printed in u.s.a. purification and characterization of bovine · pdf filepurification and...

Vol. 254, No. 23, Issue of December 10, pp. 12136-12144, 1979 Printed in U.S.A.

Purification and Characterization of Bovine Cardiac Calmodulin- dependent Myosin Light Chain Kinase*

(Received for publication, May 16, 1979, and in revised form, August 14, 1979)

Michael P. Walsh,+ Bernard Vallet, Franqoise Autric, and Jacques G. Demaille

From the Centre de Recherches de Biochimie Macromolkulaire du Centre National de la Recherche Scientifique, P. 0. Box 5051, 34033.Montpellier, France

Myosin light chain kinase, which is located primarily in the soluble fraction of bovine myocardium, has been isolated and purified approximately 1200-fold with 16% yield by a three-step procedure. The approximate content of soluble myosin light chain kinase in heart is calculated to be 0.63 PM.

The isolated kinase is active only as a ternary complex consisting of the kinase, calmodulin, and Ca’+; the apparent Z& for calmodulin is 1.3 IBM. The enzyme also exhibits a requirement for M8+ ions. Myosin light chain kinase is a monomeric enzyme with M, = 85,000. The enzyme exhibits a K,,, for ATP of 175 PM, and a Ko.5 for the regulatory light chain of cardiac myosin of 21 PM.

The optimum pH is 8.1. Kinase activity is specific for the regulatory light chain of myosin.

The specific activity of the isolated enzyme (30 nmol 32P/min/mg of protein) is considerably less than the corresponding values reported for the skeletal and smooth muscle light chain kinases. This is probably due to proteolysis during extraction of the myocardium, a phenomenon which has, as yet, proven impos- sible to eliminate. In contrast to the smooth muscle enzyme (Adelstein, R. S., Conti, M. A., Hathaway, D. R., and Klee, C. B. (1978) J. Biol. Chem. 253,8347-8350), the cardiac kinase is not phosphorylated by the catalytic subunit of CAMP-dependent protein kinase.

Regulation of vertebrate striated muscle contraction by Ca2’ ions is mediated by the troponin system (1, 2). Recent evidence from several laboratories (3-5) suggests that the mechanism of Ca” regulation of smooth muscle and non- muscle contractile systems, on the other hand, does not in- volve a troponin system, hut rather occurs via Ca’+-regulated

* This work was supported in part by grants from North Atlantic Treaty Organization, Fond&ion pour la Recherche MBdicale Fran- Gaise, Direction Generale B la Recherche Scientifique et Technique (ACC PBP and ACC Biologie et Fonction du Myocarde), Institut National de la Santb et de la Recherche MBdicale (CRL 78.4.086.1. and ATP 63.78.95 Biologie et Pharmacologic de la Fibre Musculaire Cardiaque), and Centre National de la Recherche Scientifique (ATP Structure des prot6ines and ATP Modulation de l’action des hor- mones au niveau cellulaire). Contribution No. 185 from the Centre de Recherches. A preliminary report of part of this work was presented recently (Walsh, M. P., Vallet, B., and Demaille, J. G. (1979) Fed. Proc. 38, 2952a). Simultaneously and independently, a preliminary report on the purification of canine cardiac myosin light chain kinase was presented (Rappaport, L., and Adelstein, R. S. (1979) Fed. Proc. 38, 571a). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

f Recipient of a fellowship from the Medical Research Council of Canada. To whom correspondence and reprint requests should be addressed.

phosphorylation of one of the light chains (the regulatory or P light chain, M, approximately 20,000) of myosin. Phospho- rylation of this light chain is catalyzed by a specific Ca’+- dependent kinase, referred to as myosin light chain kinase. According to this mechanism (6, 7), the kinase exists in the resting muscle as a monomer of J4, = 105,000 (6) or M, = 125,000 (8), the regulatory light chain of myosin is in the dephosphorylated state, and actin and myosin are not interacting. An appropriate stimulus triggers an increase in cytosolic Ca2+ concentration, whereupon Ca2’ hinds to calmodulin, the ubiquitous Ca2+-dependent regulator (see Ref. 9 for review). The induced conformational change in calmodulin en- ables it to interact with the apoenzyme. The resultant active complex (Ca”. calmodulin. kinase) catalyzes the phosphorylation of the regulatory light chain of myosin. Myosin can then interact with actin leading to contraction. Muscle relaxation presumably occurs through hydrolysis by a specific phosphatase (10) when the Ca2+ ion concentration decreases. A similar calmodulin-dependent myosin light chain kinase has been identified in various non-muscle tissues, such as platelets and brain (11-13).

The identification of a specific kinase in red and white skeletal muscle and cardiac muscle which catalyzes the phosphorylation of the myosin regulatory light chain (14,15) raised the question of the possible presence in sarcomeric muscles of a second Ca2+-mediated regulatory system in addition to the troponin system. The possibility of a myosin-linked regulation (16, 17) was enhanced by the recent observation (18) that rabbit skeletal muscle does indeed contain a specific calmodulin-dependent myosin light chain kinase. Similarly, we have isolated and purified a calmodulin-dependent myosin light chain kinase from hdvine heart.

EXPERIMENTAL PROCEDURES

Materials-[y-32P]ATP (300 to 400 mCi/mmol) was prepared by the method of Glynn and Chappell (19) from 32Pi (New England Nuclear). Histone II A mixture, casein, phosvitin, bovine serum albumin, and ovalbumin were obtained from Sigma Chemical Co. (St. Louis, MO.).

Purification of Proteins-Bovine brain calmodulin was isolated and purified by a modification of the procedure described by Teo et al. (20) for the purification of bovine cardiac calmodulin, as described in detail by Walsh and Stevens (21). Glycogen phosphorylase b was prepared from rabbit skeletal muscle by the method of Fischer and Krebs (22). Glycogen phosphorylase b kinase was prepared from rabbit skeletal muscle by the method of Cohen (23). The catalytic subunit (specific activity = 1.16 pmol/min/mg) of bovine heart CAMP- dependent protein kinase type II was prepared (24) and stored (25) as previously described.

Myosin light chains were prepared from bovine cardiac, turkey gizzard, and rabbit skeletal muscle myosin according to the method of Perrie and Perry (26) by ethanol precipitation from 5 M guanidine hydrochloride. The whole light chain fraction was used as substrate of myosin light chain kinase; this fraction contained sufficient contaminating calmodulin to provide maximal activation of calmodulin-free

12136

by guest on May 1, 2018

http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Cardiac Myosin Light Chain Kinase 12137

myosin light chain kinase, as shown by addition of extra calmodulin. In certain experiments (indicated in the text) a partially purified, calmodulin-free regulatory light chain fraction was prepared from the appropriate light chain fraction by ion exchange chromatography performed essentially as described by Nairn and Perry (27). This fraction was devoid of contaminating calmodulin as evidenced by its lack of activation of calmodulin-deficient myosin light chain kinase; it contained approximately 0.05 mol of acid-stable phosphate/mol, determined as indicated below. The concentration of the regulatory light chain was determined by densitometric scanning at 560-nm of a Coomassie blue-stained 0.1% sodium dodecvl sulfate, 15% nolvacrvl-

- _” ”

amide gel electrophoresis of this fraction. Preparation of Calmodulin-Sepharose-Calmodulin was coupled

to Sepharose 4B (Pharmacia Fine Chemicals Inc.) according to the method of Klee and Krinks (28), equilibrated with 40 mrvr Tris-HCl, pH 7.6, 10 mM 2-mercaptoethanol, 0.05 M NaCl, 0.05 mM CaCl2, and packed into a column of 2.5 X 25 cm. The Sepharose contained approximately 1.0 mg of calmodulin/ml of packed resin. When not in use, the resin was stored in the presence of 0.05 mM CaC12, 0.02% sodium azide.

Enzyme Assays-Myosin light chain kinase activity was measured by the amount of 32P incorporated into 0.825 mg of whole light chain fraction of bovine cardiac myosin, or 0.275 mg of partially purified regulatory light chain fraction, as indicated in the text. The assay mixture (0.07 ml) contained 35 mM Tris-HCl, pH 8, 8 mM magnesium acetate, 1.6 mM dithiothreitol, 1.5 mM [y-“‘P]ATP (50 to 100 cpm/ pmol), and either CaClz or EGTA’ at concentrations noted in the text. Reactions were initiated by addition of the enzyme and incubated at 30°C for varying periods of time. Reactions were terminated by pipetting 0.06 ml of the incubation mixture onto Whatman No. 3MM filter paper discs (23 mm 9) which were then washed as described by Corbin and Reimann (29).

For assays of myosin light chain kinase activity in fractions obtained during purification of the enzyme, sodium fluoride (70 mM) was included in order to inhibit phosphatase activitv; it was found that inclusion or omission of this inhibitor was without effect on the snecific activitv of the kinase. The initial velocitv of the kinase reaction was routinely determined in the presence”and absence of Ca*+ for each fraction obtained during purification by withdrawing aliquots from the reaction mixture at 1-min intervals up to 10 min incubation at 30°C. The use of short incubation times was designed to minimize possible interference due to phosphatase activity.

The ATPase activity of myosin light chain kinase was measured by incubating the enzyme (35 pg/ml) at 30°C in 35 mM Tris-HCl, pH 8,8 mM magnesium acetate, 1.6 mu dithiothreitol, 0.16 mu potassium phosphate, 1.6 mM [y-“‘P]ATP (50 to 100 cpm/pmol), and either 0.3 mM CaC12, 0.3 mM CaCls plus 1O-7 M calmodulin, or 10 mM EGTA. Blanks from which the kinase was omitted were assayed simultaneously. Aliquots (0.06 ml) of reaction mixtures were withdrawn at 5- min intervals up to 60 min and spotted onto Whatman 31 ET filter paper squares (1.5 x 2 cm). The liberated 32P1 was quantitated by the filter paper technique of Reimann and Umfleet (30).

Protein- bound Phosphate Determination-The amount of acid- stable phosphate bound to myosin light chain kinase was measured after ashing the protein according to the methods of Ames (31) by the malachite green method of Itaya and Michio (32). Bovine serum albumin and the nonphosphorylated regulatory light chain of bovine cardiac myosin were used as blanks in the assay.

Gel Electrophoresis-1) Polyacrylamide gel electrophoresis in the presence of 0.1% sodium dodecyl sulfate was carried out at 24 mA in 7.5% polyacrylamide slab gels employing the discontinuous buffer system described by Laemmli (33). 2) In order to preserve the activity of the isolated kinase, gel electrophoresis employing 7.5% polyacrylamide cylindrical gels was carried out in the absence of sodium dodecyl sulfate and in the presence of 2.5 mM dithiothreitol, using the buffer and gel system of Fairbanks et al. (34) as described by Daniel and Adelstein (35). Gels were pre-electrophoresed overnight to remove the ammonium persulfate prior to application of protein samples. Electrophoresis was performed at 2 mA/tube at 4°C. Gels were either stained and destained according to the method of Fairbanks et al. (34) and scanned at 560 nm, or cut into 2-mm slices for elution of the kinase. The gel slices were minced and incubated at 4°C overnight

’ The abbreviations used are: EGTA, ethylene glycol bis(b-amino- ethyl ether)-NJ’-tetraacetic acid; DEAE, diethylaminoethyl; EDTA, ethylenediaminetetraacetic acid; TPCK, L-I-tosylamido-2-phenyl- ethyl chloromethyl ketone; CDR, calcium-dependent regulator (cal modulin).

in 0.1 ml of 0.09 M Tris-HCl, pH 8, 20 mu magnesium acetate, 4 mM dithiothreitol, 5 mM calcium chloride, 3 mM [y-32P]ATP (approximately 50 cpm/pmol), and calmodulin (3 X 1O-7 M). The eluate from each slice was assayed for kinase activity as described above after addition of 0.4 mg of partially purified regulatory light chain fraction of bovine cardiac myosin.

Phosphorylation by the Catalytic Subunit of Protein Kinase- Myosin light chain kinase (166 pg/ml) was incubated at 30°C for 20 min in 50 mM potassium phosphate, pH 7, 1 mu dithiothreitol, 6 mM

magnesium acetate, 0.2 mM [y-32P]ATP (47.9 cpm/pmol), and the catalytic subunit of bovine cardiac CAMP-dependent protein kinase (2 pg/ml). The reaction was terminated by adding 30 1.11 of the reaction mixture (containing 5 pg of myosin light chain kinase) to an equal volume of 0.5 M Tris-HCl, pH 6.8, 1% sodium dodecyl sulfate, 30% glycerol, 1% 2-mercaptoethanol, 0.01% bromphenol blue, and boiled for 5 min. Samples were cooled and subjected to 7.5% polyacrylamide slab gel electrophoresis in the presence of 0.1% sodium dodecyl sulfate according to the method of Laemmli (33). After staining and destain- ing the gel was cut in two, one-half being subjected to autoradiography, while protein bands and appropriate blanks were cut out of the other half, digested in 1 ml of NCS tissue solubilizer (Amersham- Searle) at 50°C for 2 h, and radioactivity counted in 9 ml of toluene- based scintillant.

Proteolysis of Myosin Light Chain Kinase-Myosin light chain kinase (0.1 mg/ml of 40 mM Tris-HCl, pH 7.6, 10 mM 2-mercaptoethanol) was treated with trypsin or chymotrypsin (protease/kinase, 1: 100) at 22’C. Aliquots of reaction mixtures were withdrawn at various time intervals and reaction quenched with TPCK (IO-fold weight excess over chymotrypsin) or soybean trypsin inhibitor (4-fold weight excess over trypsin). Myosin light chain kinase activity was assayed immediately as described above in the presence of 0.1 mM CaC12 or 5 mM EGTA.

RESULTS

Purification of Bovine Cardiac Myosin Light Chain Ki- nase-All procedures were carried out at 4°C unless otherwise indicated. All buffers contained 0.1 mM phenylmethylsulfonyl fluoride and 10% (v/v) glycerol.

Extraction-The ventricular muscle (1 kg) of fresh beef hearts, freed of fat and fibrous tissue, was chopped into small pieces, minced, and homogenized with 3 volumes of 40 mM Tris-HCI, pH 7.6, 10 mu Z-mercaptoethanol, 4 mu EDTA (Buffer A) in a Waring blendor at top speed for 2 x 30 s, with a 15-s interval between. The pH of the extract was adjusted to 7.6 by addition of 0.5 N NaOH. The extract was centrifuged at 25,000 x g for 30 min. The supernatant was filtered fist through glass wool and then through Whatman 114 filter paper with the aid of a Buchner funnel. The myofibrillar pellet was re-extracted as above with 1% volumes of Buffer A and the supernatants were combined (“homogenate supernatant”). To verify that all the myosin light chain kinase activity was, indeed, located in the soluble fraction, myofibrillar pellets were extracted under the following conditions: (a) 1 volume of Buffer A containing Triton X-100 (0.1, 0.2, 0.3, 0.4, and 0.5% (v/v)); (b) Buffer A containing 0.3 M NaCl. Triton X-100 was found to release a small amount of Ca2+-independent myosin light chain kinase activity but absolutely no Ca’+- dependent kinase; 0.3 M NaCl released a small amount of Ca2’-dependent kinase, equivalent to 10% of the activity located in the soluble fraction. Resuspended myofibrillar pellets, after treatment with Triton X-100, 0.3 M NaCl, or no treatment at all, exhibited no myosin light chain kinase activity. In a separate series of experiments, washed myofibrils were prepared by the method of Sobieszek and Bremel (36) and extracted with Guba-Straub solution. Myosin was reprecipi- tated by dialysis against 15 mu Tris-HCl, pH 7.5, 5 mM 2- mercaptoethanol, 2 mu EDTA, 1 mM NaHC03. No Ca’+- dependent myosin light chain kinase activity was detected in the supernatant after reprecipitation of myosin, in the repre- cipitated myosin itself, or in the residue remaining after extraction of the washed myofibrils with Guba-Straub solution. It was concluded, therefore, that 290% of the endogenous


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Cardiac Myosin Light Chain Kinase

kinase was located in the homogenate supernatant. Batchwise Ion Exchange on DE-4E-cellulose-DEAE-cel-

lulose resin (1 kg wet weight Whatman DE 52), previously equilibrated with Buffer A, was added slowly to the stirred homogenate supernatant and mixed gently for 60 min. The mixture was filtered with the aid of a Biichner funnel and washed with Buffer A containing 0.05 M NaCl. The packed resin was added slowly with stirring to 1 liter 40 IIIM Tris-HCl, pH 7.6, 10 mM 2-mercaptoethanol, 4 mu EDTA, 0.3 M NaCl (Buffer B), and stirred gently for 60 min. The mixture was filtered with the aid of a Biichner funnel and the resin washed twice with 500 ml of Buffer B. The 0.3 M NaCl eluate and washings were combined; this fraction contained >50% of the Ca’+-dependent myosin light chain kinase activity. Solid ammonium sulfate was added up to 60% saturation. The mixture was stirred for 60 min, centrifuged at 25,000 x g for 30 min, and the resultant pellet was resuspended in 200 ml of 40 IIIM Tris-HCl, pH 7.6, 10 InM 2-mercaptoethanol, 4 mM EDTA, 0.02 M NaCl (Buffer C), and dialyzed overnight against two changes (8 liters each) of Buffer C.

DEAE-Sephacel Ion Exchange Chromatography-The dialyzed sample was applied to a column (5 x 20 cm) of DEAE- Sephacel (Pharmacia Fine Chemicals Inc.) previously equilibrated with Buffer C. The column was washed with Buffer C and eluted with a linear salt gradient generated from 1 liter each of Buffer C and Buffer C containing 0.4 M NaCI. Fig. 1 shows that a single peak of Ca’+-dependent kinase activity is eluted at 0.12 M NaCl. The corresponding fractions were pooled and dialyzed overnight against two changes (8 liters each) of 40 IIIM Tris-HCl, pH 7.6, 10 IIIM 2-mercaptoethanol, 0.05 M NaCl, 0.05 IIIM CaCl2 (Buffer D).

Affinity Chromatography on Calmodulin-Sepharose-The dialyzed fraction obtained after chromatography on DEAE- Sephacel was loaded onto a column of cahnodulin coupled to Sepharose 4B (see “Experimental Procedures”) previously equilibrated with Buffer D (Fig. 2). The column was washed with Buffer D, then with 40 mM Tris-HCl, pH 7.6, 10 mu 2- mercaptoethanol, 0.2 M NaCl, 0.05 mM CaClz to remove non- specifically bound proteins. Finally, the kinase was eluted with 40 mM Tris-HCl, pH 7.6, 10 mM 2-mercaptoethanol, 0.2 M NaCl, 2 mM EGTA. Those fractions containing kinase activity were pooled and dialyzed overnight against two changes (8 liters each) of 40 mM Tris-HCl, pH 7.6, 10 mu 2- mercaptoethanol. The enzyme was stored in 0.5-ml aliquots

Fraction number

FIG. 1. DEAJGSephacel ion exchange chromatography of the dialyzed 0 to 60% ammonium sulfate fraction. The column (5 x 20 cm) was equilibrated with Buffer C and eluted with a linear salt gradient (0.02 to 0.4 M NaCl) as described in the text at a flow rate of 45 ml/h. Fractions of 7 ml were collected. Myosin light chain kinase activity was measured in the presence of 0.2 mu CaC12 (O----O) or 2 mM EGTA (Cl- - -Cl) using bovine cardiac myosin whole light chain fraction (see “Experimental Procedures”) as substrate. Fractions indicated by the bar were pooled and dialyzed against Buffer D.

- 100

FIG. 2. Affinity chromatography of myosin light chain kinase on a column (2.5 x 25 cm) of Sepharose-calmodulin prepared as described under “Experimental Procedures.” Ex- cess protein was washed off the column with Buffer D, then nonspe- cifically bound protein was removed by washing with 40 mM Tris- HCl, pH 7.6, 10 mu 2-mercaptoethanol, 0.2 M NaCl, 0.05 mu CaC12. Specifically bound protein was eluted with 40 mM Tris-HCl, pH 7.6, 10 mM 2-mercaptoethanol, 0.2 M NaCl, 2 mM EGTA. The flow rate was 15 ml/h and 3-ml fractions were collected. Kinase activity was measured in the presence of 2 mu CaC12 (0- - -0) or 2 mu EGTA (O----O) using bovine cardiac myosin whole light chain fraction (see “Experimental Procedures”) as substrate. The arrows indicate the stages at which eluting buffers were changed. Fractions indicated by the bar were pooled and dialyzed against 40 mM Tris-HCl, pH 7.6,10 IIIM 2-mercaptoethanol.

at -20°C in this buffer containing 30% (v/v) glycerol. A summary of the purification is shown in Table I. Myosin

light chain kinase was purified approximately 1200-fold, with a yield of 16%, which represents 6 mg of protein obtained from 1 kg of bovine ventricular muscle. This indicates a cellular content of 37.5 mg/kg or 53.6 mg/liter of intracellular water (assumed at 70% of wet weight (38)). This corresponds to a concentration of 0.63 pM assuming M, = 85,000 for the kinase (see below).

Purity of the Isolated Myosin Light Chain Kinase and Molecular Weight Determination-When the purified kinase obtained after affinity chromatography was subjected to electrophoresis under nondenaturing conditions in 7.5% polyacrylamide gels, a single strongly staining band and three faint bands were observed (Fig. 3). In order to verify that the major band seen on electrophoresis, which represents ~70% of the stained protein, is indeed the myosin light chain kinase, a duplicate electrophoresis was performed, the gel was sliced, and the slices assayed for kinase activity as described under “Experimental Procedures” (Fig. 3). It is clear that the myosin light chain kinase activity co-migrates with the major protein band on the gel. Furthermore, when this major band was cut out of the nondenaturing gel and subjected to 0.1% dodecyl sulfate, 7.5% polyacrylamide gel electrophoresis, a single protein-staining band was observed with M, = 85,000 (Fig. 4).

When the purified kinase was subjected to gel filtration on a calibrated column of Sepharose 6B under nondenaturing conditions it exhibited a Stokes radius of 43.7 A assuming a native M, = 85,000. A globular rotein of M, = 85,000 would exhibit a Stokes radius of 36.0 w (39). These data, therefore, indicate that myosin light chain kinase is an elongated monomeric protein of M, = 85,000.

The Effect of Ca” and Calmodulin on Myosin Light Chain Kinase-Fig. 5 demonstrates that, in the presence of a fixed amount of Ca2+ (0.2 mM), increasing concentrations of bovine brain calmodulin activate myosin light chain kinase. When the data are plotted according to the method of Scatchard (40), as shown in Fig. 5, the apparent Kd of the kinase for cahnodulin is calculated to be 1.3 X lo-’ M, i.e. half-maximal


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


TABLE I Purification of bovine cardiac myosin light chain kinase

Purification step Total vol- Protein

ume concentra- Total protein” Activity/ml’ Tot$$ctiv- Activity/mg Yield Purification tion”

ml r&ml m9 units/ml units X IO-' units&q 90 -fold

Homogenate supernatant 4470 10.2 45,594 253.3 11.3 24.8 100 DEAE-cellulose batch (0.05-0.3 M 2580 4.1 10,578 223.3 5.76 54.5 51 2.2

NaCl eluate) 60% (NH&S04 385 7.1 2,734 1266.7 4.88 178.4 43 7.2 DEAE-Sephacel 234 1.2 280.8 696.7 1.63 580.6 14.4 23.4 Affinity chromatography on cal- 60 0.10 6.0 2980.0 1.788 29,800 15.8 1202

modulin-Sepharose

n Determined by the method of Bradford (37). * One unit is defined as that amount of enzyme which catalyzes the incorporation of 1 pmol of 32P into cardiac myosin regulatory light

chain/min under the standard conditions described under “Experimental Procedures.”

FIG. 3. Gel electrophoresis and elution profile of myosin light chain kinase. Duplicate samples of myosin light chain kinase (100 pg) were electrophoresed in 7.5% polyacrylamide gels; one gel was subsequently stained with Coomassie Brilliant Blue G250, destained, and scanned at 560 mn (-); the other gel was sliced into 2-mm slices, protein eluted as indicated under “Experimental Proce- dures,” and the eluates assayed for myosin light chain kinase activity (O- - -0).

stimulation is obtained for 91 fmol/assay, or 1.5 ng. These observations have enabled us to develop an assay for calmodulin, based on its activation of calmodulin-deficient bovine cardiac myosin light chain kinase, which is approximately 5 times more sensitive! and operationally easier than the classi- cal calmodulin-dependent CAMP phosphodiesterase assay (41). This assay has proved particularly useful for quantitating calmodulin in samples which are available in very small amounts. For example, we have used this assay to quantitate cahnodulin in chick embryonic muscle extracts during muscle differentiation?

It is also apparent from Fig. 5 that calmodulin, in the absence of Ca2+, has no stimulatory effect whatsoever on myosin light chain kinase. It appears, therefore, that the mechanism of activation of myosin light chain kinase by calmodulin is similar to the mechanism of calmodulin activation of CAMP phosphodiesterase (for review, see Ref. 9) and adenylate cyclase (42,43): activation is initiated by the binding of Ca2+ to cahnodulin; the resultant complex is capable of interacting with the kinase to form an active ternary complex composed of Ca*+, calmodulin, and myosin light, chain kinase. Evidence for a Ca’+-dependent interaction between cahnodulin and myosin light chain kinase is also provided by the fact that the kinase binds quantitatively to the cahnodulin-Seph- arose affinity column in the presence of Ca2+, and is eluted with EGTA (Fig. 2).

’ C. J. Le Peuch, C. Ferraz, M. P. Walsh, J. G. Demaille, and E. H. Fischer, submitted for publication.

+

dY@ 1

0 0.2 a4 0.6 0.8 1.0

Relatw Mobility

FIG. 4. Determination of the subunit molecular weight of myosin light chain kinase. Myosin light chain kinase (MUX) (10 pg) and various molecular weight marker proteins (10 pg of each) were subjected to electrophoresis on 7.5% polyacrylamide slab gels in the presence of 0.1% sodium dodecyl sulfate as described under “Experimental Procedures.” The marker proteins used were as follows: 1, 2, 3, 4 = (Y, (Y’, p, and y subunits, respectively, of glycogen phosphorylase b kinase; 5 = glycogen phosphorylase 6; 6 = bovine serum albumin; 7 = ovalbumin.

bmol ‘*PAnhl/kalmodulinkl~*

>lP

,/ A ‘\

\

\\\

0 H!-: . I .

0010 9 8 7 p calmodulin

FIG. 5. The effect of calmodulin on the activity of bovine cardiac myosin light chain kinase. The kinase (1.2 pg/ml) was incubated for 20 min at 30°C in the presence of 25 mM Tris-HCl, pH 7.6,8 mM magnesium acetate, 1.6 mu dithiothreitol, 2 mM [y-32P]ATP (51.5 cpm/pmol), 0.275 mg of partially purified regulatory light chain fraction of bovine cardiac myosin, and either 0.2 mM CaClz (o--O) or 2.4 mM EGTA (w). The amount of bovine brain calmodulin was varied as shown. Apparent Kd of calmodulin for myosin light chain kinase was determined from the reciprocal of the slope of the plot (A- - -A) activity in the presence of Ca*+/(calmodulin) versus activity in the presence of Ca2+. Linear regression was performed by the least squares method using a minicomputer Olivetti P6060. r* = 0.96, slope = -7.74 x 10’.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

12140 Cardiac Myosin Light Chain Kinase

, -5 0 5 10 15

'QATpj hM)-'

0 0.5 1 2

[ATPI imMi FIG. 6. Determination of the K,,, of myosin light chain kinase

for substrates. A, determination of the K, for ATP. Myosin light chain kinase (20 ag/ml) was incubated for 20 min at 30°C in the presence of 35 rnM Tris-HCl, pH 88 mM magnesium acetate, 1.6 mM dithiothreitol, 0.1 m&r CaCl*, 1.4 x 10m6 M calmodulin, 0.275 mg of partially purified regulatory light chain fraction of bovine cardiac myosin, and various concentrations of [y-32P]ATP (55 cpm/pmol), as indicated. The inset shows the Lineweaver-Burk representation of the data:linear regression was performed by the least squares method

Determination of the K,,, Values for Substrates-Fig. 6 illustrates the results of determination of the K, of the isolated myosin light chain kinase for ATP (Fig. 6A) and the regulatory

light chain of bovine cardiac myosin (Fig. 6B). The K, for ATP was calculated to be 175 pM, which is comparable to the values reported for the rabbit skeletal muscle enzyme (l&44). On the other hand, the dependence of reaction velocity on regulatory light chain concentration (shown in Fig. 6B) does not obey Michaelis-Menten kinetics, since the data do not provide a linear Lineweaver-Burk plot. Hence, a K,,, value for the regulatory light chain substrate cannot be calculated. However, a KO.s value of 21 pM is obtained from the data in Fig. 6B; this value corresponds to the K, values reported for the rabbit skeletal muscle enzyme (18, 44).

The Effect of Mg’+ on Kinase Actiuity-Fig. 7 shows the effect of increasing concentrations of Mg” ions on myosin light chain kinase activity assayed under optimal conditions in the presence of 1.5 mM ATP as described under “Experi- mental Procedures.” Maximal enzymatic activity occurs at 4.5 mM Mg’+, indicating that the enzyme requires free Mg2+ in addition to MgZf bound in the form of MgATP as substrate. Slight inhibition of kinase activity is observed at higher Mg2+ concentrations. This may reflect competitive binding of Mg2+ and Ca2+ to calmodulin at relatively high Mg’+ concentration (45), Mg”calmodulin being less capable of activating the kinase than the Ca2+-bound form of the protein. Alternatively, the kinase may exhibit another ion binding site which is inhibitory.

The Effect of Ionic Strength on Kinase Actiuity-The effect of increasing the ionic strength of the environment on the activity of myosin light chain kinase is shown in Fig. 8. Maximal activity is observed in the ionic strength range 0.1 to 0.25, above which the activity declines gradually. This decrease in activity at higher ionic strengths may be due to a decrease in the affinity of the kinase for calmodulin or for the regulatory light chain.

OptimumpH-The results of determination of the optimum pH of myosin light chain kinase are presented in Fig. 9. The enzyme exhibits an optimum pH of 8.1, the activity falling off quite rapidly with rise or fall in pH. The enzyme is essentially inactive below pH 5.5. Moreover, if the enzyme is maintained for a short period of time (30 to 60 min, for example) at pH 5

25 50 75 “25

I Regulatory Light Chain 1 'pM 1

using a minicomputer Olivetti P6060. r2 = 0.983, intercept on abscissa: -5.718. B, determination of the K,,, for the regulatory light chain (RX). Myosin light chain kinase (20 pg/ml) was incubated for 20 min at 30°C in the presence of 35 mM Tris-HCl, pH 8, 8 mu magnesium acetate, 1.6 mru dithiothreitol, 1 mM CaC12, 2.4 mM [y-32P]ATP (49 cpm/pmol), 1.5 x lo-” M calmodulin, and various concentrations of partially purified regulatory light chain fraction of bovine cardiac mvosin. as indicated. The interceut shows the Line- weaver-Burk representation of the data.

0 10 I

20 30 %I

lMg%M) FIG. 7. The effect of M8+ ions on myosin light chain kinase

activity. The kinase (20 ag/ml) was incubated for 30 min at 30°C in the presence of 35 mM Tris-HCl, pH 8, 1.6 mM dithiothreitol, 1.4 mM [y-32P]ATP (58 cpm/pmol), 0.2 mM CaC12, 0.825 mg of whole light chain fraction of myosin, and the indicated concentrations of Mg” ions.

0 0.5 1 1.5 2

IJ

FIG. 8. Effect of ionic strength on myosin light chain kinase. The kinase (20 ag/ml) was incubated for 30 min at 30°C in the presence of 35 mM Tris-HCl, pH 8,8 XTIM magnesium acetate, 1.6 mM dithiothreitol, 0.1 mu CaCL, 1.6 mM [y-32P]ATP (44.1 cpm/pmol), whole light chain fraction of bovine cardiac myosin (11.8 mg/ml), and various concentrations of KC1 to provide the ionic strengths indicated.

and then readjusted to pH 8, only about 50% of enzymatic activity is recovered.

Substrate Specificity-Various known phosphorylatable proteins, in addition to the regulatory light chain of myosin, were examined as possible substrates for the bovine cardiac myosin light chain kinase, namely casein, phosvitin, histone


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


6 I 8 9

PH

FIG. 9. Determination of the optimum pH of myosin light chain kinase. The kinase (20 pg/ml) was incubated for 30 min at 30°C in the presence of 1.6 mru dithiothreitol, 8 mu magnesium acetate, 1.6 mru ]$‘P]ATP (62.5 cpm/mol), 0.2 mM CaC12, 0.825 mg of whole light chain-fraction of myosin, and one of the following buffers (35 mM) depending on the pH desired: piperazine, pH 5.5 to 6.0; imidazole, pH 6.1 to 7.9; Tris, pH 8.0 to 8.5.

TABLE II

Substrate specificity of myosin light chain kinase P-acceptor activity”

Substrate +CDR + Ca’+ -cc;5 + +CDR -

Ca’+

% % % 96

P light chain of 100 11 0 0 bovine cardiac myosin

Casein 0.2 0 0 0 Phosvitin 0.1 0.4 0 0.6 Histone IIA 0.6 1.0 3.5 1.6 Glycogen phos- 2.5 0 0 0

phorylase b kinase

Glycogen phos- 0.9 0.4 0 0.1 phorylase b

Waterb 11.3 10.0 N.D.’ 17.5

n Myosin light chain kinase (20 pg/ml) was incubated for 20 min at 30°C in the presence of 35 mM Tris-HCl, pH 8, 8 mM magnesium acetate, 1.6 mM dithiothreitol, 0.8 mu [y-3ZP]ATP (52.0 cpm/pmol), 5 mg/ml of substrate, 0.2 mM CaC12, or 5 mu EGTA, with or without 2 x lo-’ M calmodulin, as indicated. “P incorporation was assayed as described under “Experimental Procedures.”

“ATPase activity was measured by the filter paper technique of Reimann and Umfleet (30) as described under “Experimental Proce- dures.”

’ Not determined.

IIA mixture, phosphorylase kinase, and phosphorylase b (Ta- ble II). The regulatory light chain of myosin is the only suitable substrate for the kinase of those proteins tested. The value of 11% obtained using the regulatory light chain as substrate in the presence of Ca2+ but without the addition of calmodulin reflects the presence of a trace of contaminating calmodulin in the regulatory light chain fraction (approximately 0.6 ppm w/w).

The last line in Table II indicates that the isolated myosin light chain kinase exhibits ATPase activity which is equivalent to approximately 10% of the kinase activity and which is, furthermore, independent of Ca”+ and calmodulin. Another protein kinase, CAMP-dependent protein kinase of bovine heart, has been shown to possess ATPase activity (46). It may

be that all protein kinases have the capacity to transfer the y-phosphoryl group from ATP to water as well as to protein substrates, albeit at a much lower rate.

We also compared the regulatory light chain of bovine cardiac myosin with the regulatory light chains of smooth muscle (turkey gizzard) and rabbit skeletal muscle as substrates of the bovine cardiac kinase. The enzyme was assayed at 3-min intervals up to 12 min, as described under “Experi- mental Procedures,” in the presence of 0.1 mM CaC12 and 6 X 1O-7 M calmodulin and an excess of the appropriate regulatory light chain (10 mg/ml). V,,, values obtained for 32P incorporation into the various regulatory light chains were as follows: bovine cardiac muscle = 79.2 pmol/min; smooth muscle = 81.4 pmol/min; rabbit skeletal muscle = 91.7 pmol/min. Thus, the rabbit skeletal muscle regulatory light chain appears to be a slightly better substrate than the cardiac or smooth muscle proteins.

Proteolysis of Myosin Light Chain Kinase-We considered the possibility that the low specific activity of the isolated myosin light chain kinase (see above) may be due to phosphorylation of a serine or threonine residue close to either ter- minus of the molecule; such phosphorylation sites are known to exist in, for example, glycogen synthase (47, 48), smooth muscle myosin light chain kinase (8), cGMP-dependent protein kinase (49), and many other proteins. It should be possible, therefore, to cleave off a small phosphorylated fragment by mild proteolysis. Such treatment may be expected to relieve the inhibition due to phosphorylation, resulting in an augmentation of kinase activity which would subsequently decline upon further digestion of the molecule. As shown in Fig. 10, mild proteolysis with trypsin or chymotrypsin does not induce such an augmentation of activity but rather results in a gradual loss of kinase activity. In each case, the residual kinase activity remains CaZc-dependent throughout the digestion, in contrast to the effect of mild proteolysis on calmodulin- dependent cyclic nucleotide phosphodiesterases which are rendered Ca2+- and calmodulin-independent (50, 51).

The lack of activation upon mild proteolysis may be due to loss of the phosphorylation site during purification of the enzyme. This possibility is suggested by the relative sizes of the cardiac (Mr = 85,000) and smooth muscle (Mr = 125,000

Time I mn 1

FIG. 10. Mild proteolysis of myosin light chain lcinase with trypsin and chymotrypsin. Myosin light chain kinase (0.1 mg/ml of 40 mM Tris-HCl, pH 7.6, 10 mM 2-mercaptoethanol) was incubated at 22°C with trypsin (0, 0) or chymotrypsin (0, n ) at a protease/ kinase ratio of 1:lOO. Aliquots (0.015 ml) of reaction mixtures were withdrawn at the indicated times and reactions quenched by addition to 0.055 ml of 35 mM Tris-HCl, pH 8, 8 mM magnesium acetate, 1.6 mM dithiothreitol, 2 mru [y-32P]ATP (52.4 cpm/pmol), 15 mg/ml of whole light chain fraction of bovine cardiac myosin, 2 pg/ml of soybean trypsin inhibitor (0,O) or 1.3 pg/ml of TPCK (0, n ), and 0.1 mu CaCL (0,O) or 5 mM EGTA (0, ml. Quenched reaction mixtures were incubated for 30 min at 30°C at which time the amount of 32P incorporated into the light chain substrate was determined as described under “Experimental Procedures.”


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


(8)) kinases. Furthermore, we have observed that the cardiac myosin light chain kinase is highly susceptible to proteolysis during purification: if attempts are made to further purify the enzyme after affinity chromatography by gel filtration on Sepharose 6B, kinase activity is completely lost, concomitant with proteolysis of the enzyme; similarly, if the gel filtration is effected before affinity chromatography, only -50% of the kinase activity loaded on the Sepharose 6B column is recovered, and this declines gradually to zero over a period of 3 to 4 days as a result of proteolysis.

Furthermore, we have demonstrated3 the presence of a protease associated with cardiac myofibrils which is capable of digesting smooth muscle (turkey gizzard) myosin light chain kinase.4 We have made several attempts to find extraction conditions which prevent proteolysis of cardiac myosin light chain kinase. For example, it is well known that human serum is a very effective anti-protease cocktail. When extraction of the myocardium was carried out with buffers made 1% (v/v) in human serum, no enhancement of myosin light chain kinase activity was observed in any of the fractions obtained from the tissue extract and the kinase was once more isolated as an M, = 85,000 species.

Phosphorylation of Myosin Light Chain Kinase-It has been shown recently (8) that smooth muscle myosin light chain kinase can be phosphorylated by the catalytic subunit of CAMP-dependent protein kinase with incorporation of 1 mol of phosphate/m01 of kinase and results in a 50% diminution of activity. To determine whether or not a similar regulatory mechanism exists in the cardiac enzyme, we attempted to phosphorylate the isolated cardiac myosin light chain kinase as described under “Experimental Procedures.” The reaction product was examined by 0.1% dodecyl sulfate, 7.5% polyacrylamide slab gel electrophoresis: one set of samples was subjected to autoradiography, while myosin light chain kinase bands and appropriate controls were cut out of duplicate electrophoresed samples, digested, and the radioactivity counted. The autoradiogram indicated no incorporation of 32P into the myosin light chain kinase while counting of the radioactivity in the gel slices revealed incorporation of an insignificant amount of “P (-6.8 x 10m4 mol/mol of kinase). Simultaneous treatment of smooth muscle (turkey gizzard) myosin light chain kinase under identical conditions, followed by autoradiography, revealed that the smooth muscle enzyme was phosphorylated by the catalytic subunit of CAMP-dependent protein kinase.4

The lack of phosphorylation of the cardiac myosin light chain kinase may be due to the absence of the phosphorylation site found in the smooth muscle enzyme, probably as a result of proteolysis during purification, as suggested above. On the other hand, acid-stable phosphate, apparently resistant to phosphatase activity, is still present in variable proportions in the isolated enzyme: the phosphate contents of two prepara- tions of the kinase were determined to be 3.03 and 1.35 mol of phosphate/m01 by the malachite green method after ashing of the protein as described under “Experimental Procedures.”

DISCUSSION

A kinase which catalyzes the phosphorylation of the M, = 19,000 regulatory light chain of bovine cardiac myosin has been isolated and purified from bovine myocardium. Studies of the localization of myosin light chain kinase in myocardium revealed that the enzyme could be quantitatively extracted by homogenization in an EDTA-containing buffer, no kinase

‘Data submitted with this manuscript for review; available to interested readers on request.

4 M. P. Walsh, J. C. Cavadore, B. Vallet, and J. G. Demaille, submitted for publication.

activity being detectable in the myofibrillar pellet, purified myofibrils, myosin, or native tropomyosin fractions. This observation is somewhat at variance with the methods of isola- tion of myosin light chain kinase from other tissues. Thus, for example, the smooth muscle kinase has been prepared from washed myofibrils (8) or native tropomyosin (6); the skeletal muscle enzyme has been isolated from washed myofibrils (18), myosin (52) or the soluble fraction obtained after homogenization of the muscle in EDTA-containing buffer (44).

The procedure described here for the purification of the bovine cardiac kinase exploits the biological affinity of the enzyme for calmodulin. The initial stages of purification, batchwise ion exchange and ion exchange chromatography in the presence of EDTA, serve to separate the apoenzyme and endogenous calmodulin, and also provide considerable purification of the apoenzyme. The major purification step, however, involves affinity chromatography on a column of calmodulin coupled to Sepharose 4B: the kinase is adsorbed quantitatively to the affinity column in the presence of Ca” ions and is eluted with an EGTA-containing buffer. This indicates a Ca’+-dependent interaction between the kinase and calmodulin similar to that known to occur in the case of various other myosin light chain kinases (8, 11, 18,27), as well as other cahnodulin-dependent enzymes such as phosphodiesterase and adenylate cyclase (for review, see Ref. 9). The purification procedure detailed above provides approximately a 1200-fold purification of the kinase (Table I).

Bovine cardiac myosin light chain kinase is absolutely dependent on Ca2+ and calmodulin, the active form of the enzyme presumably being a ternary complex composed of Ca*+, calmodulin, and the apoenzyme. The mechanism of Ca2+ activation of the kinase presumably occurs via a mechanism similar to that regulating the activities of phosphodiesterase and adenylate cyclase (for review, see Ref. 9): at low Ca2+ ion concentrations the kinase exists in dissociated form and is inactive. Activation is triggered by an increase in cytosolic Ca2+ concentration whereupon Ca2+ binds to calmodulin, thereby inducing a conformational change in the calciprotein and enabling it to interact with the kinase. The resultant ternary complex (the actual stoichiometry of which remains to be elucidated) represents the active form of the kinase. Inactivation presumably occurs via the reverse process as a result of a decrease in cytosolic Ca2+ concentration.

Bovine cardiac myosin light chain kinase exhibits a considerable degree of substrate specificity: various known phosphorylatable proteins (casein, phosvitin, histones, phosphorylase b, and phosphorylase kinase) proved not to be substrates of the isolated kinase. A similar observation has been reported for skeletal muscle (44) and human platelet (35) myosin light chain kinases; however, Waisman et al. (53) reported a broader specificity for the skeletal muscle enzyme which may perhaps be explained by contamination with another protein kinase. The relatively high K, for ATP (175 pM) of the isolated cardiac kinase, compared with the K, values for ATP of CAMP-dependent protein kinases, appears to be characteristic of calmodulin-dependent protein kinases: rabbit skeletal muscle myosin light chain kinase has been assigned K, values for ATP of 200 to 400 pM (44) and 280 pM

(18); glycogen phosphorylase b kinase (glycogen synthase kinase-II) also exhibits a high K, for ATP of 400 pM (54). Similarly, the K0.5 for the regulatory light chain substrate of the cardiac myosin light chain kinase (21 pM) is of the same order as the K, values reported for the rabbit skeletal muscle enzyme: 24 PM (18) and 40 to 50 pM (27).

From the yield of 16% and M, = 85,000, the concentration of myosin light chain kinase in the heart is calculated to be 0.63 PM. This is comparable to the concentration of the enzyme


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


in smooth muscle (0.57 pM) calculated from the data of Adel- stein et al. (8) assuming a yield of 50%. The cardiac content of the substrate, the regulatory light chain of myosin, is 100 PM based on a content of cardiac troponin of 50 pM (55). Together with the specific activity of the isolated kinase (30 nmol 32P/ min/mg of protein), these data indicate that the kinase could not catalyze the phosphorylation of the light chain substrate at a sufficient rate to constitute a mechanism of actin-myosin regulation on the time scale of a single muscle twitch, since the enzyme would catalyze the incorporation of only approximately 2 ~01 of phosphate/100 pmol of regulatory light chain/min. On the other hand, the very presence of a specific phosphorylation-dephosphorylation system regulating the phosphorylation state of the regulatory light chain suggests its importance in the regulation of actin-myosin interactions.

If phosphorylation of the regulatory light chain of myosin catalyzed by calmodulin-dependent myosin light chain kinase constitutes a mechanism of regulation of cardiac muscle contraction, then the isolated enzyme must be a modified form of the native enzyme, the latter exhibiting much higher specific activity, probably in the same range as reported for the smooth (27) and skeletal (28) muscle kinases. In this context, two possible modifications to the native enzyme appear likely to account for the low specific activity of the isolated kinase: phosphorylation or proteolysis of the native myosin light chain kinase. It has been demonstrated by Adelstein et al. (8) that the smooth muscle kinase phosphorylation by the catalytic subunit of CAMP-dependent protein kinase is accompanied by an approximately 2-fold decrease in specific activity. This decrease in catalytic activity is due to a decrease in the affinity of the kinase for calmodulin as a result of phosphorylation (56). The possibility that the low specific activity of the cardiac kinase is due to the phosphorylation state of the isolated enzyme seems unlikely: while the isolated kinase contains a variable amount of phosphate which survives throughout purification of the enzyme, the enzyme binds calmodulin with high affinity (Kd = 1.3 nM) and, furthermore, the kinase exhibits a low specific activity even in the presence of saturating amounts of calmodulin.

On the other hand, the possibility that the isolated kinase is a proteolytic fragment of the native enzyme appears much more likely. We have presented evidence that the kinase is highly susceptible to proteolysis due to endogenous proteases as well as trypsin and chymotrypsin. Furthermore, we have demonstrated that bovine myocardium contains a protease which digests turkey gizzard myosin light chain kinase. In addition, comparison of the tryptic peptides generated by limited digestion of the smooth muscle and cardiac kinases by the method of Cleveland et al. (57) reveals the presence of many common peptides, there being more peptides derived from the heavier smooth muscle enzyme.4 This indicates that the cardiac enzyme is homologous to a fragment of the smooth muscle kinase. These observations suggest that the isolated cardiac myosin light chain kinase is a proteolytic fragment of the native enzyme, the latter presumably exhibiting a much higher specific activity.

It is apparent from Table I that at no stage during the purification procedure is there a considerable loss of kinase activity, suggesting that the proteolysis occurs during the initial extraction and homogenization. This possibility is further suggested by comparing the specific activity of the kinase in the cardiac homogenate supernatant (25 pmol 32P/min/mg of protein, Table I) with that of the rabbit skeletal muscle enzyme in a tissue homogenate (25,000 pmol 32P/min/mg of protein (44)). Thus, it is apparent that proteolysis of cardiac myosin light chain kinase occurs between the death of the

animal and the completion of homogenization. Several attempts to prevent this proteolysis, e.g. the inclusion of anti- proteases in extraction buffers and mild extraction conditions, have failed. In order to minimize the time between killing the animal and completing homogenization of the myocardium myosin light chain kinase was purified from the heart of a dog killed immediately prior to homogenization of the myocardium; 0.1 mM phenylmethylsulfonyl fluoride and 1% (v/v) human serum were included in the extraction buffer. Once again, low specific activity kinase was obtained in the homogenate, and the enzyme obtained after affinity chromatography co-migrated with bovine cardiac myosin light chain kinase on 0.1% dodecyl sulfate, 7.5% polyacrylamide gel electrophoresis.

In contrast to the smooth muscle (turkey gizzard) myosin light chain kinase (8), the bovine cardiac enzyme is not phosphorylated by the catalytic subunit of CAMP-dependent protein kinase. The difference may be due to loss of the phosphorylation site in the cardiac enzyme as a result of the proteolysis which apparently occurs during extraction of the myocardium.

The physiological significance of a specific system regulating the phosphorylation and dephosphorylation of the regulatory light chain of myosin in cardiac muscle remains obscure. However, the existence of such a system under the control of Ca2+ ions indicates the importance of such phosphorylation- dephosphorylation in regulating actin-myosin interactions. It is known from the work from numerous laboratories that the troponin C-mediated effects induced by Ca2’ ions cannot account completely for the observed effects of Ca2+ activation in vertebrate striated muscle. For example, Ca2+ ions have been shown to affect the thick filament (16,58). Ca2+ ions also influence the interaction of the thick filament with unregu- lated actin (59). The regulatory light chain of skeletal muscle myosin has been found to bind Ca2+ both in situ (60, 61) and in the isolated state (62,63). Such Ca2’ binding to the isolated regulatory light chain is accompanied by a substantial change in secondary structure and overall shape of the molecule (64). Furthermore, phosphorylation of the regulatory light chain results in considerable diminution of affinity of the light chain for Ca2+ (64). These observations suggest that regulation of regulatory light chain phosphorylation by Ca2’ ions may manifest itself through subtle changes in the tertiary structure of the regulatory light chain which are transmitted to neigh- boring myosin subunits and thereby modulate the interaction between actin and myosin and the resultant muscular contraction.

REFERENCES

1. Ebashi, S., Endo, M., and Ohtsuki, I. (1969) Q. Reu. Biophys. 2, 351-384

2. Weber, A., and Murray, J. M. (1973) Physiol. Rev. 53,612-673 3. Aksov. M. 0.. Williams. D.. Sharkev. E. M.. and Hartshorne. D.

J. (1976) Blochem. Biophys. Res. ‘commuk 69, 35-41 4. Sobieszek, A. (1977) in The Biochemistry of Smooth Muscle

(Stephens, N. L., ed) pp. 413-443, University Park Press, Bal- timore

5. Chacko, S., Conti, M. A., and Adelstein, R. S. (1977) Proc. N&l. Acad. Sci. U. S. A. 74, 129-133

6. Dabrowska, R., Aromatorio, D., Sherry, J. M. F., and Hartshorne, D. J. (1977) Biochem. Biophys. Res Commun. 78, 1263-1272

7. Dabrowska. R.. Sherrv. J. M. F.. Aromatorio. D. K.. and Hart- shorne, D. J.‘(1978) “kochemisb-y 17,253-258

8. Adelstein, R. S., Conti, M. A., Hathaway, D. R., and Klee, C. B. (1978) J. Biol. Chem. 253,8347-8350

9. Wang, J. H. (1977) in Cyclic Nucleotides: Mechanisms of Action (Cramer, H., and Schultz, J., eds) pp. 37-56, John Wiley & Sons, Inc., New York

10. Morgan, M., Perry, S. V., and Ottaway, J. (1976) Biochem. J. 157, 687-697

11. Dabrowska, R., and Hartshorne, D. J. (1978) Biochem. Biophys.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


Res. Commun. 85,1352-1359 12. Hathaway, D. R., KIee, C. B., and Adelstein, R. S. (1979) Fed.

Proc. 38,2112a 13. Hathaway, D. R., and Adelstein, R. S. (1979) Proc. Natl. Acad.

Ski. U. S. A. 76, 1653-1657 14. Pires, E., Perry, S. V., and Thomas, M. A. W. (1974) FEBS Lett.

41,292-296 15. Frearson, N., and Perry, S. V. (1975) Biochem. J. 151,99-107 16. Morimoto, K., and Harrington, W. F. (1974) J. Mol. Biol. 88,693-

709 17. Lehman, W. (1978) Nature 274,80-81 18. Yazawa, M., and Yagi, K. (1978) J. Biochem. (Tokyo) 84, 1259-

1265 19. Glynn, I. M., and Chappell, J. B. (1964) Biochem. J. 90, 147-149 20. Teo, T. S., Wang, T. H., and Wang, J. H. (1973) J. Biol. Chem.

248,588-595 21. Walsh, M., and Stevens, F. C. (1978) Can. J. Biochem. 56, 420-

429 22. Fischer, E. H., and Krebs, E. G. (1958) J. Biol. Chem. 231.65-71 23. Cohen, .P. (1973) Eur. J. Biochem. 34, 1-14 24. Demaihe. J. G.. Peters. K. A.. and Fischer. E. H. (1977) Biochem

istry l&308&3086 ' 25. Peters, K. A., DemaiIle, J. G., and Fischer, E. H. (1977) Biochem-

istry 16,5691-5697 26. Perrie, W. T., and Perry, S. V. (1970) Biochem. J. 119, 31-38 27. Nairn, A. C., and Perry, S. V. (1979) Biochem. J. 179,89-97 28. Klee. C. B.. and Krinks. M. H. (1978) Biochemistry 17. 120-126 29. Co&in, J. D., and Reimann, E. ‘M. (i974) Methods Eniymol. 38,

287-290 54. 55.

30. Reimann, E. M., and Umfleet, R. A. (1978) Biochim. Biophys. Acta 523,516-521 56.

31. Ames, B. N. (1966) Methods Enzymol. 8, 115-118 32. Itaya, K., and Michio, U. (1966) Clin. Chim. Acta 14, 361-366 33. Laemmli, U. K. (1970) Nature 227.680-685 34. Fairbanks, G., Steck, T. L., and Wallach, D. F. H. (1971) Bio-

chemistry 10,2606-2617

57.

35. Daniel, J. L., and Adelstein, R. S. (1976) Biochemistry 15, 2370- 2377

58. 59. 60.

36. Sobieszek, A., and Bremel, R. D. (1975) Eur. J. Biochem. 55,49- 61. 60 62.

37. Bradford, M. M. (1976) Anal. Biochem. 72,248-254 38. Muntwyler, E. (1968) Water and Electrolyte Metabolism and 63.

Acid-Base Balance, The C. V. Mosby Co., p. 10, St. Louis, MO. 39. Andrews, P. (1970) Methods Biochem. Anal. 18, 1-53 40. Scatchard, G. (1949) Ann. N. Y. Acad. Sci. 51,660-672

64.

41.

42.

43.

44. 45.

46.

47. 48. 49.

50.

51.

52.

53.

Thompson, W. J., Brooker, G., and Appleman, M. M. (1974) Methods Enzymol. 38,205-212

Brostrom, C. O., Huang, Y.-C., Breckenridge, B. M., and Wolff, D. J. (1975) Proc. Natl. Acad. Sci. U. S. A. 72. 64-68

Cheung, W. Y., Bradham, L. S., Lynch, T. J., Lin, Y. M., and TaUant, E. A. (1975) Biochem. Biophys. Res. Commun. 66, 1055-1062

Pires, E. M. V., and Perry, S. V. (1977) Biochem. J. 167, 137-146 Wolff, D. J., Brostrom, M. A., and Brostrom, C. 0. (1977) in

Calcium Binding Proteins and Calcium Function (Wasser- man, R. H., Corradino, R. A., Carafoli, E., Kretsinger, R. H., MacLennan, D. H., and Siegel, F. L., eds) pp. 107-116, Elsevier/ North Holland, Amsterdam

Armstrong, R. N., Kondo, H., and Kaiser. E. T. (1979) Proc. Natl. Acad. Sci. U. S. A. 76,722-725

Huang, T.-S., and Krebs, E. G. (1979) FEBS Lett. 98,66-70 Rylatt, D. B., and Cohen, P. (1979) FEBS Lett. 98, 71-75 Lincoln, T. M., Flockhart, D. A., and Corbin, J. D. (1978) J. Biol.

Chem. 253,6002-6009 Sakai, T., Yamanaka, H., Tanaka, R., Makino, H., and Kasai, H.

(1977) Biochim. Biophys. Acta 483, 121-134 Moss, J., ManganieIIo, V. C., and Vaughan, M. (1978) Biochim.

Biophys. Acta 541,279-287 Barylko, B., Kuznicki, J., and Drabikowski, W. (1978) FEBSLett.

90,301-304 Waisman, D. M., Singh, T. J., and Wang, J. H. (1978) J. Biol.

Chem. 253,3387-3390 Nimmo, H. G., and Cohen, P. (1974) FEBS Lett. 47, 162-166 StuII, J. T., Blumenthal, D. K., de Lanerolle, P., High, C. W., and

Manning, D. R. (1978) Adu. Pharmacol. Z’her. 3,171-180 Adelstein, R. S., Conti, M. A., Hathaway, D. R., Eaton, C. R., and

Klee, C. B. (1979) Int. Congr. Biochem. Abstr. 137 Cleveland, D. W., Fischer, S. G., Kirschner, M. W., and Laemmli,

U. K. (1977) J. Biol. Chem. 252, 1102-1106 Haselgrove, J. G. (1975) J. Mol. Biol. 92, 113-143 Lehman. W. (1977) Biochem. J. 163.291-296 Beinfeld; M. C., Bryce, D. A., Kochavy, D., and Martonosi, A.

(1975) J. Biol. Chem. 250,6282-6287 Bagshaw, C. R. (1977) Biochemistry 16,59-67 Werber, M. M., Gaffin, S. L., and Oplatka, A. (1972) J. Mechan-

ochem. Cell Motil. 1,91-96 Kuwayama, H., and Yagi, L. (1977) J. Biochem. (Tokyo) 82,25-

33 Alexis, M. N., and Gratzer, W. B. (1978) Biochemistry 17, 2319-

2325


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

M P Walsh, B Vallet, F Autric and J G Demaillemyosin light chain kinase.

Purification and characterization of bovine cardiac calmodulin-dependent

1979, 254:12136-12144.J. Biol. Chem.

http://www.jbc.org/content/254/23/12136Access the most updated version of this article at

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here

http://www.jbc.org/content/254/23/12136.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/content/254/23/12136

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;254/23/12136&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/254/23/12136

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=254/23/12136&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/254/23/12136

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/254/23/12136.full.html#ref-list-1

http://www.jbc.org/

printed in u.s.a. purification and characterization of bovine · pdf filepurification and...

Documents