Adenosine 3':5'-Cyclic Monophosphate

Transcription

1 Proc. Nat. Acad. Sci. USA Vol. 68, No. 4, pp , April 1971 Role of the Receptor in the Mechanism of Action of Adenosine 3':5'-Cyclic Monophosphate GORDON N. GLL AND LEONARD D. GARREN Department of Medicine, Division of Endocrinology, School of Medicine, University of California at San Diego, La Jolla, Calif Communicated by Nathan. Kaplan, January 25, 1971 ABSTRACT Highly purified camp-dependent protein phosphokinase from adrenal-cortical tissue contains camp-receptor activity. n activating the kinase, camp binds to the receptor and causes it to dissociate from its complex ith the kinase. The kinase, freed of receptor, is fully activated and no longer stimulable by camp. Kinase can be similarly activated by differentially denaturing the receptor ith heat. Addition of receptor suppresses kinase activity; this suppression can be overcome by camp. After dissociation of receptor, to molecular forms of the activated kinase exist. The camp receptor thus functions as a repressor of the protein kinase; binding of camp to receptor causes it to dissociate from the kinase, hich is then fully activated. Adenosine 3':5'-cyclic monophosphate (camp), hich mediates the action of a number of hormones and biogenic amines, exerts regulatory control over various cellular functions. Several lines of evidence suggest that in each of these events camp acts by a common mechanism. We described a campreceptor protein in the adrenal-cortical cell (1), hich subsequently as shon to be present in many organs (2), and postulated that the binding of the nucleotide to this receptor as the initial event in the action of camp. A camp-receptor protein has since been identified in bacteria and shon to be essential in the stimulation of mrna transcription by the nucleotide (3, 4). The possibility as considered that the receptor as involved in the camp activation of a protein phosphokinase (EC ) because it appeared that camp activated the enzyme directly (5), and that the enzyme phosphorylated many substrates (5-9) and as ubiquitous in its distribution (1). These attributes of this camp-dependent enzyme appeared to satisfy the physiological requirements for the site of action of camp. Our previous investigations into the functional interrelationship of the camp receptor ith the camp-dependent protein phosphokinase (kinase) in the adrenal-cortical cell indicated a regulatory role for the receptor, suggesting a mechanism of action for camp (11, 12). The present studies demonstrate that purified protein kinase from the adrenal cortex contains both the receptor and the kinase activities. The receptor protein inhibits the protein kinase hen complexed to the enzyme moiety. camp binds to the receptor and causes it to dissociate from the protein kinase; the protein kinase free of receptor is then fully active. These investigations indicate that at least one major function of the camp-receptor protein is the inhibitory control of protein kinase activity. Abbreviations: TMG buffer, 1 mm Tris (ph 7.4)-6 mm 2- mercaptoethanol-1% glycerol; TCA, trichloroacetic acid. METHODS Assays Receptor Activity. Binding of [3H]cAMP to receptor protein is measured by incubation of the nucleotide ith receptor, then isolation of the bound complex on a cellulose-ester filter (Millipore;.45 jam) (13). Specific activity measurements (pmol camp bound per mg of protein) ere performed at saturating concentrations of camp. After electrophoresis in polyacrylamide gels, the receptor protein band as identified by stirring the gel in 1-7 M ['HlcAMP in 5 mm Tris (ph 7.4)-S mm theophylline buffer at 4VC for 4 hr, rinsing the gel in ice ater for 8 hr to remove unbound camp, then slicing it into 2-mm sections and determining the [3H]cAMP bound, as described belo. ncubation of protein ith saturating concentrations of [3H]cAMP (5 X 1-7 M) prior to electrophoresis allos quantitation of the effects of the binding of camp to receptor on the receptor-kinase complex. Free camp migrates ith the tracking-dye front. To determine the bound [3H]cAMP, gel slices ere incubated overnight at 45C in 15 ml of scintillator solution, hich consisted of 145 ml of NCS (Amersham/Searle), 165 ml of Liquifluor (Ne England Nuclear), and 1 gal (3.8 liters) of toluene; radioactivity as then determined in a liquid scintillation counter. Protein Kinase. Kinase activity as assayed as described by Walsh et al. (5), using conditions previously described for the adrenal protein kinase (11). Specific activity measurements [nmol of 32p incorporated into trichloroacetic acid (TCA)-insoluble material per mg of enzyme protein per 1 min] ere performed at saturating concentrations of ['y-82p]atp, either 2.4 mg/ml of histone or 1.7 mg/ml of protamine as substrate (as indicated in the text), and 2 X 1-7 M camp. Activity as linearly responsive to increasing amounts of enzyme protein and time in all experiments. Kinase activity of material separated on acrylamide gels as measured by incubation of 2-mm gel slices in reaction mixtures of 2,A; at the end of the reaction, 1,ul as ithdran and carried through the same TCA precipitation and ash procedures as the standard reactions. Protein Concentration as determined by the method of Lory et al. (14). Acrylamide Gels. Polyacrylamide gel electrophoresis as performed under standard conditions, using system 398 described by Rodbard and Chrambach (15). The ratio (C) of cross-linking agent (methylene bisacrylamide) to total gel concentration (T) as 3; electrophoresis as at C at 2 ma per tube. Gels ere sliced into 2-mm sections and ere assayed 786

2 Proc. Nat. Acad. Sci. USA 68 (1971) for either kinase or receptor activity. Measured mobilities (Rf) of the reference proteins at different total gel concentrations (T) ere analyzed by a computer (15) to obtain the standard curve for molecular eight estimations of the proteins under study. Soybean trypsin inhibitor, ovalbumin, fetuin, bovine serum albumin (monomer and dimer), lactic dehydrogenase (EC ), and glucose-6-phosphate dehydrogenase (EC ) ere run as standard protein markers. Analytical Ultracentrifugation. The sedimentation constant of the purified protein kinase-receptor complex as determined in a model L analytical ultracentrifuge equipped ith scanner and monochromator optics. Protein as sedimented at a concentration of.275 mg/ml in 5% glycerol, buffered ith.1 Tris(pH 7.4), at 17.5C and 5,74 rpm. Absorbance as scanned at 28 nm at 8-min intervals. MATERALS ['y-32p]atp as prepared by the method of Glynn and Chappel (16). Purity as estimated by paper chromatography, using a solvent of.1 M phosphate (ph 6.8)-ammonium sulfate-n-propanol 1:6:2. [3H]cAMP (2.35 Ci/mmol) as from Scharz BioResearch. Purified protein markers used as standards for acrylamide gel electrophoresis ere obtained commercially, except for haddock LDH, hich as a gift of Dr. George Sensabaugh. RESULTS Purification Bovine adrenal-cortical tissue as used. All steps ere performed at 4 C. nitial purification procedures, including subcellular fractionation, ammonium sulfate precipitation, and adsorption to and elution from calcium phosphate gel, resulted in parallel enrichment of camp receptor and campactivated protein kinase activities as described (11). The calcium phosphate gel eluate as adjusted to ph 5.9 ith.1 M acetic acid, and the precipitate formed as removed by centrifugation. The supernatant as dialyzed against buffer containing 1 mm Tris (ph 7.4)-6 mm 2-mercaptoethanol-1% glycerol (TMG), and then resolved on a DEAEcellulose column into three peaks (Fig. 1): Peak contained both protein kinase and receptor activity; the kinase activity as markedly camp-dependent. Both activities sedimented in a sucrose gradient in a single peak at 7 S. Peak contained receptor relatively free of kinase activity; the receptor sedimented at 4 S. Peak, predominantly kinase, but containing a shoulder of receptor activity, demonstrated less camp-dependent kinase activity; both receptor and kinase sedimented at 7 S on sucrose gradient sedimentation. Pooled protein kinase from peaks and (15 mg) as then purified on a 44-ml isoelectric focusing column, using a 2.3% ampholine gradient of ph 3-6. Both protein kinase and receptor appeared together in a single cloudy band ith an isoelectric point of ph Similar results ere obtained ith peak alone. After neutralization, the active band as sedimented at 39, rpm for 18 hr on a 5-2% linear sucrose gradient containing 5 mm Tris(pH 7.4)-6 mm 2-mercaptoethanol. The receptor and kinase activities ere present in a single peak sedimenting at 7 S. On polyacrylamide gel electrophoresis, this complex gave a single band containing both activities. The complex contained camp-activated protein kinase purified approximately 64-fold over the 1, X g c. a. E. Q a- D Q. m Function of the camp Repressor TUBE NUMBER FG. 1. DEAE-cellulose chromatography of the camp receptor O-O and camp-activated protein kinase A-A. 2.8 g of protein in TMG buffer as applied to a 4 X 4 cm DEAEcellulose 52 column equilibrated ith TMC buffer and then eluted ith a 16-ml linear (.4 to.4 M) NaC gradient in this same buffer (the gradient is represented by the straight line). Receptor and kinase activities ere assayed and the 1-ml fractions ere pooled as indicated by the Roman numerals. supernatant (337 nmol of 32p incorporated into protamine per mg of enzyme protein per 1 min at 3 C) and receptor purified 24-fold over the 1, X g supernatant (649 pmol of camp bound per mg protein). Sedimentation of the purified receptor-kinase complex in the analytical ultracentrifuge revealed a single front, ith an 82, of 7.4. The S value of the complex is in agreement ith that obtained by sucrose gradient centrifugation and suggests a minimal molecular eight of 152,, hich is similar to that obtained by acrylamide gel analyses described subsequently. DEAE-cellulose peak as further purified by gel filtration on Sephadex G-2 (85 X 1.5-cm column, TMG buffer) to give receptor ith a specific activity of 471 pmol of camp bound per mg protein. This material is the peak receptor in the folloing experiments. Acrylamide gel analysis of kinase and receptor activities Both receptor and kinase can be identified after electrophoresis in polyacrylamide gels. By determining the mobility (Rr) of the activity under study at different gel concentrations (T), estimates of physical-chemical identity, molecular eight, molecular radius, and free mobility can be obtained, even in impure protein mixtures (15, 17, 18). Both the retardation coefficients (K,) and the actual mobilities at each T value can be used as tests of identity. Kinase and receptor activities migrate in the same band in the acrylamide gels (Fig. 2A,C). As shon in Fig. 3, the plot of log Rr as a function of the total gel concentration (T) yields a straight line, ith a slope (Kr) that can be related to the molecular eight of the protein; the calculated molecular eight of the receptor-kinase complex is 144,. dentical Rr values are obtained at each T concentration ith DEAEcellulose peaks,, a mixture of peaks and, and the purified preparation. Both receptor and kinase activities are present in the same protein band hen assayed separately (K,.13 for each). Peak of the DEAE-cellulose column,._ Q E c: 8 fr,. z a-

3 788 Biochemistry: Gill and Garren a 3.- z O 2.- m, 1.- m O 2.- E a:, CL u A R K At Ahn flb,4, incubation ith camp r-4 K dye front D K 111 *14 -l pr incubation ith camp gil p, L,, i332 SLCE NUMBER FG. 2. Migration in polyacrylamide gels. After electrophoresis in 6% polyacrylamide gels, 2-mm slices ere assayed for kinase and receptor activity. All Rf values have been corrected for a dye front in slice No. 3. Each gel slice as assayed and background as subtracted. Receptor activity is indicated by the solid line, kinase by the dashed line. (A) Peak, 68,gg protein/gel. (B) Peak, as in (A), incubated for 3 min on ice ith 5 X 1-7 M [3H calvp prior to electrophoretic separation. (C) Peak, 5,g protein/gel. (D) Peak, as in (C), incubated for 3 min on ice ith 5 X 1-7 ['H] camp prior to electrophoretic separation. Results identical to (C) and (D) are obtained using a mixture of and. (E) Peak, 23 jsg protein/gel, assayed for receptor activity only. hich contains the receptor essentially freed of kinase activity, has a different mobility in the gels than does the receptor complexed to kinase from DEAE-cellulose peaks and (Fig. 2E, Fig. 3). The estimated molecular eight of the peak receptor is 92,. This is slightly larger than that estimated from the sucrose gradient sedimentation value of 4 S (19). Effect of camp on the receptor-kinase complex ncubation of the receptor-kinase complex ith 5 X 1-7 M ['H]cAMP prior to electrophoresis resulted in dissociation of the camp-bound receptor from the activated kinase, as shon previously (11) ith sucrose gradients (Fig. 2B,D; Fig. 3). The dissociated camp-bound receptor no longer migrated ith kinase, but instead migrated identically ith the purified receptor from peak (Fig. 2E). To molecular forms of the activated kinase have been observed after the dissociation of the receptor by camp. The camp-activated kinase derived from peak migrates ith changed Rf values and has an estimated molecular eight of 6,5 (Fig. 2A,B; Fig. 3). The sum of the estimated molecular eights of the dissociated receptor (92,) and kinase (6,5) suggests that there is one of each moiety in the complex. The receptor dissociated from the complex ith peak kinase by camp Proc. Nat. Acad. Sci. USA 68 (1971) also migrates identically ith the isolated receptor from peak and ith the receptor dissociated from the peak complex by camp (Fig. 2D, Fig. 3). The kinase derived from peak after camp addition, hoever, migrates ith different Rf values from those observed ith peak. The K7 value derived from the log Rf versus T plot indicates a significantly larger form of kinase, ith an estimated molecular eight of 145,. After the addition of camp, no residual receptor can be demonstrated ith either form of activated kinase. Mixing of peaks and yields additive kinase activity and a single band on gels containing kinase and receptor activities. After camp-induced separation of the receptor from the complex, the kinase migrates as a single band, ith the Rf of the activated kinase of peak (Fig. 2D, Fig. 3). No evidence of activated kinase ith the Rf of peak is observed after mixing ith peak. Properties of kinase after receptor removal Removal of receptor from either peaks or results in fully activated kinase that is no longer stimulable by camp (Fig. 4). A phenomenon in line ith this finding is observed in response to heating the receptor-kinase complex (Fig. 5). Heating at 42C causes a progressive loss of receptor activity; concomitantly, there is an activation of the basal kinase assayed in the absence of camp. The total kinase assayed in the presence of camp is only slightly decreased by heat. f e raise the temperature to 44C for 3 min, 93% of receptor activity is lost. Under these conditions, there is a 38% increase in basal kinase activity and a 3% decrease in total kinase activity; camp no longer stimulates the enzyme. The temperature-induced loss of receptor activity concomitant ith stimulation of kinase supports the concept that the receptor functions as a suppressor of kinase activity. Effects of the addition of receptor to kinase f the camp-induced dissociation of receptor from kinase results in activation of the kinase, then the addition of receptor ithout camp should suppress basal kinase activity. Combination of proteins present in gel slices appeared physically difficult; experiments ere therefore performed ith partially purified receptor from peak and the kinase fraction that contained relatively less receptor (peak ). Each fraction as further purified on sucrose gradients into 4S and 7S peaks, respectively. Addition of increasing amounts of receptor to kinase resulted in increased suppression of the basal kinase activity; up to 6% suppression as observed. The suppression as completely overcome by camp. Thus, the more the kinase as suppressed in response to the added receptor, the more dependent the enzyme became on camp for activity. Addition of receptor to kinase activated by heat treatment also resulted in an inhibition of the basal kinase activity. camp similarly completely overcame this suppression. DSCUSSON Analyzing cruder components on sucrose gradients, e demonstrated that camp activated protein kinase by dissociating an inhibitory receptor from the enzyme (11). n the present investigation, electrophoresis of purer enzyme preparations in acrylamide gels at several different concentrations alloed the identification and estimation of the molecular eights of the protein kinase and receptor in specific bands. These studies support our previously proposed mechanism of action of camp.

4 Proc. Nat. Acad. Sci. USA 68 (1971) Function of the camp Repressor 789 2C N f 8 loo ~~ TOTAL GEL CONCENTRATON (T) FG MNUTES AT 42' C FG. 4 FG. 5 FG. 3. Mobilities of the camp receptor and camp-activated protein kinase at different gel concentrations. Proteins ere electrophoresed at various gel concentrations as described in Fig. 2 and assayed for activity. Points are the averages of at least four gels at each T value. *-* Kinase activity of peak,, or plus ; Receptor activity - of peak,, or plus.-4 ; Receptor activity (incubation ith [3H] camp prior to electrophoresis) of peak,, or ; A-A Kinase activity of peak (incubation ith camp prior to electrophoresis); O-O Kinase activity of peak or plus (incubation ith camp prior to electrophoresis). FG. 4. Effect of camp on kinase after receptor removal. Pooled protein kinase from peaks and as electrophoresed on 6% polyacrylamide gels. 5,ug of protein as run on each of 12 gels. Six of the gels ere incubated ith 1-6 M camp prior to electrophoresis. After electrophoresis, gel slices corresponding to the knon Rf of the protein kinase ere pooled from three identical gels and ere assayed for protein kinase activity (in.6 mnl) ith protamine as substrate as follos: No camp in assay; * 1-6 M camp added to assay; El ncubation ith camp prior to electrophoresis, no camp in assay; ncubation ith camp prior to electrophoresis, 1f6 AM camp added to assay. Addition of camp prior to electrophoresis is indicated belo the columns and addition of camp to the assay is indicated above each column. 1-,l aliquots ere ithdran at 3, 6, and 12 min, and the TCA-insoluble material ashed and counted as in the standard kinase assay. Binding experiments demonstrated removal of the receptor bound to camp from the kinase hen incubated ith camp prior to electrophoresis, and the absence of free camp from the kinase area of the gel. The left side of each panel demonstrates that camp stimulability can be readily demonstrated in the gel. Removal of receptor shon on the right of each panel results in a fully activated protein kinase that is no longer stimulated by camp. Similar results are obtained ith peak using the kinase area of the gel, free of receptor (Fig. 2B). FG. 5. Effect of temperature on receptor and kinase activities. Peak (68 ug of protein/assay) as mixed ith assay buffer (ph 6.) and incubated in a shaken ater bath at 42C for the times indicated. Aliquots ere ithdran at each time point and placed on ice until assayed for receptor O-O, kinase ithout camp and kinase ith 2 X 1-7 M camp A-A. The camp receptor and camp-dependent protein kinase are present in a complex, as shon by its sedimentation as a single front in the analytical ultracentrifuge, and by its electrophoresis as a single band, containing both activities, at several gel concentrations. The binding of camp to the receptor causes this protein to dissociate from the kinase and move as a separate band in polyacrylamide gels. The activated kinase, free of receptor activity, is no longer stimulable by camp. Differential inactivation of the receptor by heat also results in kinase activation. Chromatography on DEAE-cellulose results in to peaks ( and ) containing camp-dependent protein phosphokinase activity. The addition of camp to either peak dissociates the receptor, resulting in a fullyactivated kinase(s), no longer stimulable by camp; the addition of receptor suppresses kinase activity in each peak. Certain differences, hoever, are observed ith the enzymes obtained from the to peaks. The protein kinase activity of peak is more dependent on camp than is that of peak. The phenomenon may be explained by a greater ratio of receptor to kinase in peak as compared to peak. After camp-induced dissociation from receptor, the activated kinase from peak demonstrates a different mobility on acrylamide gel electrophoresis than does the activated kinase of peak. A similar phenomenon is observed on sucrose gradient sedimentation (data not shon). The molecular eight estimates of the camp-induced dissociated receptor and kinase from peak is in reasonable agreement ith the sum of the components contained in the complex. Removal of the receptor from the protein kinase of peak by camp results in the formation of an activated kinase of about tice the molecular eight (estimated by acrylamide gels or sucrose gradient sedimentation) of the activated kinase of peak. The failure to demonstrate residual receptor activity ith the larger activated kinase of peak argues against incomplete separation of the receptor-kinase complex in response to camp as an explanation for the observation. Although receptor protein no longer able to bind camp could still be complexed to the kinase, the suppression of kinase by the addition of receptor argues against this. Each peak may contain a distinct species of camp-dependent protein kinase. Such a suggestion has been made for a similar finding in reticulocytes (2). Another explanation for the observation is that dissociation of the peak receptor from the kinase results in the rapid association of kinase subunits to form an activated kinase dimer. n line ith this explanation are the results of mixing peaks and. Here, receptor removal results in the formation of only the larger activated kinase. Further, purification of the mixture of peaks and results in a single complex. These data tend to favor the suggestion that the campdependent protein kinases of peaks and are the same, and

5 79 Biochemistry: Gill and Garren that the kinase of peak is formed after much of the receptor is removed into peak during DEAE-cellulose chromatography. Why, after the addition of peak to peak, camp induces only the activated kinase observed ith peak alone is not understood. The camp receptor, present in both peaks, functions as a repressor of the enzyme activity. Removal of the receptor from the kinase by specific binding to camp, or by inactivation ith heat, results in the activated kinase. Addition of receptor inhibits the activated kinase enzyme. This provides a mechanism by hich the intracellular concentrations of camp regulate protein phosphokinase activity. A similar mechanism has been suggested also by Tao, Salas, and Lipmann (2). Protein-protein regulatory interactions have been documented in several systems (21, 22). The diverse manifestations of camp action in different tissues ould result from the particular substrate phosphorylated by the activated enzyme of that tissue. The regulation of both glycogen synthetase and phosphorylase kinase by camp-dependent protein kinase supports this postulate (23). n the present studies, protamine and histone ere utilized as substrates because of their ready availability. n the adrenal cortex, here it has been postulated that ACTH modulates protein synthesis at the level of translation (24), e have demonstrated that ribosomes serve as a substrate for the camp-activated protein kinase (25). NOTE ADDED N PROOF After the manuscript as submitted for publication, evidence supporting this model of camp action in liver and skeletal muscle appeared (26, 27). We thank Mrs. June Kalstrom for expert technical assistance, Mr. David Kemper for analytical ultracentrifuge analysis, and Dr. David Rodbard for computer analysis of the acrylamide gel electrophoresis data. Portions of this ork ere presented at the 62nd Annual Meeting of the American Society for Clinical nvestigation, May 197; The Laurentian Hormone Conference, September 197; The Ne York Academy of Sciences, November 197; and ere published in abstract form (12). This ork as supported in part by USPHS grants AM and AM G. N. G. as the recipient of a Helen Hay Whitney Foundation Felloship. Proc. Nat. Acad. Sci. USA 68 (1971) 1. Gill, G. N., and L. D. Garren, Proc. Nat. Acad. Sci. USA, 63, 512 (1969). 2. Garren, L. D., G. N. Gill, H. Masui, and G. M. Walton, Recent Progr. Horm. Res. (197), in press. 3. Emmer, M., B. de Crombrugghe,. Pastan, and R. Perlman, Proc. Nat. Acad. Sci. USA, 66, 48 (197). 4. Zubay, G., D. Schartz, and J. Beckith, Proc. Nat. Acad. Sci. USA, 66, 14 (197). 5. Walsh, D. A., J. P. Perkins, and E. G. Krebs, J. Biol. Chem., 243, 3763 (1968). 6. Langan, T. A., Science, 162, 579 (1968). 7. Jergil, B., and G. H. Dixon, J. Biol. Chem., 245, 425 (197). 8. Wieland, O., and E. Siess, Proc. Nat. Acad. Sci. USA, 65, 947 (197). 9. Huttunen, J. K., D. Steinberg, and S. E. Mayer, Proc. Nat. Acad. Sci. USA, 67, 29 (197). 1. Kuo, J. F., and P. Greengard, Proc. Nat. Acad. Sci. USA, 64, 1349 (1969). 11. Gill, G. N., and L. D. Garren, Biochem. Biophys. Res. Commun., 39, 335 (197). 12. Gill, G. N., and L. D. Garren, J. Clin. nvest., 49, 34a (197). 13. Walton, G. M., and L. D. Garren, Biochemistry, 9, 4223 (197). 14. Lory,. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem., 193, 265 (1951). 15. Rodbard, D., and A. Chrambach, Anal. Biochem., in press. 16. Glynn,. M., and J. B. Chappell, Biochem. J., 9, 147 (1964). 17. Ferguson, K. A., Metabolism, 13, 985 (1964). 18. Hedrick, J. L., and A. J. Smith, Arch. Biochem. Biophys., 126, 155 (1968). 19. Martin, R. G., and B. N. Ames, J. Biol. Chem., 236, 1372 (1961). 2. Tao, M., M. L. Salas, and F. Lipmann, Proc. Nat. Acad. Sci. USA, 67, 48 (197). 21. Sartz, M. N., N.. Kaplan, M. E. Frech, Science, 123, 5 (1956). 22. Gerhart, J. C., and H. K. Schachman, Biochemistry, 4, 154 (1965). 23. Soderling, T. R., J. P. Hickenbottom, E. M. Reimann, F. L. Hunkeler, D. A. Walsh, and E. G. Krebs, J. Biol. Chem., 245, 6317 (197). 24. Garren, L. D., W. W. Davis, G. N. Gill, H. L. Moses, R. L. Ney, and R. M. Crocco, in Progress in Endocrinology, ed. C. Gual (Excerpta Medica Foundation, Amsterdam, 1969), pp Walton, G. M., G. N. Gill,. B. Abrass, and L. D. Garren, Proc. Nat. Acad. Sci. USA (1971), in press. 26. Kumon, A., H. Yamamura, and Y. Nishizuka, Biochem. Biophys. Res. Commun., 41, 129 (197). 27. Reimann, E. M., C.. Brostrom, J. D. Corbin, C. A. King, and E. G. Krebs, Biochem. Biophys. Res. Commun., 42, 187 (1971). ("