e d w i t h alkaline p h o s p h a t a s e ( e n z y m e : substrate r a t i o o f 1:300) a n d 2 5 % o f t h e 3~p was released after 20 rain. F u r t h e r a d d i t i o n o f a l k a l i n e p h o s p h a t a s e i n c r e a s e d t h e e n z y m e : substrate r a t i o to 1:40, a n d a n increase in the r a t e o f release o f 8sp was observed. Seventy-five p e r c e n t o f t h e szp was released after 90 rain ( F i g u r e 4). T h e s e results suggest t h e presence o f a p h o s p h a t e ester linkage b e t w e e n t h e p r o t e i n a n d t h e p h o s p h o r y l g r o u p o f ATP. 0.2

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FIGURE 3. Time-dependent incorporation of 32P-ATP into the cardiac myosin light chain fraction. The reaction medium for phosphorylation using protein kinase and cyclic A M P consisted of 20 rr~ (MES) (pH 7.0), 1.25 rnM [y-- asp] ATP, 3 m_Mmagnesium acetate, 2 mMEGTA, 25 n ~ NaF, 2.0 mM dithiothreitol, 200 ~g of protein kinase and 10-sM cyclic AMP, 2.0 mg myosin light chain fraction in a total volume of 2.0 ml. The reaction medium for phosphorylation using phosphorylase b kinase comisted of 20 rnM (MES), 3 m~ magnesium acetate, 25 rnM NaF, 2.0 mM dithiothreitol, 85 ~g of phosphorylase b kinase, 0.5 mM CaCls (pH 8.0), 2.0 nag of myosin light chain fraction in a total volume of 2.0 ml. 200 ~d aliquots were removed at the indicated times and the reaction was stopped with 0.5 vol. of 15% TCA, and the resulting precipitate was diluted with the equal volumes of 5% TCA containing 2.5 mM ATP. The precipitate was collected by centrifugation at 2000 rev/ rain, for 5 rain at 5~ This process was repeated twice. The precipitate was dissolved in 0.5 ml of 0.5 s NaOH and diluted with 10 ml of glass distilled water. Aliquots of the material were taken for protein estimation. (O---O---O) Phosphorylation by protein klnase; (C)--O--C) ) phosphorylase b kinase.

500

Y . S . REDDY, B. J. R. PITTS AND A. SGHWARTZ

Myosin light chains were phosphorylated and the gels were cut into 2 m m slices and counted in a scintillation counter. Most o f the counts appeared in the L C s c o m p o n e n t (Figure 5). F r o m this experiment, it is clear that the L C s (20 000) light chain fraction is phosphorylated by cyclic A M P - d e p e n d e n t protein kinase and phosphorylase b kinase.

4. D i s c u s s i o n

I n the past 2 years there have been several reports showing that protein kinase m a y be associated with sarcolemma [2, 6, 15], sarcoplasmic reticular m e m b r a n e s [43], cardiac regulatory proteins [32] and platelet myosin [1]. We, therefore, attempted to eliminate the contamination of myofibrils with the membrane-associated protein

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FIGURE 4. Time course of release of 32P from the phosphorylated light chains fraction by E. to/i alkaline phosphatase. 1.5 mg ofphosphorylated light chain fraction was dissolved in 200 rms KOI + 100 m.MTris-HC1 (pH 7.5). The phosphorylated protein was incubated with alkaline phosphatase with an enzyme: substrate ratio of 1 : 300. Samples were taken at times shown and acid-soluble sip was determined. Where indicated by the arrow, a further addition of alkaline phosphatase was made to increase the enzyme: substrate ratio to 1 : 40.

PHOSPHORYLATION

OF CARDIAC

MYOSIN

509

LIGHT CHAINS

kinase by treatment with Triton X-100 [36]. Such myofibrillar preparations still contain endogenous protein kinase activity, as revealed by the cyclic AMPdependent phosphorylation of regulatory proteins in the absence of added protein kinase (data not shown). In the absence of added protein kinase, there was very little pH dependent-incorporation of a2p, and cyclic AMP had no effect, suggesting that there is no cyclic AMP-dependent protein kinase associated with the cardiac light chain fraction. Perrie et al. [27] pointed out that phosphorylation of the DTNB light chain with crude protein kinase (from skeletal muscle) was higher than with purified protein kinase, suggesting that an enzyme system might be present in the crude preparations which is specific for light chains. Subsequently, Pires et al. [29] isolated an enzyme from the crude preparation which was specific for myosin light chains and was Ca~+-dependent but not cyclic AMP-dependent. Cardiac myosin isolated from the purified myofibrils showed two light chains of molecular weights 30 000 (LCz) and 20 000 (LCa) in close agreement with the

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F I G U R E 5. SDS gel electrophoresis of phosphorylated light chain fraction. Phosphorylation was conducted as described in Figure 3. U p to 150 ttg of phosphorylated protein were loaded onto the gels. A t the end of the run, the gels were frozen in dry ice for 5 mln and cut into 2 m m pieces which were dissolved in 30% HgO~ containing 0.2% NHa at 80~ (It took 1 or 2 h to dissolve the gels.) (--) Optical density recorded by a Gilford Automatic Densitometer; ( - - - - ) corresponds to ct/min; ( O - O - O ) phosphorylase b kinase; ( A - & - & ) protein kinase. Inset in this figure represents a standard graph calibrated by the use of proteins of known molecular weights. A, B, C, D, correspond to albumin, ovalbumin, pepsin and myoglobin, respectively. Incubation time for phosphorylation was 120 min.

510

Y . S . REDDY, B, J. R. PITTS AND A. SCHWARTZ

reported values in the literature [18, 21, 42]. However, Perrie et al. [27] reported the existence of light chains in myosin from rabbit heart with molecular weights of 19 000 and 24 000 dahons. Neither chain was phosphorylated by phosphoryleme b kinase but a very low incorporation of ssp into a minor band of a 28 000 dalton component was reported [27]. The authors suggested that this might be a proteolytic digestion product of TN-T. As shown in the present study, LC2 of myosin light chains was phosphorylated by cyclic AMP-dependent protein kinase and phosphorylase b kinase. The phosphorylated form of the cardiac myosin light chain fraction was dephosphorylated by E. coli alkaline phosphatase similar to the dephosphorylation of phosphorylated TN-T and DTNB light chains of skeletal muscle reported recently [27, 28]. Petrie et al. [27] have suggested that there may be a protein phosphata~ which specifically hydrolyzes phosphorylated myosin light chains, and in at least one case an enzyme specific for the phosphorylated protein has been reported [35]. Phosphorylation of troponin and its components has been reported by various investigators [3, 31, 32], but no physiological significance has been attributed to this event. Kendrick-Jones [16] reported that the DTNB light chain of rabbit skeletal muscle binds to the desensitized myosin from scallop (myosin that has lost Ca *+ sensitivity upon the removal of EDTA-releasable light chain) and restores Ca s+ sensitivity, suggesting that the DTNB light chain might have some type of regulatory function. It has been suggested that this may be mediated through a Ca z+ dependent kinase associated with the DTNB light chain [29]. There is apparently no DTNB-releasable light chain in cardiac myosin [18]. Weeds and Frank [40] have shown that subfragment-I of cardiac myosin prepared by papain treatment contains LC1 but no LC~ when examined by SOS gel electrophoresis. Since this subfragment-I contains all the enzymatic properties of myosin, it appears that LC~ is not required for the myosin ATPase activity. Phosphorylatlon of cardiac LCz resembles that of DTNB light chain in skeletal muscle. In one instance, it was reported that dibutyryl cyclic AMP increased the myosin ATPase [12] activity, which suggests that stimulation of phosphorylation of light chain may Occur.

It should bc emphasized that while the phosphorylation of the cardiac light chain fraction is significant,the amount incorporated is about ~ that rcported for skeletalmuscle lightchain. Immunological and chemical differencesin lightchalm from various types of muscles have been reported [22, 34].

Acknowledgements This work was supported by USPHS grants HL 16915 (YSR), HL 07906 (A.S.) and NIH Contract 71-2493 (A.S.). We thank Mr Raymond Givens for his expert technical assistance.

PHOSPHORYLATION OF CARDIACMYOSINLIGHT CHAINS

511

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512 21. 22. 23. 24. 25. 26.

27. 28. 29. 30. 31.

32.

33. 34. 35. 36. 37. 38. 39.

Y.s. REDDY, B. j. R. Prrrs AND A. SCHWARTZ MAre, R. S. & KAY, C. M. Some physiochemical properties of light chains isolated from skeletal cardiac myosin. Can~ian~ou~lofBiodz#mist~ 51, 178-181 (1973). MASAKI,T. Irnmunochcmicai comparison ofmyosins from chicken cardiac, fast white, slow red and smooth muscle, o7apaneseoTournalof Biochemistry76, 441-449 (I 974). MACFARLAtCO,B. H. & INESI, I. Studies of solubilized sarcoplasmic reticulum. Biochemicaland BiophysicalResearchCommunications30, 83-88 (1968). MePHERSON,J., F~Nmm, G., SMITH, D. J., MASON, D. T. & WXKMAN-CO~LT, J. Identification of in vivo phosphorylated myosin subunits. Federation of European BiochemicalSocietiesLetters47, 149-154 (1974). MEISlmR, M. H. & LA~GAN, T. A. Characterization of a phosphatase specific for phosphorylated histones and protamine. Journal of Biological Chemistry244, 4961-4968 (1969). MIX'AMOTO,E., KUO, J. F. & GRE~.N~Am~, P. Cyclic nueleotide-dependent protein kinases I I I Purification and properties of adenosine 3', 5'-monophosphate-dependentprotein kinase from bovine brain, aTournal of Biological Chemistry 2441, 6395--6420 (1969). P~, W. R., SMxta~t~,L. B., P~m~y, S. V. A phosphorylated light-chain component of myosin from skeletal muscle. BiochemicaloTournal135, 151-164 (1973). PEmty, S. V. & CIOLE,H. A. Phosphorylation of the 37 000 component of the troponin complex (Troponin-T). BiochemicalJournal 131,425-428 (1973). PinEs, E., PERRY, S. V. & THOUAN, M. A. W. Myosin light chain kinases, a new enzyme from striated muscle. Federation of European Biochemical Sodeties 41, 292-296 (1974). Frrl~, B.J.R., LANE, L. K. & SCH'W'ARTZ,A. Nature of the transport ATPase-digitalh complex VI : Purification of and ouabain binding to a highly active cardiac Na +, K +ATPase. Biochemicaland BiophysicalResearchCommunications53, 1060-1066 (1973). PRATJE,E. & HEILMEYER,L. M. G. Phosphorylation of rabbit muscle troponin and actin by a 3', 5"-cAMP-dependent protein kinase. Federation of European Biochemical SodetiesLetters27, 89-93 (1972). R.EDDY,Y. S., BALLARD,D., GIRX,N. Y. & SCHWARTZ,A. Phosphorylation of cardiac native tropomyosin and troponin: inhibitor effect of actomyosin and possible presence of endogenous myofibrillar located cyclic AMP-dependent protein kinase, oT0urna/of Molecularand CeUularCardiology5, 461-471 (1973). REDDY, Y. S. & SCaiWARTZ, A. Phosphorylation of cardiac contractile proteins by cyclic AMP-dependent protein kinase. Abstract. Federation Proceedings33, 406 (1974). SAmCAR,S., Sm~amn,F. A. & GEROELY,J. Light-chaius ofmyosins from white, red, and cardiac muscles. Proceedingsof the National Academyof Sciemes 68, 946--950 (1971). Smss, E. A. & W~.mAm~,O. H. Purification and characterization ofpyruvate-dehydrogenase phosphatase from pig heart muscle. EuropeanJournal of Biochemistry 26, 96-105 (1972). Solamo, R.J., PASO, D. C. & Bmc.os, F. N. The purification of cardiac myofibrils with Triton X- I00. Biochimicaet biophysicaacta245, 259-262 (1971). STULL,J. T., BROSTnOM, C. O. & KRXnS, E. G. Phosphorylation of the inhibitory component of troponin by phosphorylase kinase. Journal of Biological Chemistry 247, 5272-5274 (1972). Vm~P.s, G. A. & ZInGIeR, F. D. Azide inhibition of mitochondrial ATPase. B/odwrtdtalandBiophysicalResearchCommunications30, 83--88 (1968). WEBER, K. & OSBORN, M. The reliability of molecular weight-determinationt by dodecyl sulfate polyacrylamide gel eleetrophoresis, oTournalof Biological Chemla~ 244, 4406 Aa.12 (1967).

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40. WEEDS,A. G. & FRANKS,G. Structural studies on the light chains of myosin. ColdSpring Harbor Symposiumon QuantativeBiology 37, 9-14 (1972). 41. WEEDS,A. G. & Low~v, S. Substructure of the myosin molecule II; the light chains of myosin. Journal of MolecularBiology 61, 701-725 (1971). 42. W ~ - C O F F E L T , J., FEN~ER, C., SmitH, A. & MASON,D. T. Myosin chains of myocardial tissue--1. Purification and immunological properties of myosin heavy chains. Biochemicaland Biopkysical ResearchCommunications51, 1097-1104 (1973). 43. W~Y, L. H., GravY, R. R. & OLSSON,R. A. Cyclic adenosine 3', 5'-monophosphate stimulated protein ldnase and a substrate associated with cardiac sarcoplasmic reticulum.3ournalofBiological Chemistry248, 1496-1498 (1973).

Addendum These studies were completed in 1974 (Reddy and Schwartz, Federation Proceedings 33, 406, 1974) but have been in revision since that time. We originally found very low but significant phosphorylation of cardiac myosin light chains. We refined the procedures of isolation so that phosphorylation was augmented but still much lower than skeletal muscle counterparts. Frearson and Perry have recently published a paper on this same subject (Biochemical jTournal 151, 99-107, 1975) in which 19 000 and 27 000 dalton light cham_s derived from cardiac muscle are phosphorylated to the same level found by ourselves, by a "myosin light chain kinase". There were no data on cyclic AMP.

PLATE 1. SDS gel electrophoresis of: A, cardiac myosin (20 ~g) ; B, actin (20 ~xg); C, light chain fraction (30 ~.g); D, light chain fraction (10 ~g). Electrophoresis was carried out as described in Methods.

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