Catalysis of the Phosphorylase Kinase Activation Reaction*

TIE JOURNAL OF BIO~~ICAL CHEMISTRY

Vol. 246, No. 7, Issue of April 10, pp. 1968-1976, 1971

hinted in U.S.A.

Catalysis of the Phosphorylase Kinase Activation Reaction*

(Received for publication, August 28, 1970)

DONAL A. WALSH,$ ,§ JOHN P. PERKINS,~ CHARLES 0. BROSTROM,[[ ESTER S. Ho, AND EDWIN G. KREBS$

From the Department of Biological Chemistry, School of Medicine, University of California, Davis, California 95616, and the Department of Biochemistry, University of Washington, Seattle, Washington 98105

SUMMARY

Activation of skeletal muscle phosphorylase kinase, which occurs when the purified enzyme is incubated with ATP,

Mg’+, and cyclic adenosine 3’,5’-monophosphate (cyclic AMP), has been shown to involve two catalytic components. One of the catalysts is a cyclic AMP-dependent protein kinase and the other is phosphorylase kinase itself. The process was elucidated in part through a study of the effects of inhibitors on the activation process. One of the inhibitors that was used is a heat-stable protein from skeletal muscle which was shown to block the cyclic AMP-dependent protein kinase component of the reaction, and the other inhibitor was ethylene glycol bis(/I-aminoethyl ether)-IV, N-tetraace- tate which inhibits phosphorylase kinase. Clarification of the mechanisms operating at the phosphorylase kinase activation step makes it possible to describe more precisely how glycogenolysis is regulated by hormones, and for the first time to identify a specific site of action of cyclic AMP in a physiological process.

Phosphorylase kinase (ATP : phosphorylase phosphotransferase, EC 2.7.1.38, the enzyme which catalyzes the conversion of phosphorylase b to phosphorylase a, exists in phosphorylated and nonphosphorylated forms that differ in their catalytic properties (1, 2). The nonphosphorylated form, referred to as nonactivated phosphorylase kinase, is essentially inactive below pH 7 but manifests appreciable activity at higher pH values. Phosphorylated phosphorylase kinase is an activated form of the enzyme which is much more active than the nonactivated form below pH 7 and slightly more active than the latter form at high pH values (3, 4). An increase in the ratio of enzyme activity at pH 6.8 to activity at pH 8.2 has

* This study was supported by a grant from the Muscular Dys- trophy Association of America and by Grant AM12842 from the United States Public Health Service.

f Present address, Department of Biological Chemistry, School of Medicine, University of California, Davis, California 95616.

5 Recipient of Postdoctoral Fellowship of the American Cancer Society.

lj Postdoctoral Fellow of the United States Public Health Service. Present address, Department of Pharmacology, Uni- versity of Colorado, Denver, Colorado 80220.

/I Postdoctoral Fellow of the United States Public Health Service.

been used as an indication of phosphorylase kinase activation which occurs in wivo under conditions in which tissue levels of cyclic adenosine 3’, 5’-monophosphate are elevated (5-9). Non- activated phosphorylase kinase can be converted to the activated form in vitro by incubating the purified enzyme with MgZt--ATP (2). This reaction is accelerated by cyclic AMP1 but does not show an absolute requirement for this nucleotide. The covalently bound phosphate in activated phosphorylase kinase is not transferable to phosphorylase b (2). Thus phosphorylation and dephosphorylation of the kinase is not simply a step in the phosphotransferase function of the enzyme but is analogous to the reversible phosphorylation of phosphorylase itself that is manifested by an altered catalytic potential of the enzyme. A phosphatase catalyzing the dephosphorylation and inactivation of phosphorylase kinase has been described (10) ; this phosphatase is not, identical with phosphorylase phosphatase.

In the absence of cyclic AMP, the phosphorylase kinase activation reaction is slow, and the reaction rate increases with time. This behavior has been interpreted as indicating that the reaction is autocatalytic, i.e. that phosphorylase kinase catalyzes its own activation (2). In the presence of cyclic AMP, the activation reaction is more rapid, and under these conditions no lag phase is apparent. A possible explanation that was considered for these phenomena is that, two catalysts for the reaction might be acting. One of these would be phosphorylase kinase, acting independently of cyclic AMP, and the other might be a phosphorylase kinase-activating enzyme, i.e. a phosphorylase kinase kinase, which requires the cyclic nucleotide. The two mechanisms are given in Equations 1 and 2. Indirect support

Nonactivated phosphorylase kinase

activated phosphorylase kinase activated

+ phosphorylase 0) ATP, Mg2+ kinase

phosphorylase ki-

Nonactivated phosphorylase kinase

nase-activating enzyme activated

+ phosphorylase (2) ATP, Mg2+, cyclic kinase

AMP

for the concept that a cyclic AMP-requiring enzyme involved in activation might be a protein other than phosphorylase kinase itself comes from the finding that preparations of the latter en-

1 The abbreviations used are: cyclic AMP, cyclic adenosine 3’,5’-monophosphate; EGTA, ethylene glycol his@-aminoethyl ether)-N ,N’-tetraacetic acid; KAF, phosphorylase kinase-activating factor (22).

1968

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Issue of April 10, 1971 Walsh, Perkins, Brostrom, Ho, and Krebs 1969

zyme bind only traces of cyclic AMP (2) ; this would be expected if a separate cyclic AMP-requiring activating enzyme were present as a minor contaminant in the phosphorylase kinase preparation.

The present study was undertaken in the hope of resolving the uncertainties existing concerning catalysis of the phosphorylase kinase activation reaction. Such an investigation was deemed important both because of the importance of phosphorylase kinase activation per se in the regulation of glycogenolysis, and also because this system appeared to offer an oppor- tunity for determining the mechanism of action of cyclic AMP at the molecular level. An outgrowth of the study was the discovery that crude skeletal muscle extracts are a rich source of a cyclic AMP-dependent enzyme answering all the requirements for the phosphorylase kinase-activating enzyme postulated in Equation 2. In the communication announcing the discovery of this enzyme (II), it was referred to as a “cyclic AMP-dependent protein kinase,” rather than a phosphorylase kinase-activating enzyme, since it was shown to be capable of catalyzing the phosphorylation of casein and protamine as well as phosphorylase kinase, and it was anticipated that the enzyme might have a wider spectrum of function than would be implied either by the terms phosphorylase kinase-activating enzyme or phosphorylase kinase kinase.

Accompanying this paper, portions of which have been presented in preliminary reports at meetings (12-15), are several related papers. Of these papers, one (16) is concerned with the requirement of phosphorylase kinase for Cat+ ions, a property of importance in considering the phosphorylase kinase activation reaction as well as the conversion of phosphorylase b to phosphorylase a. Another paper (17) describes the purification and properties of a heat-stable protein inhibitor of cyclic AMP- dependent protein kinase, and two other papers (18, 19) extend the study of cyclic AMP-dependent protein kinases in skeletal muscle and the use of this enzyme in assaying for cyclic AMP.

EXPERIMENTAL PROCEDURES

Heat-stable Protein Inhibitor of Phosphorylase Kinase Activation Reaction-This inhibitor was purified as described in an accompanying paper (17). The method for purifying the inhibitor was in the course of development at the same time that the present study was being carried out, and some of the experiments described herein were performed utilizing intermediate fractions rather than the extensively purified material. In the experiment of Table II and Fig. 6 the inhibitor preparation used had not been subjected to the DEAE-cellulose chromatography step. In the experiment of Fig. 5, the inhibitor obtained at Step 2 of the procedure (17) was dialyzed for 2 hours against 0.05 M glycerol-P buffer, pH 6.8, containing 2 mM EDTA and used without further purification. The units of activity of inhibitor are defined in the accompanying manuscript (17).

Other Mater&&-The preparation and assay of phosphorylase kinase were performed as described earlier (1, 2). The purification of the cyclic AMP-dependent protein kinase was carried out as described previously (11) with the exception that the final chromatography step on Sephadex G-200 was omitted. Y-~~P-AI’P was prepared by a modification of the method of Glynn and Chappell (20). The incubation, containing 60 pmoles of ATP in 10 ml (20), was terminated by the addition of 1 ml of 1 N hydrochloric acid followed by the addition

of 250 mg of Norite (Pfansteil). This suspension was filtered through two 0.45-p Millipore filters and the charcoal washed with 20 ml of distilled water. Y-~~P-ATP was eluted from the Norite with 30 ml of 0.15 M ammonium hydroxide in 50% ethanol. The latter two compounds were removed from the nucleotide preparation by rotary evaporation. The preparation had a final specific activity of 6.0 x IO8 cpm per ,umole and contained less than 1% contamination by ADP or 32Pi. All other chemi- cals were obtained as described elsewhere (I, 2).

Treatment of Crude Skeletal Muscle Extract to Remove Phos- phoylase Kinase and Cyclic AMP-The crude fraction from rabbit skeletal muscle in which the phosphorylase kinase-activating enzyme was first detected was prepared as follows. The neutralized supernatant solution from the pH 6.1 acid precipitation step of a standard phosphorylase kinase purification (2) was dialyzed for 2 hours against 0.05 M glycerol-P buffer, pH 6.8, containing 0.002 M EDTA. This solution was then passed through a column of Norite (3 x 22 mm) equilibrated with the same buffer and the resultant protein fraction was collected.

Other Methods--The content of 32P bound to protein in enzy- matic phosphorylation reactions was determined by the following protocol. All steps were performed at 0’. To aliquots (0.1 ml) containing azP-protein and other reactants were added 0.2 ml of bovine serum albumin (6.25 mg per ml) and 1.5 ml of 6.7% trichloracetic acid, w/v. The precipitated protein suspension was kept at 0” for 10 min and then centrifuged in a clinical centrifuge for 10 min. The supernatant solution was discarded, and the protein precipitate was dissolved in 1 ml of 0.1 N NaOH and then rapidly reprecipitated by the addition of 1 ml of 10% trichloracetic acid‘(w/v). Complete precipitation was ensured by allow- ing the suspension to stand for 10 min before centrifugation. The protein precipitate obtained by centrifugation was washed twice in 5% trichloracetic acid, dissolved in 98% formic acid, and then counted in a dioxane-naphthalene based scintillant.

RESULTS

E$ect of Varying Magnesium and ATP on Phosphoylase Kin- use Activation Reaction-Previous studies of the phosphorylase kinase activation reaction carried out at relatively high concentrations of Mg* and ATP showed that when [ATP] exceeded [Mg*] the activation rates fell off sharply (1). This was found to be true whether or not cyclic AMP was included in the activation reaction mixtures. Mg*+ ions appeared to have a stimulatory effect on the reaction in addition to being essential for the formation of the substrate, presumably the Mg*-ATP complex. The stimulatory effect of magnesium was more prominent without cyclic AMP than in the presence of this nucleotide. Huijing and Larner in an analysis of these data (21) pointed out that the behavior was compatible with a mechanism for phosphorylase kinase activation involving two separate enzymes as catalysts for the reaction.

Further studies on the effects of varying Mgz+ and ATP concentrations on phosphorylase kinase activation have now shown that with low levels of these components2 the reaction becomes completely dependent on cyclic AMP (Fig. lA, Curves I and 11).

* Throughout the paper the concentration of Mgz+ given for this system is that added to the reaction; the total amount of Mg*+ either free or bound to ATP will be less than this due to the presence in the reaction mixture of EDTA which is added by addition of the phosphorylase kinase preparation.


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1970 Phosphoqdase Kinase Activation Vol. 246, No. 7

TIME (MINUTES)

FIG. 1. Effect of Mgz+ and ATP concentrations on the activation (A) and phosphorylation (B) of phosphorylase kinase. Non- activated phosphorylase kinase (0.275 mg) was incubated at 30” in a reaction mixture containing: glycerol-P buffer, pH 6.8, 7 pmoles; &mercaptoethanol, 20 pmoles; with either magnesium acetate, 6 pmoles, and Y-~~P-ATP, 2 /Imoles (Curves ZZZ and IV); or magnesium acetate, 0.27 pmole, and +2P-ATP, 0.09 pmole (Curves Z and ZZ) ; in the presence (Curves ZZ and IV) or absence (Curves Z and III) of 1 mpmole of cyclic AMP in a total volume of 1 ml. The presence of EDTA in the phosphorylase kinase preparation resulted in a concentration of 2 X lo-” M of this chelator in the incubation mixture.2 At intervals aliquots were either diluted in cold (0’) 10 mM glycerol-P buffer containing 45 EIM fi-mercaptoethanol and assayed for phosphorylase kinase activity at pH 6.8 (A) or used for the determination of r*P incorporation into protein as described in the text (Z?).

At higher levels of Mg2+ and ATP, cyclic AMP still stimulates the reaction but activation is also marked in the absence of this nucleotide (Curves III and ZV). The effect of varying these reaction components on the uptake of 32P by phosphorylase kinase (Fig. 1B) more or less parallels the results obtained in the activation studies, but a direct correlation between activation and phosphorylation is not observed. This is particularly striking if a comparison is made of the strong effect of cyclic AMP on enzyme activity (Curves I and II, Fig. 1A) to its effect on phosphorylation of the kinase (Curves Z and II, Fig. 1B) at low levels of Mg* and ATP. The lack of close correlation between phosphorylase kinase activation and phosphorylation has been noted before (2). In general, the results of the experiment of Fig. 1 would support the idea of the existence of two catalysts for the reaction. One of these would be an enzyme effective at low levels of Mg* and ATP, but requiring cyclic AMP, and the other would be an enzyme requiring higher concentrations of Mgz+ and ATP and independent of cyclic AMP!

3 A complete study of the kinetics of the phosphorylase kinase activation reaction over a wide range of MgZ+ and ATP concentrations and with varying ratios of these components has not been carried out. Thus, it cannot be said with certainty how much of the effect seen in Fig. 1 is due to lowering the concentration of the Mgzf-ATP complex from 2 mM to about 0.1 mM or from lowering free [Mg”f]. Judging from the effects of magnesium seen in the earlier study (l), it is probable that the latter may be a major factor. It should be noted, however, that the K, for Mgz+.ATP for phosphorylase kinase in the phosphorylase 6 to phosphorylase a reaction (1) is considerably higher than the K, for Mgz+.ATP for the cyclic AMP-dependent protein kinase (18), so this may also be an important factor.

Effect of Calcium and EGTA on Phosplwrylase Kinase Activa- tion Reaction-Phosphorylase kinase is strongly inhibited by chelating agents but its activity can be restored by addition of Ca* (22). The specificity for calcium in the restoration of activity, together with the finding that EGTA is a much stronger inhibitor of the enzyme than EDTA, led to the conclusion that phosphorylase kinase is a specific Ca#-requiring enzyme. It has been estimated (16) that a Ca* concentration of the order of 10e7 to 10e6 M is sufficient for half-maximal activity of phosphorylase kinase. In the assay system for determining phosphorylase kinase activity, i.e. the phosphorylase b to phosphorylase a conversion reaction, the amount of contaminating CaZf contributed by the various reagents that are used is sufficient to saturate the enzyme with respect to this metal, so that no effect of added calcium salts is noted unless extensive precautions are used to eliminate this ion (16). In contrast, it has been noted that addition of small amounts of Ca* to phosphorylase kinase activation reactions4 usually does affect this process. A typical experiment is illustrated in Fig. 2. Here added Ca2f increased the rate of activation in the presence or absence of cyclic AMP, particularly under the latter condition in which the reaction ex- hibited a prolonged lag period and kinet,ics characteristic of autoactivation. At times there is no effect of added Caz+ on the activation reaction which is probably due to an uncontrolled increase in trace levels of the metal in reaction mixtures. Con- sistent with this inferred level of contamination by Ca* is the observation that the base-line activation of phosphorylase kinase is considerably increased under these latter conditions.

The chelating agent, EGTA, is effective in nullifying the stimulatory effect of added Ca* on the phosphorylase kinase activation reaction as shown in the experiment of Fig. 3 carried out in the absence of cyclic AMP. In those instances in which appreciable cyclic AMP-independent activation of phosphorylase kinase occurs without added Ca+, EGTA is also an effective inhibitor (Experiment I of Table I). In phosphorylase kinase activation reactions carried out in the presence of cyclic AMP, EGTA is only partially effective in blocking the reaction (Experiment II of Table I).

Effect of Heat-stable Protein Inhibitor of Phosphorylase Kinme Activation Reaction-Extracts of rabbit skeletal muscle contain a heat-stable protein that interferes with assays for cyclic AMP carried out using the phosphorylase kinase activation system (23,24). This inhibitor has been purified as described in accompanying paper (17), and was thus made available for use in the present study either as the most purified fraction or at earlier stages of the purification procedure. The addition of the inhibitor to the phosphorylase kinase activation reaction was found to inhibit only that component of activation that requires cyclic AMP (Table II). In activation reactions carried out at low concentration of magnesium and ATP this effect is manifested by an almost complete inhibition of activation. In contrast, in the activation reactions occurring at high magnesium-ATP concentra-

4 The reaction mixtures commonly used in studying the phosphorylase kinase activation reaction differ from those used in measuring phosphorylase kinase activity in that the activation reactions contain no phosphorylase b; the concentration of buffer is much lower in the activation reactions but the concentration of phosphorylase kinase is much higher than in the phosphorylase b to a reaction. A significant concentration of EDTA introduced with phosphorylase kinase is present in activation reaction mixtures, whereas this component is effectively diluted out in the phosphorylase b to a reaction.


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TIME (MINUTES)

FIG. 2. (left). Effect of Ca*+ on the ATP-dependent activation of phosphorylase kinase. The activation of phosphorylase kinase was investigated in a system essentially identical with that presented in the legend of Fig. 1, Curves ZZZ and IV, with the exception that the phosphorylase kinase concentration was 0.55 mg per ml, EDTA, 0.55 mM, and glycerol-P, 18 mM. The concentration of cyclic AMP, where added (Curves ZZZ and IV), was 1 PM. The calcium chloride, where added (Cuurves ZZ and IV), was 0.35 mM. (The extent of carry over of Ca 2+ from the respective reaction mixtures into the phosphorylase b to a conversion would have no effect on this reaction which was performed at saturating levels of this ion.)

TABLE I

Effect of EGTA on activation of phosphorylase kinase

Nonactivated phosphorylase kinase was incubated with 6 mM magnesium acetate and 1.8 mM ATP in the absence (Experiment I) or presence (Experiment II) of 0.01 mM cyclic AMP for 6 min at 30”, diluted, and then assayed for phosphorylase kinase activity at pH 6.8. The concentration of phosphorylase kinase in the activation reaction mixtures was 0.25 mg per ml. Activity before incubation was 0.6 X lo3 units per mg. Other conditions as in the legend of Fig. 1.

Experiment

I

II

EGTA concentration

Increase in phosphorylase kinase activity

M x 106

0 1 3 6

10 20

units/rig x 10-a

3.5 2.5 0.9 0.1 0.0 0.1

0 15.9 1 14.8 3 15.4 5 12.1

10 9.9 50 8.9

I I I I I

5 IO 15 20

TIME (MINUTES)

FIQ. 3. (right). The effect of calcium and EGTA on the cyclic AMP-independent activation of phosphorylase kinase. The activation of phosphorylase kinase (0.3 mg per ml) was investigated under conditions described in the legend of Fig. 1, utilizing the activation conditions of Curve ZZZ (2 X W8 M ATP; 6 X 10-* M magnesium acetate, in the absence of cyclic AMP). In addition the complete incubation mixtures contained 1.6 X 1CY M calcium chloride. O-O, complete system; O-0, complete system minus calcium chloride; O-O, complete system plus 1 X 10-d M EDTA; +B, complete system plus 2 X 10-e M EDTA; A-A, complete system plus 4 X lO+ M EDTA.

TABLE II

Effect of protein inhibitor on activation of phosphorylase kinase

Nonactivated phosphorylase kinase (870 units per mg, 0.3 mg per ml) was incubated for 15 min in the presence of cyclic AMP under essentially the conditions of activation described in Fig. 1. High Mg2+ and ATP refers to concentrations of 6 mM and 1.8 mM, respectively, in the activation reaction mixture; low Mgz+ and ATP refers to concentrations of 0.6 mM and 0.18 mM, respectively. The inhibitor was prepared as described in the text.

Inhibitor added - I

units/ml None

0.5 1.0 2.0 4.0 8.0

None (Cyclic AMP omitted)

--

Increase in phosphorylase kinase activity when activation reactions

carried out with

Iigh Mgz+ and ATP Low MgZ+ and ATP

29.1 24.7 18.4 11.9 10.0 8.1 7.1

6.23 3.48

1.02 0.65 0.80 0.39


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FIG. 4. The combined effect of cyclic AMP, EGTA, and the protein inhibitor on the activation of phosphorylase kinase. The activation of phosphorylase kinase (0.3 mg per ml) was investigated under conditions essentially identical with those presented in Fig. 1 (Curves ZZZ and ZV) at concentrations of Mgz+ and ATP of 6 mM and 1.8 mM, respectively. Where added, cyclic AMP was at a concentration of 10 PM; [EGTA] was 1 X 1V M, and the protein inhibitor concentration was 5.3 units per ml. A----A, plus cyclic AMP; A--A, plus cyclic AMP and EGTA; O--U, plus cyclic AMP, EGTA, and protein inhibitor; O-O, plus cyclic AMP and protein inhibitor; +W, minus cyclic AMP; O- - -0, minus cyclic AMP plus EGTA; A- - -A, minus cyclic AMP plus EGTA and protein inhibitor; O--O, minus cyclic AMP plus protein inhibitor.

tion, the inhibitor depressed the activation that occurs in the presence of cyclic AMP to the level that occurred in the absence of cyclic AMP.

In order to gain further information concerning catalysts of the phosphorylase kinase activation reaction, the combined effects of the protein inhibitor and EGTA on this process were examined (Fig. 4). In this experiment, carried out with high concentrations of Mg*-ATP, the protein inhibitor and EGTA were used at levels that would give a maximal effect for each component (see Tables I and II). The protein inhibitor alone eliminated the cyclic AMP-dependent component of the activation reaction, as seen before, but had no signifmant effect on phosphorylase kinase activation in the absence of cyclic AMP. EGTA caused partial inhibition of the reaction with cyclic AMP and essentially complete inhibition of the reaction without cyclic AMP, as seen before, but EGTA plus the protein inhibitor essentially blocked activation completely even in the presence of cyclic AMP.

Identi$cattin of Cyclic AMP-dependent Enzyme Catalyxing Phosphoylase K&se Activation Reaction-When it became apparent that purified phosphorylase kinase behaved as though it contained traces of a different enzyme, i.e. a cyclic AMP-dependent, phosphorylase kinase-activating enzyme or phosphorylase kinase kinase, efforts were made to determine whether larger amounts of this enzyme could be found in muscle extract. Ear- lier experiments (4) had suggested that the enzyme was present in crude extracts, but these were complicated and difficult to interpret because of the presence of phosphorylase kinase phosphatase as well as phosphorylase kinase. In the present study fluoride was added to control the phosphatase and phosphorylase kinase was removed by acid precipitation at pH 6.1. After this treatment the muscle extract was found to have a definite stimulatory effect on phosphorylase kinase activation (Fig. 5A). This experiment provided evidence for the existence of a phosphorylase

INCUBATION TIME g $;I;

I II Ill IV v VI VII VIII

I = COMPLETE SYSTEM VI = PLUS 0.2ml PROTEIN

II = MINUS CYCLIC AMP INHIBITOR

111: PLUS 0.03ml TREATED VII= PLUS O.lml TREATED

EXTRACT EXTRACT AND O.lml

IV= PLUS O.lml TREATED INHIBITOR

EXTRACT VIII= PLUS 0.03ml TREATED

V = PLUS O.lml PROTEIN EXTRACT AND 0.2ml

INHIBITOR INHIBITOR

FIG. 5. Stimulation of the activation and phosphorylation of phosphorylase kinase by a fraction prepared from rabbit muscle extract. The activation (A) and phosphorylation (B) of phosphorylase kinase that occurs in 10 and 25 min (as shown) in the presence of muscle extract was investigated in a system that was essentially the same as that used for the experiment presented in Fig. 1, Curve ZZ, except for the inclusion of sodium fluoride, higher Mgz+ needed to overcome the EDTA added with the protein fractions, and a 2-fold increase in the concentration of ATP. The components of the reaction mixtures were: glycerol-P buffer, pH 6.8, 22 pmoles; fi-mercaptoethanol, 20 pmoles; sodium fluoride, 20 @moles; EDTA, 0.8 pmole; magnesium acetate, 1.2 pmoles; -@2P- ATP, 0.2 pmole; cyclic AMP, 0.1 pmole; phosphorylase kinase, 0.3 mg; in a total volume of 1.05 ml. Where indicated, reaction mixtures contained treated extract from which phosphorylase kinase had been removed, and a solution of the protein inhibitor. See “Experimental Procedures” for a description of these two solutions. The initial phosphorylase kinase activity was 0.12 X 10” units per mg of enzyme.

kinase-activating enzyme, but even stronger support for this hypothesis was obtained by adding the protein inhibitor to the phosphorylase kinase activation reaction mixture. The effect of this protein inhibitor (0.1 ml) was to reduce the endogenous activation rate that occurs with purified phosphorylase kinase in the absence of any other added fraction. Under these conditions, addition of the treated muscle extract stimulated the activation rate g-fold. The addition of high levels of inhibitor (0.2 ml) to the activation mixture not only eliminated endogenous activation


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CONTROL

OlOl Oh O.b3 0.b4

AMOUNT OF TREATED EXTRACT ADDED (ml)

FIG. 6. The assay of phosphorylase kinase-activating enzyme. The extent of activation of phosphorylase kinase (ordinate) in 15 min was determined as a function of the concentration of the treated extract containing phosphorylase kinase-activating enzyme (abscissa) in the absence (O-O) and the presence of 1.3 units (0-0) (Curve A) and 3.35 units (O---6) (Curve B) of the protein inhibitor. The conditions of the activation reaction were identical with those given in the legend of Fig. 5, i.e. low concentrations of Mgz+ and ATP. Th e original kinase activity was 1.03 X 1Oj units per mg of protein. The preparation of the solutions of the inhibitor and of treated muscle extract are given under “Experimental Procedures.” Inset, increasing concentrations of the protein inhibitor were added to the phosphorylase kinase activation reaction carried out in the presence of cyclic AMP with low concentrations of Mgz+ and ATP as described in Table II. Percentage of inhibition of activation rates is plotted against inhibitor concentration. The arrows refer to the amounts of inhibitor used in Curves A and B.

of phosphorylase kinase but also inhibited the activation of the phosphorylase kinase catalyzed by the component of muscle extract. In addition to the effect of muscle extract and the inhibitor on the activation of phosphorylase kinase, these components also affected the phosphorylation of this protein (Fig. 5B).

Development of Assay System for Phosphorylase Kinuse-activating Enzyme-The experiment of Fig. 5 suggested that the protein inhibitor could be used to good advantage in the development of an assay for the phosphorylase kinase-activating enzyme, since it was possible to overcome what amounted to almost complete inhibition of the activation reaction by addition of the enzyme. In order to determine how much of the inhibitor should be used, the phosphorylase kinase activation reaction was first titrated with increasing amounts of the inhibitor as is shown in the inset of Fig. 6. Two concentrations (Points A and B) were selected, and the amounts of inhibitor present in these reactions were then used in trial assays of the activating enzyme; a control was run in the absence of inhibitor. The results of the experiment are given in Fig. 6. The activation reaction that gave the best over-all response in terms of linearity and suitability for use in an assay system for the activating enzyme was the one in which the lower level of inhibitor was employed (Curve A, Fig. 6). With the larger amount of inhibitor a sigmoidal response curve (Curve B, Fig. 6) was obtained, presumably due to an excess of available inhibitor at that point. In the absence of any inhibitor (Control Curve, Fig. B), only a limited response of added activating enzyme was seen as would be expected since endogenous activat-

I I I I

5 IO 15

TIME (b4iid~TES)

FIG. 7. The effect of purified cyclic AMP-dependent protein kinase on the activation of phosphorylation of phosphorylase kinase Nonactivated phosphorylase kinase (3.68 mg) was incubated at 20” in a reaction mixture containing: glycerol-P, 7 Fmoles; @-mereaptoethanol, 20 pmoles; EGTA, 0.3 amole; EDTA, 0.8 pmole; magnesium acetate, 1.3 pmole; r-**P-ATP, 0.2 pmole; cyclic AMP, 1 mpmole ; in the presence (open symbols) or absence (closed symbols) of 365 pg of purified cyclic AMP-dependent protein kinase in a total volume of 1 ml at pH 6.8. At intervals aliquots were removed for determination of phosphorylase kinase activity (circles) and also for protein-bound 32P (squures). The ordinate on the left-hand side refers to kinase a&&y in’units per mg of protein in the activation reaction mixture, and that on the right-hand side refers to moles of phosphate incorporated into phosphorylase kinase. Control experiments indicated that there was no significant uptake of phosphate by the cyclic AMP-dependent protein kinase.

ing enzyme gave nearly half-maximal activation under these circumstances.

PuriJication of Phosphorylase Kinase-activating Enzyme-With the availability of an assay system for the phosphorylase kinase- activating enzyme it was possible to purify the enzyme from crude skeletal muscle extract and to obtain it completely free of phosphorylase kinase activity. The method for achieving this purification was described in a preliminary communication together with evidence that the activating enzyme also catalyzes a cyclic AMP-dependent phosphorylation of casein and protamine (11). Because the enzyme did not appear to be specific for phosphorylase kinase, it was called a “cyclic AMP-dependent protein kinase.” Similar enzymes have since been detected in a number of other tissues through the utilization of this property (25-31). The rabbit skeletal muscle enzyme has been further purified and characterized, as is reported in the accompanying paper (18), and has been shown to be separable into at least two fractions. In an extension of specificity studies on the cyclic AMP-dependent protein kinase or kinases, it has been shown that the activities of the muscle enzymes toward casein, phosphorylase kinase, and glycogen synthetase are enriched equally during purification (32).

The effect of a high concentration of the purified protein kinase on the rate and extent of phosphorylase kinase activation and phosphorylation is shown in Fig. 7. As can be seen the activation rate is very rapid under these circumstances with full activa-


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tion being achieved within a few minutes. In this experiment, a high concentration of the protein kinase was utilized in order to achieve completion of the reaction. In a previously reported experiment (11)) the effectiveness of considerably lesser amounts of this enzyme preparation on phosphorylase kinase activation has been shown. The correlation between phosphorylation and activation of phosphorylase kinase in the experiment of Fig. 7 was better than that which has been observed in the past (Z), suggesting that less nonspecific phosphorylation of the enzyme occurs under these conditions as compared to activation reaction in which the autoactivation mechanism (Equation 1) is con- tributing significantly.

DISCUSSION

The data presented in this paper support the hypothesis that two catalysts participate in the activation of phosphorylase kinase in vitro (Equations 1 and 2). One of these catalysts is phosphorylase kinase itself,5 as had been suggested by the kinetic studies of DeLange et al. (2), and the other catalyst is a cyclic AMP-dependent activating enzyme or phosphorylase kinase kinase present presumably as a contaminant in phosphorylase kinase preparations. The contribution of phosphorylase kinase to the activation process was shown to require Ca2+ and could be eliminated by addition of EGTA which binds this metal. These observations were in keeping with the known requirement of phosphorylase kinase for Ca2+ in the conversion of phosphorylase b to phosphorylase a (22, 33). The participation of a cyclic AMP-dependent activating enzyme in the phosphorylase kinase activation reaction was supported by the studies with varying concentrations of Mgzf and ATP (Fig. 1) and by the experiments utilizing the heat-stable protein inhibitor. Finally, the existence of the phosphorylase kinase-activating enzyme as an entity was established by the finding of this enzyme in crude muscle extract and its separation from phosphorylase kinase (11). It has not been possible to obtain phosphorylase kinase completely free of the cyclic AMP-dependent activating enzyme. One of the fac- tors that has made this separation difficult to achieve is that relatively mild fractionation procedures result in the partial de- naturation of phosphorylase kinase, as is indicated by increases in the pH 6.8 to 8.2 activity ratio and an increased turbidity of enzyme solutions. A complete separation may be difficult to achieve, as an interaction between the two proteins would be expected to occur, since one is a substrate for the other. The copurification of substrate and enzyme is a common occurrence, for example, as with NAD+ and glyceraldehyde-3-P dehydrogen- ase (34). Calculations show that the observed effect of cyclic AMP on the activation of purified phosphorylase kinase can be accounted for by only a trace of the protein kinase in the former enzyme (11, 32).

The identity of the cylic AMP-dependent phosphorylase kinase-activating enzyme and the cyclic AMP-dependent protein kinase which phosphorylates casein and other proteins (11) was supported by a number of findings including (a) the copurification of the two activities (32), (b) the requirement of both activities for ATP and Mg2+ (2, 11, 18), (c) the essentially identical K,

6 In addition to catalyzing the phosphorylation of phosphorylase b, phosphorylase kinase also catalyzes, at aslow rate, the phosphorylation of casein (2). The rates of phosphorylation of phosphorylase kinase and of casein to catalyzed phosphorylase kinase are approximately equivalent and are three orders of magnitude slower than the rate of phosphorylation of phosphorylase b.

POSSIBLE SCHEMES FOR THE REGULATION OF PHOSPHORYLASE g FORMATION IN

SKELETAL MUSCLE

HORMONAL m

ATP

\ ADENYL

qyy-*yFmE~ ACTIVATED (N~NACTIVATED) PHOSPHORYLASE KINASE "QQ

CYCLIC AMP 0

#c--mm

ACTIVATED .* 4 C.3" PHOS..HOiO~LASE

PHOSPHORYLASE b PHOSPHORYLASE g

NONACTIVATED PHOSPHORYLASE

KINASE

I POSTULATED AS BEING SIGNIFICANT /N V/V0

17 DEMONSTRABLE 1N VITRO BUT OF DOUBTFUL SIGNIFICANCE /N V/V0

FIG. 8. Control of phosphorylase a formation

values for cyclic AMP in both activities (2, ll), and (d) the inhibition of both activities by the heat-stable protein inhibitor

(17). The cyclic AMP-dependent protein kinase is not identical with

other catalysts of phosphorylase kinase activation that have been reported previously. A protein kinase referred to as kinase II (4) also enhanced phosphorylase kinase activation but was not stimulated by cyclic AMP. This enzyme was found in one of the fractions obtained during phosphorylase kinase purification and appeared to be present only in small amounts. Meyer, Fischer, and Krebs (22) identified a protein factor, referred to as KAF, that catalyzed a calcium-dependent activation of phosphorylase kinase.6 The mechanism of this activation was later shown by Huston and Krebs (35) to be due to proteolysis. The physiological significance of KAF in glycogenolysis is questiona- ble.

The finding of a cyclic AMP-dependent protein kinase which acts as a phosphorylase kinase-activating enzyme provides another link in the chain of events responsible for the stimulation of glycogenolysis by epinephrine (36). It would seem probable that most, if not all, of the enzymes involved in the regulatory cascade controlling this process (4) have now been identified. Referring to the left side of Fig. 8, it can be seen that these are (a) adenyl cyclase (37), (b) the cyclic AMP-dependent protein kinase (ll), (c) phosphorylase kinase (38), and (G?) phosphorylase (39). Some question still remains as to whether adenyl cyclase itself or some other membrane-bound protein is the actual receptor for epinephrine (40) ; hence, it is probable that additional components may intervene at that point. It is also of considerable interest that evidence is accumulating to support the idea that the cyclic AMP-dependent protein kinase may contain a regulatory subunit, which is the actual “receptor” for cyclic AMP, as well as a separate catalytic subunit. This concept was

6 The calcium dependent autoactivation of phosphorylase kinase cannot be attributed to contamination by KAF, all detecta- ble traces of which are removed in the final purification step (2). KAF activation is not associated with concomitant phosphorylation.


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first introduced and supported by a report on the kinetic be- 3. KREBS, E. G., GRAVES, D. J., AND FISCHER, E. H., J. Biol.

havior of the purified heart muscle cyclic AMP-dependent pro- Chem., 234, 2867 (1959).

tein kinase (25). Gill and Garren (29) then showed that a cyclic 4. KREBS, E. G., DELANGE, R. J., KEMP, R. G., AND RILEY, W.

AMP-binding protein and protein kinase activity can be dissoci- D., Pharm&ol. Rev., 18, 163 (1966).

5. POSNER, J. B., STERN, R., AND KREBS, E. G., J. Biol. Chem., ated in adrenal cortical extracts.’ In contrast to previous find- 240, 982 (1965).

ings on the activation of phosphorylase kinase in vitro (2), the 6. KRAUSE, El G.,.Ezperientia, 22, 479 (1966).

rate of activation in the presence of the cyclic AMP-dependent 7. DRUMMOND, G. I., DUNCAN, L., AND HERTZMAN, E., J. Biol.

protein kinase observed in these studies is extremely rapid and Chem., 241. 5899 (1966).

may now be favorably compared to the rapid rate of cateehola- 8. NAMM, c. H.., AND MAYER, S. E., Mol. Phczrmacol., 4,61 (1968). 9. NAMM, D. H., MAYER, S. E., AND MALTBIE, M., Mol. Phar-

mine-induced phosphorylase a formation in viva (41). These ob- macol.. 4. 522 (1968).

servations serve to identify for the first time a specific site of ac- 10. RILEY, I?. b., DELANGE, R. J., BRATVOLD, G. E., AND KREBS,

tion of cyclic AMP in a physiological process. Not included in E. G., J. BioZ. Chem., 243, 2209 (1968).

the scheme presented (Fig. 8), but of considerable significance in 11. WALSH, D. A., PERKINS, J. P., AND KREBS, E. G., J. BioZ.

Chek, 243, 3763 (1968). the total role of cyclic AMP in the control of phosphorylase a 12. WALSH. D. A.. PERKINS. J. P.. AND KREBS. E. G.. Fed. Proc..

27, 339 (1968). ’ ’ levels, are the observations of Riley and Haynes (42) and of Chelala and Torres (43) that the activity of phosphorylase a

13. WALSH, D. A., PERKINS, J. P., AND KREBS, E. G., Pharma-

phosphatase is also regulated by this nucleotide. cologist, 10, 144 (1968).

14. KREBS, E. G.. AND WALSH, D. A., Fed. Eur. Biochem. Sot. Some of the possible sites of interaction of Ca2+ and cyclic Symp., 19, i21 (1969).

AMP in the regulation of phosphorylase a formation are illus- 15. WALSH, D. A., KREBS, E. G., REIMANN, E. M., BROSTROM, M.

trated in Fig. 8. Ca*, whose intracellular fluxes mediate con- A., CORBIN, J. D., HICKENBOTTOM, J. H., SODERLING, T. R.,

traction, is also required for the conversion of phosphorylase b AND PERKINS, J. P., in P. GREENGARD AND E. COSTA (Edi-

to phosphorylase a whether phosphorylase kinase is in its ac- tars), Advances in biochemical psychopharmacology, Vol. 5, Raven Press, New York, 1970, p. 265.

tivated or nonactivated form (16) (Fig. 8, lower right), but it is 16. BROSTROM, C. O., HUNKELER, F. L., AND KREBS, E. G.. J. BioZ.

also clear that this ion is required for the autoactivation of phos- Chem., 246, 1961 (1971).

phorylase kinase which occurs in vitro (Fig. 8, middle right). 17. WALSH. D. A.. ASHBY. C. D.. GONZALES. C.. CALKINS. D..

Thus Ca2+, as well as cyclic AMP, could conceivably promote FISC&R, E. k., AND KREBS,‘E. G., J. BaoZ. bhem., 246,’ 1977

activation of the enayme. This potential interaction was first (1971).

18. REIMANN, E. M., WALSH, D. A., AND KREBS, E. G., J. BioZ. considered by Ozawa and Ebashi (44); however, under the condi- Chem., 246, 1986 (1971).

tions of their preliminary experiments no distinction could be 19. WASTIL~, W. B., S&LL, J. T., MAYER, S. F., AND WALSH, D.

made between a role for Gaff in phosphorylase kinase activation A., J. BioZ. Chem., 246, 1996 (1971).

or in the phosphorylase b to a conversion. The question should 20. GLYNN, I. M., AND CHAPPELL, J. B., Biochem. J., 90, 147 (1964). 21. HUIJING, F., AND LARNER, J., Proc. Nat. Acad. Sci. U. S. A.,

be raised as to whether autoactivation of phosphorylase kinase 66, 647 (1966). is of any physiological importance. Even under optimal condi- 22. MEYER, W. L., FISCHER, E. H., AND KREBS, E. G., Biochem-

tions the reaction is relatively slow as studied in vitro with purified istry, 3, 1033 (1964).

components (2). Furthermore, when muscle is stimulated elec- 23. POSNER, J. B., HAMMERMEISTER, K. E., BRATVOLD, G. E.,

trically, no activation of phosphorylase kinase takes place (45). AND KREBS, E. G., Biochemistry, 3, 1040 (1964).

24. APPLEMAN, M. M., BIRNBAUMER, L., AND TORRES, H. N., Since electrical stimulation of muscle does cause rapid formation Arch. Biochem. Biophys., 116, 39 (1966).

of phosphorylase a (46,47), an effect that is presumably mediated 25. BROSTROM, M. A., REIMANN, E. M., WALSH, D. A., AND KREBS,

by Ca2+ (16), one would have expected the kinase to be activated E. G., Advan. Enzyme Regulat., 8, 191 (1970).

under these circumstances if the process does occur zn vivo. 26. LANGAN, T. A., Sci&ce, 162, 579 (1968). 27. JERGIL, B.. AND DIXON. G.. J. BioZ. Chem., 246, 425 (1970).

Cyclic AMP levels are not increased by electrical stimulation of 28. JARD, S., AND BASTIDE,‘F.,‘B~~~~~~. Biophys. Res. Cbmmka.,

muscle (48). 39, 559 (1970). 29. GILL. G. N.. AND GARREN. L. D., Biochem. Biovhus. Res.

Acknowledgment.+-We wish to thank Mrs. Carol Levitt, Mrs. Coinmun., 39, 335 (1970). ’

. I

30. Diane Calkins, and Mrs. Janet Wolf for their expert technical

Kuo, J. B., AND GREENGARD, D., Proc. Nat. Acad. Sci. U. S. A., 64, 1349 (1969).

assistance. 31. CORBIN, J. D., AND KREBS, E. G., Biochem. Biophys. Res. Comrrkn., 36; 328 (1969).

_ .

REFERENCES 32. SODERLING. T. R.. HICKENBOTTOM. J. P.. REIMANN. E. M.. HUNKEL~R, F. L:, WALSH, D. A., END K~EBS, E. G.: J. Biol:

~.KREBS,E.G.,LOVE,D.S.,BRATVOLD,G.E.,TRAYSER,K.A., Chem., 246,‘6317 (1970). MEYER, W. L., AND FISCHER, E. H., Biochemistry, 3, 1022 33. OZAWA, E., HOSOI, K., AND EBASHI, S., J. Biochem. (Tokyo), (1964). 61, 531 (1967).

2. DELANGE, R. J., KEMP, R. G., RILEY, W. D., COOPER, R. A., 34. VEL&, S: F.,‘J. Biol. Chem., 203, 563 (1953). AND KREBS, E. G., J. BioZ. Chem., 243, 2200 (1968). 35. HUSTON. R. B.. AND KREBS. E. G.. Biochemistru. ‘7.2116 (1968).

36. CORI, C: F., AND CORI, G. T., J. biol. Chem., is, 309 (1928). ’ 7 Drs. Mariano Tao, Maria Salas, and Fritz Lipmann have 37. SUTHERLAND, E. W., AND RALL, T. W., Pharmacol. Rev., 12.

shown that the cyclic AMP-dependent protein kinase of erythro- 265 (1960). cytes can be separated into a cyclic AMP-binding subunit and a 38. KREBS, E. G., AND FISCHER, E. H., Biochim. Biophys. Acta, 20, catalytic subunit by density gradient centrifugation in the pres- 150 (1956). ence of cyclic AMP (personal communication). Work in this 39. CORI, G. T., AND CORI, C. F., Proc. Sot. Ezp. Biol. Med., 39, laboratory by Dr. Erwin Reimann has also shown that the skeletal 337 (1938). muscle protein kinase is separable into a cyclic AMP-binding sub- 40. BIRNB~UM~R, L., POHL, S. L., AND RODBELL, M., J. BioZ. unit and a catalytic subunit independent of cyclic AMP. Re- Chem., 244, 3468 (1969). combination of the two fractions restores the original properties 41. DANFORTH, W. H., HELMREICH, E., AND CORI, C. F., Proc. Nat. of the protein kinase. Acad. Sci. U. S. A., 48, 1191 (1962).


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42. RILEY, G. A., AND HAYNES, R. C., JR., J. Biol. Chem., 238, 1563 (1963).

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43. CHELALA, L. A., AND TORRES, H. N., Biochim. Biophys. Acta, Amer. J. Physiol., 200, 1236 (1961).

178, 423 (1969). 47. CORI, C. F., in 0. H. GAEBLER (Editor), Enzymes: units of

44. OZAWA, E., AND EBASHI, S., J. Biochem. (Tokyo), 82, 285 biological structure and function, Academic Press, New York,

(1967). 1956, p. 573.

45. DRUMMOND, G. I., HARWOOD, J. P., AND POWELL, C. A., J. 48. POSNER, J. B., STERN, R., AND KREBS, E. G., J. Biol. Chem., Biol. Chem., 244, 4235 (1969). 240, 982 (1965).


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Donal A. Walsh, John P. Perkins, Charles O. Brostrom, Ester S. Ho and Edwin G. KrebsCatalysis of the Phosphorylase Kinase Activation Reaction

1971, 246:1968-1976.J. Biol. Chem.

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