The Role of Calmodulin in the Structure and Regulation of Phosphorylase Kinase from Rabbit Skeletal Muscle

Transcription

1 Eur. J. Biochem. 100, (1979) The Role of Calmodulin in the Structure and Regulation of Phosphorylase Kinase from Rabbit Skeletal Muscle Shirish SHENOLIKAR, Patricia T. W. COHEN, Philip COHEN, Angus C. NAIRN, and S. Victor PERRY Department of Biochemistry, University of Dundee, and Department of Biochemistry, University of Birmingham (Received April 20, 1979) Phosphorylase kinase from rabbit skeletal muscle has been shown to possess the structure (a j y 6)4, where the b-subunit is identical to the calcium binding protein, termed calmodulin. The amount of calmodulin was found to be stoichiometric with the M., p and y subunits in all preparations of phosphorylase kinase, and was not dissociated from the enzyme even in the presence of 8 M urea, provided that calcium ions were present. The activity of phosphorylase kinase was increased by the addition of calmodulin to the assay, and half-maximal activation was observed at a molar ratio, calmodulin/phosphorylase kinase, of 20 : 1. At saturating concentrations of calmodulin, all preparations of phosphorylase kinase had a specific activity of 13.5 t- 1.0 U/mg at ph 8.2. In the absence of calmodulin, the specific activity ranged from 2-7 U/mg, so that the stimulation by calmodulin varied from 2-7-fold with different preparations of phosphorylase kinase. The molecular basis for this variability is discussed. The stimulation of the activity by calmodulin was prevented by the addition of troponin-i or the antipsychotic drug trifluoperazine, whereas these compounds had little effect on the calciumdependent activity in the absence of calmodulin. These results demonstrated that the activation by calmodulin was caused by the interaction of a second molecule of calmodulin with phosphorylase kinase. The existence of an additional calmodulin binding site was also indicated by the finding that phosphorylase kinase bound to calmodulin-sepharose in the presence of calcium ions. Experiments with ICR/IAn mice which lack muscle phosphorylase kinase activity and C3H/He-mg mice with normal activity, demonstrated that when muscle extracts were fractionated with ammonium sulphate, % of the calmodulin which precipitated at 0-35 % ammonium sulphate was bound to phosphorylase kinase. This information was used to show that at least 35 % of the calmodulin in low ionic strength EDTA extracts of rabbit skeletal muscle was bound to phosphorylase kinase. Muscle extracts from ICR/IAn mice and C3H/He-mg mice had identical myosin light-chain kinase activities, showing that there was not a generalized defect in calmodulin-dependent enzymes in ICR/IAn mice. The skeletal muscle extracts of ICR/IAn mice contained 60% of the calmodulin found in C3H/He-mg mice, and addition of calmodulin did not restore phosphorylase kinase activity to muscle extracts prepared from ICR/IAn mice. This indicated that the lack of phosphorylase kinase activity was not caused by an absence of the calmodulin molecule. The role of calmodulin in the regulation of muscle phosphorylase kinase activity is discussed. Phosphorylase kinase catalyses the phosphor- phosphorylation, catalysed by cyclic-amp-dependent ylation and activation of glycogen phosphorylase protein kinase [4]. and thereby stimulates the rate of glycogenolysis. Several years ago, phosphorylase kinase was The activity of phosphorylase kinase from rabbit found to contain three types of subunit [5,6] and to skeletal muscle is dependent on Ca2+ [I- 31, and can possess the structure (apy)4 where the molecular be stimulated a further fold at ph 6.8 by weights of the a, p and y subunits were , and respectively [6]. The a and p subunits Abbreviations. Cyclic AMP, adenosine 3': 5'-monophosphate; EGTA, ethyleneglycol bis(p-aminoethy1)-n,n,n',n'-tetraacetic were the components phosphorylated by acid. dependent protein kinase [6,7], but the subunit which

2 330 Calmodulin and Phosphorylase Kinase bound the Ca2+ ions that were essential for activity was not identified. Recently phosphorylase kinase was shown to contain a fourth component termed the 6 subunit [8]. This subunit is much smaller than the a, p and y subunits and due to its acidic nature, stains rather poorly with Coomassie blue. It was therefore missed for several years since it migrated as a faintly staining band with the bromophenol blue dye-front on the 5 "/, polyacrylamide gels previously used to resolve the M, fi and y subunits. The 6 subunit (molecular weight 17000) was demonstrated to be identical to a calcium-binding protein termed calmodulin [8] previously known as the modulator protein or calcium-dependent regulator protein. This protein was first identified as a factor which stimulated the activity of the high-k, cyclic nucleotide phosphodiesterase of brain tissue [9,10], but it has subsequently been implicated in the control of a number of cellular processes which are regulated by + Ca2. These include a calcium-stimulated adenylate cyclase in brain [ll], the calcium ATPase of the erythrocyte membrane which is involved in calcium transport [12,13], the depolymerisation of microtubules [14], myosin light-chain kinase in skeletal muscle [35,16] and smooth muscle [17], and NAD kinase in higher plants [18]. The identity of the 6 subunit and calmodulin was suggested by its electrophoretic migration, heat stability, elution behaviour on DEAE-cellulose and ultraviolet absorption spectrum, and confirmed by its amino acid composition and ability to reactivate calmodulin-dependent enzymes [8]. In this paper we describe further studies of the interaction of calmodulin with phosphorylase kinase. These results establish that calmodulin is an integral component of the enzyme, and demonstrate the existence of a second calmodulin-binding site on phosphorylase kinase. EXPERIMENTAL PROCEDURE Materials Trifluoperazine was a gift from Smith, Kline and French Laboratories Ltd (Welwyn Garden City, Hertfordshire, U.K.) ; cyanogen-bromide-activated Sepharose was purchased from Pharmacia (GB) Ltd (London W5, U.K.); Cyanogum 41 for polyacrylamide gel electrophoresis and bovine serum albumin (type V) were obtained from BDH Chemicals Ltd (Poole, Dorset, U.K.). Buffer A Buffer A comprised 50 mm sodium glycerophosphate/2.0 mm EDTAjl5 mm mercaptoethanol ph 7.0. Polyacrylamide Gel Electrophoresis Polyacrylamide gel electrophoresis was carried out at ph 7.2 on 7.5% or 10% disc gels in the presence of sodium dodecyl sulphate [6] or at ph 8.6 on 10% slab gels using Cyanogum 41 [19,20]. Protein Preparations All proteins were isolated from rabbit skeletal muscle unless stated otherwise. Phosphorylase b [21], phosphorylase kinase [6], myosin light-chain kinase [22], the P-light chain of myosin which was free of calmodulin [16] and troponin-i [23] were purified to homogeneity by standard procedures. Adenosine 5'-monophosphate was removed from phosphorylase b by treating the solution with Dowex AG1 resin [24]. The enzyme was then dialysed against buffer A containing 50% glycerol and stored in this buffer at - 20 "C. There was no loss of activity under these conditions for' at least one year. Phosphorylase kinase preparations were stored in buffer A at 0 "C at a concentration of 10 mg/ml. Calmodulin was isolated from purified phosphorylase kinase in the following manner. Phosphorylase kinase (5.0 mg/ml) in buffer A was heated at 90 "C for 10 min. The suspension was cooled in ice, centrifuged at x g for 10 min and the supernatant was decanted. Solid ammonium sulphate was added to bring the solution to 60 % saturation and after standing at 0 "C for 60 min, the suspension was centrifuged at x g for 30 min. The supernatant was decanted, dialysed against 5 mm Tris/HCl ph 7.5, concentrated by vacuum dialysis, and stored at - 20 "C at a concentration of 5 mg/ml. Calmodulin was also isolated from sheep brain by a modification of the procedure described by Yagi et al. [15] and was provided by Mr Zahi Damuni in this laboratory. Calmodulin isolated from skeletal muscle or brain was homogeneous by the criteria of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate or in the presence of 8 M urea. The two preparations comigrated in both electrophoretic systems. Myosin light-chain kinase was stored at - 20 "C at 0.15 mg/ml in 0.15 M sodium phosphate ph 7.0 containing 1.O mm dithiothreitol [22]. Enzyme Assays Phosphorylase Kinase. The standard assay comprised M Tris/0.125 M sodium glycerophosphate/0.6 mm CaC12 adjusted to 8.6 with HCl (0.02 ml), phosphorylase b (15 mg/ml) in buffer A (0.02 ml), phosphorylase kinase diluted in buffer A (0.01 ml) and ATP (18 mm)/magnesium acetate

3 S. Shenolikar, P. T. W. Cohen, P. Cohen, A. C. Nairn, and S. V. Perry 331 (60 mm) ph 7.0 (0.01 ml). The final ph of the reaction was 8.2. The reaction was carried out at 30 "C and initiated with the ATP-Mg solution. After 5 min, the reactions were terminated by the addition of 2.4 ml of ice-cold 0.1 M sodium maleate ph 6.5 containing 1.O mg bovine serum albumin/ml and 15 mm mercaptoethanol. Aliquots of this solution were then assayed for phosphorylase a according to Hedrick and Fischer [25]. One unit of phosphorylase kinase was that amount which catalysed the phosphorylation of 1.0 pmol of phosphorylase b per min at ph 8.2. For this calculation, the molecular weight of the phosphorylase subunit was taken as [26] and the absorbance index A:$ nm as 13.1 [27]. The specific activity of phosphorylase a was taken as 55 U/mg [27]. In some experiments the calcium chloride was replaced by 3.0 mm EGTA. Calmodulin, troponin-i and trifluoperazine, when added, were included in the Tris/sodium glycerophosphate buffer. The final concentration of phosphorylase kinase in the assay was 0.13 pg/ml unless stated otherwise. In the presence ofsaturating concentrations ofcalmodulin this quantity of enzyme converted 5% of the phosphorylase b (0.15 nmol) to phosphorylase a per min and the reaction was linear with time up to 5 min. Myosin Light Chain Kinase. This enzyme was assayed according to pires and Perry [22]. Determination of the Amount of Calmodulin Bound to Phosphorylase Kinase 1.0 ml of phosphorylase kinase at a concentration of 7.0 mg/ml in buffer A was pipetted into a pyrex test tube. The tube was placed in a boiling water bath for 10 min, and then cooled in ice. The suspension was vortexed, transferred to a microcentrifuge tube, centrifuged at x g for 2 min and the supernatant was collected. The pyrex tube was washed with two 0.5-ml portions of buffer A, and these solutions were used to extract the precipitate in the microcentrifuge tube. The supernatant obtained after each centrifugation of the microcentrifuge tube was combined with the first supernatant. Examination of the pooled supernatants by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate showed that the 6 subunit (calmodulin) was > 95 % pure as judged by densitometric analysis. The final precipitate in the microcentrifuge tube was dissolved in sodium dodecyl sulphate and did not contain detectable amounts of calmodulin when examined by polyacrylamide gel electrophoresis. The molar ratio 6/a/3-!6 was determined by measuring the concentration of phosphorylase kinase in the starting material and the volume and concentration of 6 subunit in the pooled supernatants. The molecular weights of the a, p, y and 6 subunits of phosphorylase kinase were taken as , , and respectively. The concentration of phosphorylase kinase was measured by the procedure of Lowry et al. [28] using bovine serum albumin (A:$nm = 6.50) as a standard. The values obtained either by this procedure or by using the absorbance index, A:$,,,,,, of 12.4 for phosphorylase kinase [6], were within 5% of each other. The concentration of the 6 subunit was also determined by the Lowry procedure, and the validity of this method was established by refractometric measurements in the analytical ultracentrifuge [6,29]. A solution of 6 subunit which yielded a protein concentration of 1.9 mg/ml by the Lowry procedure possessed 8.1 interference fringes, corresponding to a protein concentration of 2.0 mg/ml [6,29]. Estimation of the Proportion of Calmodulin in Skeletal Muscle that is Tightly Bound to Phosphorylase Kinase Preparation of Tissue Extracts and Fractionation with Ammonium Sulphate. All operations were carried out at 0-4 "C. The muscle was excised from the hind limbs and back of adult C3H/He-mg mice with normal phosphorylase kinase activity, ICR/IAn mice which have < 0.2% normal activity [30], and New Zealand White Rabbits. The muscle was chopped finely with scissors (or minced in the case of rabbit muscle) and homogenized in 2.5 vol. of 4.0 mm EDTAjl5 mm mercaptoethanol ph 7.0. The homogenate was centrifuged at x g for 30 min, and the supernatant was decanted (fraction 1) vol. of 90 % saturated ammonium sulphate ph 7.0 were added to bring the solution to 35 % saturation. The solution was allowed to stand for 30 min, centrifuged at xg for 30 min, and the supernatant was decanted (fraction 2). The precipitate was redissolved in buffer A (fraction 3) and fractions 2 and 3 were both dialysed against buffer A to remove ammonium sulphate. Phosphorylase kinase was precipitated quantitatively by the 35 % ammonium sulphate precipitation step. There was no detectable phosphorylase kinase activity in fraction 2, and the phosphorylase kinase activity in fraction 3 was at least 70% of that measured in fraction 1. Estimation of the Calmodulin Content offractions 1, 2 and 3. Calmodulin can be visualized by polyacrylamide gel electrophoresis of tissue extracts, since it is the fasted migrating band at ph 8.6 when gels are run in the presence of 10 mm EGTA and 8 M urea. If, however, the EGTA is replaced by Ca2+ prior to the addition of urea, the calmodulin band disappears, since complexes formed between calmodulin and the major calmodulin binding proteins in tissue extracts are not disrupted by 8 M urea in the presence of

4 332 Calmodulin and Phosphorylase Kinase + Ca2. This property greatly facilitates the identification of calmodulin in tissue extracts. Aliquots of fractions 1, 2 and 3 (2.0 ml) were made 10 mm in EGTA and 8 M in urea, and 0.1-ml portions were subjected to electrophoresis on polyacrylamide slab gels in the presence of 8 M urea. The gels were calibrated by running purified calmodulin at a range of concentrations as an internal standard. The gels were stained with Coomassie blue, destained, and the concentration of calmodulin in the various samples was estimated by eye. The values obtained from a number of different gels, for any given fraction, were averaged. Calmodulin-Sepharose Calmodulin-Sepharose was prepared by coupling calmodulin to cyanogen-bromide-activated Sepharose according to Klee and Krinks [31]. 1.0 mg of calmodium was used per g of dried Sepharose. RESULTS Molar Proportion of the 6 Subunit (Calmodulin) The 6 subunit was stoichiometric with the a, /l and 6 subunits, within experimental error, with all preparations of phosphorylase kinase tested. The molar ratio 6/xpy6 was 0.94 (standard deviation for seven preparations was 0.08). Interaction ofthe 6 Subunit with Phosphorylase Kinase in the Presence of 8 M Urea The results of these experiments are shown in Fig. 1. In the absence of Ca2+, the 6 subunit dissociated from phosphorylase kinase in the presence of 8 M urea, as judged by polyacrylamide gel electrophoresis. Under these conditions, the a, p and 6 subunits appeared as a dark mass of unresolved material near the origin. If, however, phosphorylase kinase was incubated with Ca M urea the 6 subunit was no longer visible. This indicated that the 6 subunit remained associated with phosphorylase kinase even in the presence of 8 M urea. Nevertheless, phosphorylase kinase was inactivated within 60 min fol- lowing exposure to 2 M urea in the presence or absence of + Ca2. Activation of Phosphoryluse Kinase by Calmodulin and Ca2 Calmodulin increased the activity of phosphorylated kinase when it was included in the standard assay. The degree of activation was variable, however, and ranged from two fold to seven fold with different Fig. 1. Electrophoresis of phosphorylase kinase on 10 polyacrylamide gels in the presence of 8 M urea. The gels were stained with Coomassie blue and the migration is from top to bottom. (A) Phosphorylase kinase in buffer A, made 10 mm in EGTA and then 8 M in urea (100 pg of protein was applied to the gel). (B) Phosphorylase kinase in buffer A, made 10 mm in CaCI2 and then 8 M in urea (100 pg of protein was applied to the gel). (C) Calmodulin purified from phosphorylase kinase as described under Experimental Procedure. This solution was made 10 mm in EGTA and 8 M in urea (5 pg of protein was applied to the gels). In this system, the c(, I / and y subunits appear as a dark mass of unresolved material near the origin. Some minor bands are also visible at the high loadings employed in these experiments preparations of phosphorylase kinase. The activation by calmodulin in nine preparations was 2.5, 4.3, 6.8, 5.0,4.4,4.0, 3.5,4.2 and 1.8-fold. The lower activation of 1.6-fold, reported in the initial identification of calmodulin as a subunit [8], was due to the fact that the concentration of calmodulin used in these previous assays was not saturating. The variable stimulation by calmodulin was due to the fact that different preparations of muscle phosphorylase kinase had different specific activities in the absence of calmodulin. In the presence of calmodulin all preparations had a specific activity of 13.5 f 1.0 U/mg. In the absence of calmodulin, the specific activites ranged from 2 to 7 U/mg. The molecular basis for this variability will be considered further in the discussion. In the standard assay at ph 8.2, which contained 0.13 vg phosphorylase kinase/ml, half-maximal stimulation was observed at pg calmodulin/ml for all preparations of phosphorylase kinase corresponding to a molar ratio calmodulin/phosphorylase kinase of 20: 1. The same activation constant was observed whether the calmodulin was isolated

5 S. Shenolikar, P. T. W. Cohen, P. Cohen, A. C. Nairn, and S. V. Perry 333 I calrnodulin rng/rnl Fig. 2. Effect of calmodulin on the activity of a phosphorylase kinase preparation. The assays were carried out at ph 8.2 in the presence of Ca2+ using 0.14 pg phosphorylase kinase/ml as described under Experimental Procedure, using calmodulin isolated from skeletal muscle I r a.c a troponin I mg/rnl v) I I from skeletal muscle or brain. The activation by calmodulin was completely dependent on calcium ions. If the calcium ions in the assay were replaced by EGTA, the activities of freshly prepared preparations of phosphorylase kinase at ph 8.2 were extremely low in the presence or absence of calmodulin (> 97 % inhibition). A preparation of phosphorylase kinase which was stimulated five fold by calmodulin is illustrated in Fig. 2. Evidence that the Activation by Culmodulin is Caused by the Binding of an Additional Molecule(s) to Phosphoryluse Kinase All preparations of phosphorylase kinase contained near stoichiometric quantities of calmodulin, irrespective of the degree to which the activity was stimulated by calmodulin in the assay. For example, the molar ratios 6/apy6 were 0.95 and 0.93 respectively for two preparations, although inclusion of calmodulin in the assay stimulated the activities of these preparations 4.4-fold and 1.%fold respectively. These observations showed that the variable stimulation by calmodulin was not caused by a variable content of the 6 subunit in the different preparations of phosphorylase kinase. When the concentration of phosphorylase kinase was varied from 0.02 to 0.2 pg/ml, an identical stimulation by calmodulin was observed at each enzyme concentration. This suggested that partial dissociation of the 6 subunit on dilution of the enzyme, was also not responsible for the stimulation of the activity by calmodulin. The following evidence demonstrated that the activation by calmodulin resulted from interaction of a second molecule(s) of this protein with phosphorylase kinase. g l,,, l l I c Q trifluoperazine ym Fig.3. Ejjhct of troponin-i (A) and trijluoperazine (B) on the activity of phosphorylase kinase at ph 8.2. The assays were carried out in the presence of Ca2+, 0.14 pg phosphorylase kinase/ml, and in the presence (closed circles) and absence (open circles) of calmodulin (6 pg/ml). In the absence of Ca2+ there was essentially no activity in the presence or absence of calmodulin. 100% activity corresponds to the activity in the presence of saturating concentrations of calmodulin. Under these conditions 5 % of the phosphorylase b (0.15 nmol) was converted to phosphorylase a per min Troponin-I forms a complex with calmodulin in the presence of Ca2+ [32] and the influence of troponin-i on the activity of phosphorylase kinase is illustrated in Fig.3. Troponin-I had no effect on the Ca2 +-dependent activity in the absence of calmodulin, but it inhibited the calmodulin stimulated activity. At a calmodulin concentration of 6 pg/ml (0.35 ym), 50 % inhibition required 150 pg troponin-i/ml(7.5 pm) at ph 8.2 (Fig. 3A). The antipsychotic drug trifluoperazine which binds to calmodulin specifically in the presence of Ca2+ [33], and which inhibits the calmodulin-stimulated cyclic nucleotide phosphodiesterase [34] was also tested for its ability to influence phosphorylase kinase activity. Trifluoperazine only slightly affected the Ca2+-dependent activity in the absence of calmodulin, the inhibition from preparation to preparation being /, at 0.1 mm trifluoperazine. In contrast, the calmodulin-stimulated activity was completely abol-

6 334 Calmodulin and Phosphorylase Kinase ished by trifluoperazine (Fig. 3). At a calmodulin concentration of 6 pg/ml, 50 % inhibition required 35 pm trifluoperazine at ph 8.2 (Fig. 3 B). These experiments show that trifluoperazine and troponin-i can differentiate between a more weakly bound molecule of calmodulin which stimulates the A LO fraction number Fig. 4. Chromatography of phosphorylase kinase on calmodulin- Sepharose at 4 C. Phosphorylase kinase (2.4 mg) was dialysed against 25 mm sodium glycerophosphatejl.o mm MgClzj15 mm mercaptoethanol ph 7.0. The solution was made 1.0 mm in CaClz and applied to calmodulin-sephdrose (7 x 1 cm) equilibrated in the dialysis buffer. The column was washed with this buffer, and at the positions denoted by the arrows, it was eluted with 50 mm sodium glycerophosphate/l.o mm MgC12/1.0 mm CaC12/15 mm mercaptoethanol (A) followed by 50 mm sodium glycerophosphate/2.0 mm EDTAjl5 mm mercaptoethanol ph 7.0 (B). Only traces of phosphorylase kinase emerged in the breakthrough, and the intermediate wash (A) eluted a minor impurity of mol wt which contaminates some preparations of phosphorylase kinase [6]. Phosphorylase kinase was eluted when the CaClz was replaced by EDTA, and % of the protein applied to the column was recovered in this fraction (B). activity, and the tightly bound molecule of calmodulin (the 6 subunit) which is likely to determine the dependence of phosphorylase kinase activity on Ca2+ (see Discussion). Further evidence for the presence of an additional calmodulin binding site on phosphorylase kinase was obtained by the use of calmodulin-sepharose. Phosphorylase kinase preparations which contained stoichiometric quantitites of calmodulin nevertheless bound to calmodulin-sepharose in the presence of Caz+, and could be eluted specifically if the Ca2+ in the buffer were replaced by EDTA (Fig.4). The subunit composition of the phosphorylase kinase eluted from calmodulin-sepharose was identical to the material applied to the column, as judged by polyacrylamide gel electrophoresis. Proportion qf Calmodulin to Phosphorylase Kinuse The concentration of in Skeletal Muscle Bound calmodulin was lower in muscle extracts prepared from ICRiIAn mice (which lack phosphorylase kinase activity) than it was in control C3H/He-mg mice (Fig. 5). Examination of a number of polyacrylamide gels from several different animals indicated that the muscle of ICR/IAn mice possessed 60 f 10% of the calmodulin found in C3H/He-mg mice. When the muscle extracts were fractionated from 0-35 % ammonium sulphate, the amount of calmodulin in the 35 % ammonium sulphate supernatants (which had no phosphorylase kinase activity) were very similar in ICR/IAn mice and C3H/He-mg mice Fig. 5. Electrophoresis ofmouse muscle extracts on 10 % polyacrylamide gels in the presence of 8 A4 urea. The gels were stained with Coomassie blue, and the migration is from top to bottom. Muscle extracts from ICR/IAn mice and C3H/He-mg mice were fractionated from 0-35 /, ammonium sulphate, as described under Experimental Procedure. The fractions were made 10 mm in EGTA, 8 M in urea and subjected to electrophoresis. Channel 1: muscle extracts prepared from ICR/IAn mice (IA) and C3H/He-mg mice (IB). 1 mg of extract was applied to each channel. Channel 2: 0-35% ammonium sulphate supernatant from ICRjIAn mice (2A) and C3HjHe-mg mice (2B). 0.4mg of protein was applied to each channel. Channel 3: 0-35% ammonium sulphate precipitate from ICR/IAn mice (3A) and C3HiHe-mg mice (3B). 0.2 mg of protein was applied to each channel. Channels 1A and 1 B are directly comparable since the volumes of each extract were identical and exactly 0.1-ml aliquots of each fraction were applied to the gel. Similarly, Channels 2A and 2B, and 3A and 3B, are comparable. However, Channels 1,2 and 3 were at different relative dilutions and cannot be compared to one another

7 S. Shenolikar, P. T. W. Cohen, P. Cohen, A. C. Nairn, and S. V. Perry 335 (Fig. 5). In contrast the 0-35 % ammonium sulphate precipitate prepared from C3H/He-mg mice (which contained 70 % of the phosphorylase kinase activity of the muscle extract) contained 10-fold more calmodulin than the corresponding fraction in ICR/IAn mice, as judged by gel electrophoresis (Fig. 5) and fold more calmodulin, as measured enzymatically by the ability of the fractions to activate phosphorylase kinase and myosin light-chain kinase. These experiments demonstrated that % of the calmodulin in the 0-35 % ammonium sulphate precipitate from C3H/He-mg mice was bound to phosphorylase kinase. When the different dilutions of the various fractions were taken into account, the calmodulin bound to phosphorylase kinase was found to represent approximately 40 % of the total amount in skeletal muscle. Similar experiments with rabbit skeletal muscle indicated that 35 % of the calmodulin in this tissue is bound to phosphorylase kinase. The amount of calmodulin in rabbit skeletal muscle that is bound to phosphorylase kinase was estimated to be 17 mg per 1000 g muscle by gel electrophoresis of the 0-35 % ammonium sulphate precipitate. This was in excellent agreement with the value of 19 mg/3000 g muscle predicted from the known content of phgsphorylase kinase in rabbit skeletal muscle [6,35], and suggested that gel electrophoresis is a reasonable method of quantitating calmodulin in protein samples. A calmodulin content of 50mg per 1OOOg rabbit skeletal muscle estimated in the present work, is slightly higher than the value of 30 mg per 1000 g rat skeletal muscle cited in Yagi et al. [15]. Calmodulin is often estimated by making use of its ability to activate cyclic nucleotide phosphodiesterase [36]. In such experiments, tissue extracts are heated at 100 "C for several minutes in the absence of Ca2+ to denature calmodulin binding proteins and thereby release calmodulin. However when either the muscle extract (fraction 1) or the 35% ammonium sulphate supernatant (fraction 3) were heated at 100 "C for 5-10 min and the denatured proteins were removed by centrifugation, no calmodulin could be detected either by gel electrophoresis or by testing the ability of the fractions to activate phosphorylase kinase. This demonstrated that the heat treatment had not released the calmodulin in these fractions from calmodulin binding proteins. This problem could be partially overcome by making fractions 1 and O M in NaCl prior to the heat treatment. When this was carried out, 'x of the calmodulin in the fractions was solubilized by the heat treatment, as judged by gel electrophoresis, or by the ability of the fractions to active phosphorylase kinase. The latter procedure confirmed that the 30 % ammonium sulphate supernatant of ICR/IAn mice and C3H/He-mg mice contained essentially identical amounts of calmodulin. In contrast to the muscle extract (fraction 1) and 35 % ammonium sulphate supernatant (fraction 3), the 35 % ammonium sulphate precipitate (fraction 2) behaved like purified phosphorylase kinase, in that most of the calmodulin was solubilized by heating the solution at 100 "C even in the absence of NaC1. Myosin Light-Chain Kinase and Phosphoryluse Kinase Activities in ICR/IAn Mice Myosin light-chain kinase activities in the skeletal muscle of C3H/He-mg and ICR/IAn mice were very similar. Muscle extracts of ICR/IAn mice showed 1.03 times the specific activity found in C3H/He-mg mice. If calmodulin was omitted from the assays, the activities were decreased by only 25 7:. This indicated that myosin light-chain kinase in muscle extracts was almost saturated with calmodulin in both strains of mice. Although ICR/IAn mice were reported to have only 0.2 % of normal phosphorylase kinase activity in skeletal muscle [30] calmodulin was not included in the assays. However, the phosphorylase kinase activity in the muscle extracts of ICR/IAn mice remained at 0.2% of the normal value, even when calmodulin was included in the assays at concentrations as high as 50 pg/ml. DISCUSSION The results presented in this paper establish that calmodulin is an integral component of phosphorylase kinase and that it is present in stoichiometric amounts with the a, fi and y subunits. This confirmed the previous findings, based on amino acid analysis, which indicated that the 6/apy6 ratio was at least 0.7 [8]. It is therefore concluded that phosphorylase kinase possesses the structure ( ~fiy6)~. The calmodulin is bound very tightly and is not dissociated by the addition of 8 M urea provided that Ca2+ are present. In this respect the situation resembles the interaction of troponin-c and troponin-i, which also form a complex that is resistant to dissociation by 8 M urea in the presence of Ca2+ [37]. The finding that calmodulin interacts with phosphorylase kinase in the presence of 8 M urea may facilitate the identification of the subunit with which this protein interacts. Kilimann and Heilmeyer [38] measured the binding of Ca2+ to phosphorylase kinase in 1.O mm sodium glycerophosphate buffer ph 6.8. Under these conditions each molecule of enzyme [(apy6)4, M, bound 12 Ca2+ with a dissociation constant of 1.5 x lo-* M and a further 4 Ca2+ with a dissociation constant of 6 x M. Since calmodulin binds 4 Ca2+ per mole [39], these binding studies (which were made before calmodulin was identified as a

8 336 Calmodulin and Phosphorylase Kinase subunit) strongly suggest that all the high affinity calcium binding sites on phosphorylase kinase are located on the calmodulin subunit. This in turn suggests that calmodulin is the component which confers + CaZ sensitivity to the phosphorylase kinase reaction. However Wolff et al. [39] reported that in 20mM Tris/HCl ph 7.4, calmodulin binds three CaZ+ per mol with an affinity of 2 x lo-' M and one CaZf with an affinity of 1 x M. It would therefore appear that the interaction of calmodulin with phosphorylase kinase causes Ca2+ to bind more tightly to calmodulin. Despite the fact that all preparations of phosphorylase kinase contained stoichiometric quantities of calmodulin, the addition of calmodulin to the assays produced a stimulation of the activity. Several lines of evidence suggested that this activation was caused by the binding of a second molecule of calmodulin to phosphorylase kinase. Thus the stimulation by calmodulin could be prevented by the inclusion of troponin-i or trifluoperazine in the assays, whereas these compounds had only a slight effect on the calcium dependent activity in the absence of calmodulin (Fig. 3). Furthermore, phosphorylase kinase preparations containing stoichiometric quantities of calmodulin could still bind to calmodulin-sepharose in the presence of Ca2+ (Fig. 4). The stimulation produced by the addition of calmodulin varied with the preparation of phosphorylase kinase and ranged from 1.%fold to 6.8-fold. The variation was entirely due to the fact that the specific activity of purified phosphorylase kinase in the absence of added calmodulin differed from preparation to preparation. The basis for this variation will be given in a separate publication (Philip Cohen, unpublished results). It appears to be caused by two factors. Firstly, the f calmodulin activity ratio at ph 8.2 depends on the state of phosphorylation of phosphorylase kinase. Secondly, phosphorylase kinase preparations with a low f calmodulin activity ratio ( ) revert spontaneously to forms with a higher activity ratio ( ) over a period of several days. The phenomenon may be caused by slight limited proteolysis. When muscle extracts were fractionated with ammonium sulphate, o/, of the calmodulin which precipitated at 0-35 %, ammonium sulphate was bound to phosphorylase kinase, and this information was used to show that % of the calmodulin in low ionic strength EDTA extracts of rabbit skeletal muscle or normal mouse skeletal muscle is bound to phosphorylase kinase. However only 70% of the phosphorylase kinase activity in the extracts was recovered in the 35 7; ammonium sulphate precipitate. Accordingly, 35% is probably a minimum estimate for the percentage of calmodulin that is bound to phosphorylase kinase in rabbit skeletal muscle. It should however be emphasized that phosphorylase kinase is only likely to represent a major calmodulin binding protein in fast-twitch anaerobic muscle fibres. In slow-twitch oxidative muscle fibres and in cardiac muscle, the level of phosphorylase kinase is fold lower, and it is still lower in non-muscle tissues [40,41]. Conversely calmodulin levels in non-muscle tissues are as high or higher than in muscle. ICR/IAn mice show < 0.2 % of normal phosphorylase kinase activity in their skeletal muscles, and the a, /l and y subunits are completely absent, as judged by protein chemical and immunological criteria [30, 421. We have proposed that the mice are defective in a control gene located on the X-chromosome required for the expression of the structural genes, at least one of which, the gene for the p subunit, is on an autosome [30]. These results were, however, performed before calmodulin was identified as a subunit of phosphorylase kinase, and this raised the question of the levels of calmodulin in the muscle of ICR/IAn mice. The finding that ICR/IAn mice contain calmodulin and possess normal myosin light-chain kinase activity, shows that the defect is not in calmodulin itself and that it does not affect other calmodulin-dependent enzymes. The failure of calmodulin to restore any phosphorylase kinase activity to the muscle extracts prepared from ICR/IAn mice is consistent with this conclusion. It is however of interest that the concentration of calmodulin in ICR/IAn mice is 60 of that found in normal mice, and that the decrease can be entirely accounted for by the calmodulin that is bound to phosphorylase kinase in C3H/He-mg mice (Fig. 5). The failure to detect significant amounts of calmodulin in the 0-35 o/, ammonium sulphate precipitate from ICR/IAn mice (Fig. 5) is consistent with the previous work which showed that the other subunits (a, p and y) are absent [30]. The reduced level of calmodulin in skeletal muscle from ICR/IAn mice further suggests that the protein is synthesized in sufficient amounts to saturate calmodulin-dependent enzymes, and that there exists a mechanism for preventing further accumulation of calmodulin beyond this concentration. This work wds supported by project grants from the Medical Research Council (to Philip Cohen and Patricia T. W. Cohen) and a programme grant (to S. V. Perry). We are grateful to CIBA Laboratories Ltd for providing A. C. Nairn with a studentship and research expenses. Philip Cohen was the recipient of a Wellcome Trust Special Fellowship (January December (1978). REFERENCES 1. Ozawa, E., Hosoi, K. & Ebashi, S. (1967) J. Biochem. (Tokyo) 61, Heilmeyer, L. M. 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9 S. Shenolikar, P. T. W. Cohen, P. Cohen, A. C. Nairn, and S. V. Perry Walsh, D. A,, Perkins, J. P., Brostrom, C. O., Ho, E. S.& Krebs, E. G. (1971) J. Bid. Chem. 246, Hayakawa, T., Perkins, J. P. & Krebs, E. G. (1973) Biochemistry, 12, Cohen, P. (1973) Eur. J. Biochem. 34, Hayakawa, T., Perkins, J. P. & Krebs, E. G. (1973) Biochemistry, 12, Cohen, P., Burchell, A., Foulkes, J. G., Cohen, P. T. W., Vanaman, T. C. & Nairn, A. C. (1978) FEBS Lett. 92, Kakiuchi, S., Yamazaki, R. & Nakajima, H. (1970) Proc. Jpn. Acad. 46, Cheung, W. Y. (1970) Biochem. Biophys. Res. Commun. 38, Brostrom, C. O., Huang, Y. C., Breckenridge, B. M. & Wolff, D. J. (1975) Proc. Nut1 Acad. Sci. U.S.A. 72, Gopinath, R. M. & Vicenzi, F. F. (1977) Biochem. Biophys. Res. Commun. 77, Jarrett, H. W. & Penniston, J. J. (1977) Biochem. Biophys. Res. Commun. 77, Marcum, J. M., Dedman, J. R., Brinkley, B. R. & Means, A. R. (1978) Proc. Nut1 Acad. Sci. U.S.A. 75, Yagi, K., Yazawa, M., Kakiuchi, S., Oshima, M. & Uenishi, K. 16. Nairn, A. C. & Perry, S. V. (1979) Biochem. J. 179, Drabowski, R., Aromatoril, 0. D., Sherry, J. M. F. & Hartshorne, D. J. (1977) Biochem. Biophys. Res. Commun. 78, Anderson, J. M. & Cormier, M. J. (1978) Biochem. Biophys (1978) J. Biol. Chem. 253, Res. Commun. 84, Perrie, W. T. & Perry, S. V. (1970) Biochem. J. 119, Perrie, W. T., Smillie, L. B. & Perry, S. V. (1973) Biochem. J. 135, Fischer, E. H. &Krebs, E. G. (1958)J. Bid. Chem. 231, Pires, E. & Perry, S. V. (1977) Biochem. J. 167, Perry, S. V. &Cole, H. A. (1974) Biochem. J. 141, Gilboe, D. P., Larson, K. L. & Nuttall, F. Q. (1972) Anal. Biochem. 47, Hedrick, J. L. & Fischer, E. H. (1965) Biochemistry, 4, Titani, K., Koide, A,, Hermann, J., Ericsson, L. H., Kumor, S., Wade, R. D., Walsh, K. A., Neurath H. & Fischer, E. H. (1977) Proc. Nut1 Acad. Sci. U.S.A. 74, Cohen, P., Duewer, T. & Fischer, E. 1. (1971) Biochemistry, 10, Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193, Babel, J. & Stellwagen, E. (1969) Anal. Biochem. 28, Cohen, P. T. W., Burchell, A. & Cohen, P. (1976) Eur. J. Biochem. 66, Klee, C. B. & Krinks, M. H. (1978) Biochemistry, 17, Amphlett, G. W., Vanaman, T. C. & Perry, S. V. (1976) FEBS Lett. 72, Levin, R. M. & Weiss, B. (1977) Molec. Pharmacol. 13, Levin, R. M. & Weiss, B. (1976) Molec. Pharmacol. 12, Cohen, P. (1978) Curr. Top. Cell. Regul. 14, Singer, A. L., Dunn, A. & Appleman, M. M. (1978) Arch. Biochem. Biophys. 187, Head, J. F. & Perry, S. V. (1974) Biochem. J. 137, Kilimann, M. & Heilmeyer, L. M. G. (1977) Eur. J. Biochem. 73, Wolff, D. J., Poirier, P. G., Brostrom, C. 0. & Brostrom, M. A. (1977) J. Bid. Chern. 252, Burchell, A., Cohen, P. T. W. & Cohen, P. (1976) FEBS Lett. 67, Burchell, A,, Foulkes, J. G., Cohen, P. T. W., Condon, G. D. & Cohen, P. (1978) FEBS Lett. 92, Daegelen-Proux, D., Alexandre, Y. & Dreyfus, J. C. (1978) Eur. J. Biochem. 90, S. Shenolikar, P. T. W. Cohen, and P. Cohen*, Department of Biochemistry, University of Dundee School of Medicine, Medical Sciences Institute, Dundee, Great Britain, DDI 4HN A. C. Nairn and S. V. Perry, Department of Biochemistry, University of Birmingham, P.0. Box 363, Birmingham, Great Britain, B15 2TT * To whom correspondence should be addressed. Note Added in Proof: It has recently been found that troponin C can substitute for the second molecule of calmodulin in the activation of phosphorylase kinase [Cohen, P., Picton, C. & Klee, C. B. (1979) FEBS Lett. 104, 25-30].