The Structural Effects of Endogenous and Exogenous Ca2+/Calmodulin on Phosphorylase Kinase

The activity of phosphorylase bkinase (PbK) is stimulated by Ca2+ ions, which act through its endogenous calmodulin subunit (δ), and further stimulated by the Ca2+-dependent binding of exogenous calmodulin (δ′). In contrast to their highly characterized effects on activity, little is known regarding the structural effects on the (αβγδ)4 PbK holoenzyme induced by Ca2+and δ′/Ca2+. We have used mono- and bifunctional chemical modifiers as conformational probes to compare how the two effectors influence the structure of the catalytic γ subunit and the interactions among all of the subunits. As determined by reductive methylation and carboxymethylation, Ca2+ increased the accessibility of the γ subunit; it also increased the formation by phenylenedimaleimide of an αγγ conjugate that is characteristic of activated conformations of PbK (Nadeau, O. W., Sacks, D. M., and Carlson, G. M. (1997) J. Biol. Chem. 272, 26196–26201); however, Ca2+ also had structural effects that were clearly distinct from other activators. Moreover, similar structural effects of Ca2+ were observed with PbK that had been activated by phosphorylation, consistent with the fact that such activation does not eliminate the catalytic dependence of the enzyme on Ca2+. Our results suggest tiers of conformational transitions in the activation of PbK, with the most fundamental being induced by Ca2+. Analysis of the various cross-linked conjugates formed in the presence of Ca2+ byo-phenylenedimaleimide orm-maleimidobenzoyl-N-hydroxysuccinimide ester showed that the binding of Ca2+ to the δ subunit triggers changes in the interactions among all subunits, including between protomers, indicating an extensive communication network throughout the PbK complex. Most of the structural effects of δ′/Ca2+were qualitatively similar to, but quantitatively greater than, the effects of Ca2+ alone; but δ′/Ca2+ also had distinct effects, especially involving cross-linking of the δ subunit. The activity of phosphorylase bkinase (PbK) is stimulated by Ca2+ ions, which act through its endogenous calmodulin subunit (δ), and further stimulated by the Ca2+-dependent binding of exogenous calmodulin (δ′). In contrast to their highly characterized effects on activity, little is known regarding the structural effects on the (αβγδ)4 PbK holoenzyme induced by Ca2+and δ′/Ca2+. We have used mono- and bifunctional chemical modifiers as conformational probes to compare how the two effectors influence the structure of the catalytic γ subunit and the interactions among all of the subunits. As determined by reductive methylation and carboxymethylation, Ca2+ increased the accessibility of the γ subunit; it also increased the formation by phenylenedimaleimide of an αγγ conjugate that is characteristic of activated conformations of PbK (Nadeau, O. W., Sacks, D. M., and Carlson, G. M. (1997) J. Biol. Chem. 272, 26196–26201); however, Ca2+ also had structural effects that were clearly distinct from other activators. Moreover, similar structural effects of Ca2+ were observed with PbK that had been activated by phosphorylation, consistent with the fact that such activation does not eliminate the catalytic dependence of the enzyme on Ca2+. Our results suggest tiers of conformational transitions in the activation of PbK, with the most fundamental being induced by Ca2+. Analysis of the various cross-linked conjugates formed in the presence of Ca2+ byo-phenylenedimaleimide orm-maleimidobenzoyl-N-hydroxysuccinimide ester showed that the binding of Ca2+ to the δ subunit triggers changes in the interactions among all subunits, including between protomers, indicating an extensive communication network throughout the PbK complex. Most of the structural effects of δ′/Ca2+were qualitatively similar to, but quantitatively greater than, the effects of Ca2+ alone; but δ′/Ca2+ also had distinct effects, especially involving cross-linking of the δ subunit. Phosphorylase b kinase (PbK) 1The abbreviations used are: PbK, phosphorylaseb kinase; CaM, calmodulin; mAb, monoclonal antibody; BtCaM, biotinylated calmodulin; o-PDM,o-phenylenedimaleimide; MBS,m-maleimidobenzoyl-N-hydroxysuccinimide ester; mdPDM,1′-(methylenedi-4,1-phenylene)bismaleimide; ANB·NOS,N-5-azido-2-nitrobenzoyloxysuccinimide; PAGE, polyacrylamide gel electrophoresis. is a regulatory enzyme of glycogenolysis that integrates metabolic, hormonal, and neural signals (for review, see Refs. 1Pickett-Gies C.R. Walsh D.A. Boyer P.D. Krebs E.G. 3rd Ed. The Enzymes. 17. Academic Press, Orlando, FL1986: 395-459Google Scholar and 2Heilmeyer Jr., L.M.G. Biochim. Biophys. Acta. 1991; 1094: 168-174Crossref PubMed Scopus (50) Google Scholar). In skeletal muscle, its dependence on Ca2+ ions for activity couples contraction with energy production (3Brostrom C.O. Hunkeler F.L. Krebs E.G. J. Biol. Chem. 1971; 246: 1961-1967Abstract Full Text PDF PubMed Google Scholar). The enzyme has four copies each of four different subunits (αβγδ)4. The γ subunit, with a mass of 44.7 kDa (4Reimann E.M. Titani K. Ericsson L.H. Wade R.D. Fischer E.H. Walsh K.A. Biochemistry. 1984; 23: 4185-4192Crossref PubMed Scopus (98) Google Scholar), is catalytic; and the α, β, and δ subunits, with masses of 138.4, 125.2 (5Zander N.F. Meyer H.E. Hoffmann-Posorske E. Crabb J.W. Heilmeyer Jr., L.M.G. Kilimann M.W. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2929-2933Crossref PubMed Scopus (75) Google Scholar, 6Kilimann M.W. Zander N.F. Kuhn C.C. Crab J.W. Meyer H.E. Heilmeyer Jr., L.M.G. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 9381-9385Crossref PubMed Scopus (74) Google Scholar), and 16.7 (7Grand R.J.A. Shenolikar S. Cohen P. Eur. J. Biochem. 1981; 113: 359-367Crossref PubMed Scopus (82) Google Scholar) kDa, respectively, are regulatory. The δ subunit is an endogenous molecule of tightly bound calmodulin (CaM) (8Cohen P. Burchell A. Foulkes J.G. Cohen P.T.W. Vanaman T.C. 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Full Text PDF PubMed Google Scholar), but of the activated of the enzyme the to stimulated by have been a of on the of Ca2+ to activity, little is known the of Ca2+ ions on the structure of the activated communication has that Ca2+ the accessibility of the of the γ subunit of enzyme M. Biochem. Biophys. PubMed Scopus Google Scholar). In to the of Ca2+ by the endogenous which is bound to PbK, Ca2+ is also for the binding of exogenous to a different on the holoenzyme S. Cohen P.T.W. Cohen P. Nairn A.C. Eur. J. Biochem. PubMed Scopus Google Scholar, FEBS Lett. PubMed Scopus Google Scholar, E.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). exogenous is and it in a of Cohen P. Eur. J. Biochem. PubMed Scopus Google Jr., L.M.G. Meyer H.E. Biochem. Scopus Google Scholar). binding of δ′/Ca2+ further activity that with Ca2+ especially for PbK E.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar, P. Eur. J. Biochem. PubMed Scopus Google Scholar). on cross-linking and binding Cohen P. Eur. J. Biochem. PubMed Scopus Google Scholar, Jr., L.M.G. Meyer H.E. Biochem. Scopus Google Scholar, P. Cohen P. E. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar), the and subunits to the binding for it is the γ subunit that is the binding for δ and are distinct and on different subunits, skeletal for δ in γ subunit Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) and in the holoenzyme P. Eur. J. Biochem. PubMed Scopus Google P. FEBS Lett. PubMed Scopus Google Scholar, K. M. M. S. Eur. J. Biochem. Scopus Google Scholar). The structural effects induced by the binding of δ′/Ca2+ to PbK are characterized the effects of Ca2+ has that δ′/Ca2+ the binding to PbK D. A. of the and of Phosphorylase of Scholar) of a monoclonal for an D.A. J. Biol. PubMed Scopus Google Scholar) that the of the binding of the γ subunit A.C. Full Text Full Text PDF PubMed Scopus Google Scholar) and also the of that subunit by J. Biol. Chem. Full Text PDF PubMed Google Scholar). As in the of the structural influence of Ca2+ M. Biochem. Biophys. PubMed Scopus Google Scholar), of effects were as of increased accessibility of of the γ subunit induced by the binding of δ′/Ca2+ D. A. of the and of Phosphorylase of Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). as the used in that the of δ′/Ca2+ on the structure of γ that it greater that by Ca2+ The of the and subunits also by with of and increased J. Biol. Chem. Full Text PDF PubMed Google Scholar). the on to by the binding of the on of a conformational in that subunit induced by δ′/Ca2+. In have used mono- and bifunctional as conformational probes to compare the effects of Ca2+ δ′/Ca2+ on the structure of the have further the of changes in the of the γ subunit induced by the two activators. The bifunctional have a for changes in the interactions of all subunits by by the binding of Ca2+ to the δ subunit of δ′/Ca2+ to the subunits. conformational probes have also been used to activation of the enzyme through other the structural changes induced by Ca2+ and δ′/Ca2+. The results Ca2+ has structural effects characteristic of other it also has distinct effects, and are observed with and activated δ′/Ca2+ for the most to the structural changes by Ca2+ of has been J. Scopus Google Scholar). and PbK used in were in the J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). were a of different PbK Phosphorylase b and were as J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), as were the four and their as Biochem. Biophys. PubMed Scopus Google Scholar). by with and by the of and Vanaman Vanaman T.C. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The of PbK, phosphorylase CaM, and were determined as J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Vanaman T.C. J. Biol. Chem. Full Text PDF PubMed Google Scholar). methylation of PbK for as PubMed Scopus Google Scholar). in the PbK protomers, and The enzyme also but in the presence of of PbK with for in the PbK protomers, and of Ca2+ and to used in the reductive methylation were also in the and of the kinase subunits were by of an of the an of by for the were on PubMed Scopus Google Scholar) and with were in and The of the determined on a and by in of for The and which of polyacrylamide and were with of and for cross-linking were by and to formation of of complexes of for the formed were of in a in to ester and the cross-linking for with the of In the of the it with PbK for in the and cross-linking by with for the of the other in the PbK and were used to the effects of Ca2+ in of and for the in used the with and the subunits were by as As observed with J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), cross-linking of PbK by and as by of the conjugates with enzyme on not not in of cross-linked by as J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of cross-linking for determined and to with in and in The used to of the various conjugates in a of CaM, of its the of BtCaM, to and with an from the masses of cross-linked were determined from with the of on J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). the of known by cross-linking PbK with J. Biol. Chem. Full Text PDF PubMed Google Scholar), used in formed by the chemical and as the mass to the masses of with masses greater that of the The for the phosphorylase activity of PbK, with and were as in the J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). for of the catalytic γ subunit induced by the binding of Ca2+ to endogenous by the binding of exogenous to the PbK with chemical modifiers as conformational probes with with δ′/Ca2+ to and the of the γ subunit by which is for and reductive methylation by which is for were used for the which were the has little activity, and it is a of the γ subunit for not which by Ca2+ and respectively, by and and by and reductive methylation of the γ subunit increased with Ca2+ its by and by however, for conformational δ′/Ca2+ had little that of Ca2+ suggest that of the activity of the enzyme as by Ca2+ the accessibility of and on the γ subunit which is consistent with the fact that catalytic activity is Ca2+-dependent with different and were for their to changes in subunit interactions to that were induced by Ca2+ and δ′/Ca2+. the effects of δ′/Ca2+ to on of Ca2+ with the Ca2+ the δ subunit. of enzyme by and in the formation of and mass cross-linked of known subunit of and of mass all four subunits, but in and b in and As J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), the subunit and of cross-linked conjugates were determined by their masses and The δ′/Ca2+ changes in the of subunit cross-linking of PbK by all of the and the cross-linking of α, β, and γ increased their of with of and with the and In with δ′/Ca2+ the subunit from with of being and of Ca2+ and δ′/Ca2+ on the cross-linking of phosphorylase the cross-linking PbK cross-linked with in the of effectors in the presence of Ca2+ to and for to the of cross-linking and with the with In to the of δ′/Ca2+ also the of subunit with cross-linked formed that with and with and and In all such the of δ in as is in a were to between the two by as As a throughout conjugates are as δ δ with the two are in a conjugate on cross-linking the conjugate is as In for the of a given that δ gel but not different gel the is also The regarding the presence in conjugates of is of for cross-linking in the presence of not Ca2+ the δ subunit is cross-linking by and the most changes in to on as probes for interactions in the holoenzyme by δ and which to the effects of Ca2+ and δ′/Ca2+ on subunit characterized as as the cross-linking of PbK by and cross-linking the for cross-linking were as J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), and the cross-linked formed by a of the were as of and of an αγγ and a with the mass of an but which with As J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 1984; Full Text PDF PubMed Google Scholar), the presence of most results from cross-linking the and subunits. In to the cross-linked the of that in in of the enzyme P. Eur. J. Biochem. PubMed Scopus Google Scholar) also showed the to with its as with the mAb, but it also showed highly with the of on mass and with the different have the from further The in the to in the in all by the with the subunit, with the enzyme cross-linked with cross-linking the of the cross-linked complexes the subunit The formed were a and an and in a and an conjugate that and δ and with a mass of kDa, The mass and of not to of subunits. other complexes the subunit that were in had masses that most to a but not with the of the subunit; in the the subunit. The cross-linking of the subunit, in that such cross-linking has been to of subunit J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 1984; Full Text PDF PubMed Google Scholar). complexes not with the and a of of subunit cross-linking are are other of subunits that are consistent with the observed masses and of the by the mAb, which is the of Biochem. 1991; PubMed Scopus Google Scholar), is in the of complexes the δ subunit, the of the four PbK subunits. of Ca2+ two changes in the cross-linking of PbK by a in the formation of αγγ which formed in in the of and formation of a as an with the and it with the formed in the presence of δ′/Ca2+. structural effects of as as of are in by Ca2+ of the formation of the αγγ a for conformations induced by other of PbK J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), also observed as the not however, the clearly observed with the as it the of in J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). changes induced by Ca2+ in the cross-linking by were increased formation of conjugates the a in formation of the The results with that the binding of Ca2+ by the δ subunit of PbK a conformational in the holoenzyme as a interactions among the α, and δ subunits. Ca2+ also interactions involving the subunit, cross-linking with which conjugates of and PbK by Ca2+ and and γ γ structural changes induced by Ca2+ and δ′/Ca2+ in a given conjugate in the γ subunit are with for the two of the The for the effects of Ca2+ is the of the for the effects of δ′/Ca2+ is the presence of The the changes observed the with its an in with a greater formation of conjugate not observed in different of the δ subunit conjugate not a in with a further exogenous a of the and not regarding the of each conjugate were on as in a The structural changes induced by Ca2+ and δ′/Ca2+ in a given conjugate in the γ subunit are with for the two of the The for the effects of Ca2+ is the of the for the effects of δ′/Ca2+ is the presence of The the changes observed the with its an in with a greater formation of conjugate not observed in different of the δ subunit conjugate not a in with a further exogenous a of the and not regarding the of each conjugate were on as Ca2+ in the cross-linking with changes in the of the cross-linked two of the observed changes are not of two is a that in the of the as that were from further of its The the conjugate the and δ subunits, which the cross-linking Ca2+. The changes in the cross-linked δ the and subunits. The of is an formation is increased by for the Ca2+ an in The in which with the mAb, the cross-linking in the of Ca2+. results suggest that Ca2+ a conformational that results in the cross-linking of different of δ to the subunit, the on δ for the with the results observed with Ca2+ including δ′/Ca2+ in the cross-linking of PbK by changes were and were changes in the of other especially in the an in the of the to and as as of and a in the of the formed in the presence of δ′/Ca2+ that between the subunit and the which as were not a The complexes from different of and of the subunit in the the in the complexes δ endogenous exogenous used as the for a Vanaman T.C. J. Biol. Chem. Full Text PDF PubMed Google Scholar), which the PbK holoenzyme in with not the of the endogenous δ subunit for exogenous has been to especially in the presence of Ca2+ Cohen P. Eur. J. Biochem. PubMed Scopus Google Scholar), the with PbK for to cross-linking to eliminate further of in the presence of in the cross-linking and the as were observed with as with that especially given that it is not observed in the of exogenous that were observed to in the of exogenous CaM, such as with the formed in the presence of that δ′/Ca2+ the cross-linking of from δ to that and In the which is formed in the presence and of not to with that it is of is that δ′/Ca2+ a Ca2+ in the of observed with it a in the of that with results suggest that in the different of δ are cross-linked in the presence of δ′/Ca2+ in the presence of Ca2+ that exogenous the endogenous of In contrast to its effects on the cross-linking by δ′/Ca2+ with Ca2+ changes in the cross-linking by The two most changes were a in the of formed and in the of observed in the In the however, the mass and of the other and not their As with of as not however, the and formed in its presence not with the not that not the formation of by and the cross-linked in and greater with the In the J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) that PbK is cross-linked by in the presence of the and it activated of the effectors to the were by In formation of αγγ increased with Ca2+ and δ′/Ca2+ are that also increased formation of αγγ cross-linking in the presence of effectors the enzyme in an enzyme cross-linked in the presence of Ca2+ δ′/Ca2+ and a and respectively, in its activity, of the effectors In the enzyme not activation with of Ca2+ and δ′/Ca2+ the activation in to cross-linking results from the of on the Ca2+ and δ′/Ca2+ complexes of with which does not αγγ not PbK its Ca2+ not with different the that formation of the αγγ by is a for of PbK J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The activation of PbK is through its Walsh D.A. J. Biol. Chem. Full Text PDF PubMed Google Scholar), but activity which that effects on which were observed with PbK, also with We the of Ca2+ and δ′/Ca2+ to the of the γ subunit of enzyme to the results with the enzyme Ca2+ and the of the γ subunit by and however, in that observed with the enzyme not cross-linking as the conformational Ca2+ and δ′/Ca2+ had the effects on the cross-linking of and PbK, increased formation of αγγ and and of as with PbK, the two complexes were formed in the presence of as not with that in and in PbK, has similar effects on as the the of and PbK but with The cross-linking in the of effectors were the and Ca2+ and δ′/Ca2+ had the influence on the cross-linking of of the with the in the for the and complexes formed in the presence of Ca2+ In Ca2+ the structure of the γ subunit of PbK, as by the accessibility by carboxymethylation, and the cross-linking of the effects are similar to observed with PbK and that the effects of Ca2+ on PbK are of its of The results with δ′/Ca2+ that does not the binding of exogenous its being cross-linked to the regulatory subunits its to in the cross-linking of the δ subunit however, the influence of δ′/Ca2+ on the chemical of the γ subunit with the The influence of Ca2+ ions on the of the and bifunctional chemical probes used in to PbK extensive between the δ subunit and the subunits of the of its of the activity of the holoenzyme by the γ subunit is Ca2+-dependent and interactions between the γ and δ subunits have been J. Biol. Chem. Full Text PDF PubMed Google Cohen P. Eur. J. Biochem. PubMed Scopus Google Scholar), the of Ca2+ to the of the catalytic γ subunit, as by chemical and reductive methylation of γ were increased by Ca2+. Ca2+ also the formation of cross-linked complexes γ that the increased of γ increased accessibility of subunit, increased of increased accessibility of γ in to Ca2+ is also consistent with that Ca2+ the binding to the holoenzyme of of the γ subunit M. Biochem. Biophys. PubMed Scopus Google Scholar) and the of PbK for its phosphorylase b D.A. Biochemistry. PubMed Scopus Google Scholar). the bifunctional Ca2+ the formation of cross-linked the and subunits to the subunit, Ca2+ increased formation of αγγ and the conjugate by and a influence by Ca2+ on the interactions among the α, and δ subunits an activation for the holoenzyme that a in which the binding of Ca2+ to δ interactions by for such a from the of and J. Biol. Chem. Full Text PDF PubMed Google Scholar), that the activity of the showed a for Ca2+ the complex. however, that Ca2+ also the subunit, as by the results with by the of as a of J. Biol. Chem. Full Text PDF PubMed Google Scholar). Ca2+ the to other of PbK of formation by of the αγγ it also has effects that are distinct from of the other activators. formation of the by J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) stimulated of the γ subunit J. Scholar) the of PbK for phosphorylase b D.A. Biochemistry. PubMed Scopus Google Scholar). were the structural effects of Ca2+ observed in were the for and similar of of the γ subunit and similar of cross-linking by the Ca2+ has structural effects that with and activated PbK is consistent with the fact that all other of the enzyme its Ca2+-dependent activity but not eliminate the for Ca2+. a of conformational transitions to the activation of PbK, with the most fundamental being that induced by the binding of Ca2+ to the δ subunit. transitions by to the subunits and to and C.R. Walsh D.A. Boyer P.D. Krebs E.G. 3rd Ed. The Enzymes. 17. Academic Press, Orlando, FL1986: 395-459Google binding of to Cohen P. Eur. J. Biochem. PubMed Scopus Google Scholar, Jr., L.M.G. Meyer H.E. Biochem. Scopus Google Scholar, P. Cohen P. E. J. Biol. Chem. 1991; Full Text PDF PubMed Google binding of to FEBS Lett. PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google and binding of in to γ J. Biol. Chem. Full Text PDF PubMed Google Scholar, Biochemistry. 1991; PubMed Scopus Google the The conformations of the holoenzyme different of activity, the different of on the γ subunit from among the of the α, β, and δ subunits. dependence on Ca2+ is not by the extensive communication network between the δ subunit and the subunits that in that the Ca2+ to and by the subunits. a activation in been by Cohen P. Eur. J. Biochem. PubMed Scopus Google Scholar), that and the a for Ca2+. has been that the activation by of the γ subunit is not to the of but also to an of γ by J. Biol. Chem. Full Text PDF PubMed Google Scholar), which of the other known the Ca2+ dependence of the PbK In contrast to the of the δ subunit, which is to through the γ subunit, that the binding for δ′/Ca2+ of the and subunits Cohen P. Eur. J. Biochem. PubMed Scopus Google Scholar, Jr., L.M.G. Meyer H.E. Biochem. Scopus Google Scholar, P. Cohen P. E. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). Our cross-linking results with and as in that and were We that δ′/Ca2+ the cross-linking of and on the and subunits to an but were to that in J. Biol. Chem. Full Text PDF PubMed Google Scholar). the of conjugates the δ subunit, most of the effects of δ′/Ca2+ on as by were qualitatively similar to, but quantitatively greater than, induced by Ca2+ consistent with effects with and enzyme indicating that δ′/Ca2+ to PbK and conformational changes that are characteristic of its effects on the activity of enzyme is by δ′/Ca2+ E.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar, P. Eur. J. Biochem. PubMed Scopus Google Scholar). its effects on activity, that of the of the γ subunit by with Ca2+ with PbK with In other with PbK, δ′/Ca2+ also has been to the of the γ subunit by J. Biol. Chem. Full Text PDF PubMed Google Scholar) and to the binding of an monoclonal D. A. of the and of Phosphorylase of Scholar). results the of γ and its interactions with and are consistent with an in its accessibility induced by the binding of an greater that by Ca2+ its effects on the γ subunit δ′/Ca2+ also the formation of cross-linked conjugates involving all four subunits of the PbK holoenzyme In the of the δ subunit, results suggest that different of it to the and subunits in the presence of δ′/Ca2+ in the presence of Ca2+ the that exogenous influence the of the endogenous of