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• W1995314021 abstract "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. Nairn A.C. FEBS Lett. 1978; 92: 287-293Crossref PubMed Scopus (400) Google Scholar) that is undoubtedly responsible for the Ca2+ dependence of the enzyme activity, given that complexes containing the δ subunit (γδ, αγδ, and PbK) are stimulated by Ca2+ (9Chan K.-F.J. Graves D.J. J. Biol. Chem. 1982; 257: 5948-5955Abstract Full Text PDF PubMed Google Scholar), whereas the free γ subunit is not (10Kee S.M. Graves D.J. J. Biol. Chem. 1986; 261: 4732-4737Abstract Full Text PDF PubMed Google Scholar). Two distinct, high affinity binding domains for CaM/Ca2+ have been identified near the COOH terminus of the γ subunit (11Dasgupta M. Honeycutt T. Blumenthal D.K. J. Biol. Chem. 1989; 264: 17156-17163Abstract Full Text PDF PubMed Google Scholar); the δ subunit has been shown to interact with γ in the holoenzyme (12Picton C. Klee C.B. Cohen P. Eur. J. Biochem. 1980; 111: 553-561Crossref PubMed Scopus (71) Google Scholar); and, CaM/Ca2+ stimulates the activity of free isolated γ subunit (10Kee S.M. Graves D.J. J. Biol. Chem. 1986; 261: 4732-4737Abstract Full Text PDF PubMed Google Scholar). Thus, the primary site of interaction for the δ subunit within the holoenzyme is presumed to be on the catalytic γ subunit. PbK can be activated through a variety of mechanisms, including phosphorylation (13Walsh D.A. Perkins J.P. Brostrom C.O. Ho E.S. Krebs E.G. J. Biol. Chem. 1971; 246: 1968-1976Abstract Full Text PDF PubMed Google Scholar), proteolysis (14Cohen P. Eur. J. Biochem. 1973; 34: 1-14Crossref PubMed Scopus (508) Google Scholar), and allosterically by ADP (15Cheng A. Fitzgerald T.J. Carlson G.M. J. Biol. Chem. 1985; 260: 2535-2542Abstract Full Text PDF PubMed Google Scholar), but none of the variously activated forms of the enzyme loses the capacity to be stimulated by Ca2+ions. Although there have been a large number of studies on the relationship of Ca2+ to activity, little is known about the effect of Ca2+ ions on the structure of the holoenzyme, either activated or nonactivated. A recent communication has suggested that Ca2+ increases the accessibility of specific loci within the COOH-terminal region of the γ subunit of nonactivated enzyme (16Wangsgard W.P. Dasgupta M. Blumenthal D.K. Biochem. Biophys. Res. Commun. 1997; 230: 179-183Crossref PubMed Scopus (4) Google Scholar). In addition to the stimulatory effect of Ca2+ mediated by the endogenous CaM (δ subunit), which is essentially bound irreversibly to PbK, Ca2+ is also required for the reversible binding of exogenous CaM to a different site on the holoenzyme (17Shenolikar S. Cohen P.T.W. Cohen P. Nairn A.C. Perry S.V. Eur. J. Biochem. 1979; 100: 329-337Crossref PubMed Scopus (167) Google Scholar, 18DePaoli-Roach A.A. Gibbs J.B. Roach P.J. FEBS Lett. 1979; 105: 321-324Crossref PubMed Scopus (32) Google Scholar, 19Walsh K.X. Millikin D.M. Schlender K.K. Reimann E.M. J. Biol. Chem. 1980; 255: 5036-5042Abstract Full Text PDF PubMed Google Scholar). This exogenous CaM is termed δ′, and it binds in a stoichiometry of one δ′ molecule/each αβγδ protomer (12Picton C. Klee C.B. Cohen P. Eur. J. Biochem. 1980; 111: 553-561Crossref PubMed Scopus (71) Google Scholar,20Heilmeyer Jr., L.M.G. Gerschinski A.M. Meyer H.E. Jennissen H.P. Mol. Cell. Biochem. 1993; 127/128: 19-30Crossref Scopus (8) Google Scholar). This binding of δ′/Ca2+ further stimulates activity past that obtained with Ca2+ alone, especially for nonactivated PbK (19Walsh K.X. Millikin D.M. Schlender K.K. Reimann E.M. J. Biol. Chem. 1980; 255: 5036-5042Abstract Full Text PDF PubMed Google Scholar, 21Cohen P. Eur. J. Biochem. 1980; 111: 563-574Crossref PubMed Scopus (101) Google Scholar). Based on cross-linking and peptide binding studies (12Picton C. Klee C.B. Cohen P. Eur. J. Biochem. 1980; 111: 553-561Crossref PubMed Scopus (71) Google Scholar, 20Heilmeyer Jr., L.M.G. Gerschinski A.M. Meyer H.E. Jennissen H.P. Mol. Cell. Biochem. 1993; 127/128: 19-30Crossref Scopus (8) Google Scholar, 22James P. Cohen P. Carafoli E. J. Biol. Chem. 1991; 266: 7087-7091Abstract Full Text PDF PubMed Google Scholar), both the α and β subunits apparently contribute to the binding site for δ′/Ca2+, although it is again the γ subunit that is ultimately stimulated. Even though the binding sites for δ and δ′ are distinct and on different subunits, skeletal muscle troponin C can substitute for both δ in activating isolated γ subunit (23Paudel H.K. Carlson G.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7285-7289Crossref PubMed Scopus (16) Google Scholar) and δ′ in activating the holoenzyme (21Cohen P. Eur. J. Biochem. 1980; 111: 563-574Crossref PubMed Scopus (101) Google Scholar,24Cohen P. Picton C. Klee C.B. FEBS Lett. 1979; 104: 25-30Crossref PubMed Scopus (47) Google Scholar, 25Yoshikawa K. Usui H. Imazu M. Takeda M. Ebashi S. Eur. J. Biochem. 1983; 136: 413-419Crossref Scopus (7) Google Scholar). The structural effects induced by the binding of δ′/Ca2+ to PbK are only slightly more fully characterized than the effects of Ca2+ alone. This laboratory has found that δ′/Ca2+ increases the binding to nonactivated PbK (26.Wilkinson, D. A. (1993) Immunochemical Studies of the Structure and Function of Phosphorylase Kinase. Ph.D. dissertation, pp. 107–108, University of Tennessee, Memphis.Google Scholar) of a monoclonal antibody specific for an epitope (27Wilkinson D.A. Norcum M.T. Fitzgerald T.J. Marion T.N. Tillman D.M. Carlson G.M. J. Mol. Biol. 1997; 265: 319-329Crossref PubMed Scopus (33) Google Scholar) that occurs at the base of the peptide binding lobe of the γ subunit (28Owen D.J. Noble M.E.M. Garman E.F. Papageorgiou A.C. Johnson L.N. Structure. 1995; 3: 467-482Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar) and also increases the incorporation of putrescine into that subunit by transglutaminase (29Nadeau O.W. Carlson G.M. J. Biol. Chem. 1994; 269: 29670-29676Abstract Full Text PDF PubMed Google Scholar). As in the case of the structural influence of Ca2+ alone cited above (16Wangsgard W.P. Dasgupta M. Blumenthal D.K. Biochem. Biophys. Res. Commun. 1997; 230: 179-183Crossref PubMed Scopus (4) Google Scholar), both of these effects were interpreted as manifestations of increased accessibility of particular regions of the γ subunit induced by the binding of δ′/Ca2+ (26.Wilkinson, D. A. (1993) Immunochemical Studies of the Structure and Function of Phosphorylase Kinase. Ph.D. dissertation, pp. 107–108, University of Tennessee, Memphis.Google Scholar, 29Nadeau O.W. Carlson G.M. J. Biol. Chem. 1994; 269: 29670-29676Abstract Full Text PDF PubMed Google Scholar). Inasmuch as the transglutaminase used in that study required Ca2+, the effect of δ′/Ca2+ on the structure of γ that it detected was necessarily greater than that caused by Ca2+ alone. The incorporation of putrescine into the α and β subunits was also influenced by δ′/Ca2+, with modification of α decreased and β increased (29Nadeau O.W. Carlson G.M. J. Biol. Chem. 1994; 269: 29670-29676Abstract Full Text PDF PubMed Google Scholar). Although the effect on α could be due to direct steric inhibition caused by the specific binding of δ′/Ca2+, the stimulatory effect on modification of β indicates a conformational change in that subunit induced by δ′/Ca2+. In this study, we have used mono- and bifunctional modifying agents as conformational probes to compare the effects of Ca2+ aloneversus δ′/Ca2+ on the structure of PbK. Using the monofunctional reagents, we have further addressed the issue of relative changes in the conformation of the γ subunit induced by the two activators. The bifunctional reagents have allowed a screening for relative changes in the interactions of all subunits (as detected by cross-linking) initiated by the binding of Ca2+ to the δ subunit or of δ′/Ca2+ to the α/β subunits. These conformational probes have also been used to evaluate whether activation of the enzyme through other mechanisms alters the structural changes induced by Ca2+ and δ′/Ca2+. The results obtained indicate that, although Ca2+ has structural effects characteristic of other activators, it also has distinct effects, and these are observed with both nonactivated and activated enzyme; δ′/Ca2+ appears, for the most part, to amplify the structural changes brought about by Ca2+ alone. A preliminary account of this work has been published (30Nadeau O.W. Sacks D.B. Carlson G.M. FASEB J. 1995; 9 (abstr.): 1304Crossref Scopus (25) Google Scholar). Nonactivated and autophosphorylated PbK used in this study were described in the accompanying report (31Nadeau O.W. Sacks D.B. Carlson G.M. J. Biol. Chem. 1997; 272: 26196-26201Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). All experiments described herein were repeated a minimum of three times using three different PbK preparations. Phosphorylase b and bovine serum albumin were obtained as described (31Nadeau O.W. Sacks D.B. Carlson G.M. J. Biol. Chem. 1997; 272: 26196-26201Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar), as were the four mAbs and their detection conjugates. Bovine brain CaM was isolated as described previously (32Gopalakrishna R. Anderson W.B. Biochem. Biophys. Res. Commun. 1982; 104: 830-836Crossref PubMed Scopus (718) Google Scholar). Biotinylated CaM (at Lys-94) was prepared by treatment with N-hydroxysuccinimidyl biotin at pH 6.0 and purified over DEAE-Spherogel by the procedure of Mann and Vanaman (33Mann D.M. Vanaman T.C. J. Biol. Chem. 1989; 264: 2373-2378Abstract Full Text PDF PubMed Google Scholar). The concentrations of PbK, phosphorylase b, bovine brain CaM, and BtCaM were determined as described (31Nadeau O.W. Sacks D.B. Carlson G.M. J. Biol. Chem. 1997; 272: 26196-26201Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 33Mann D.M. Vanaman T.C. J. Biol. Chem. 1989; 264: 2373-2378Abstract Full Text PDF PubMed Google Scholar). Reductive methylation of PbK was carried out for 30 min at 30 °C essentially as described (34Jentoft N. Dearborn D.G. Methods Enzymol. 1983; 91: 570-579Crossref PubMed Scopus (174) Google Scholar). Final concentrations in the reaction were: 1.73 μm PbK αβγδ protomers, 50 mm Hepes, pH 6.8 or 8.2, 1.0 mm EDTA, 2.5 mmNaCNBH3, and 2.92 mm[3H]CH2O (1.28 Ci/mol, American Radiochemicals, St. Louis). The enzyme was also modified under identical conditions, but in the presence of 1.25 mmCaCl2 ± 1.73 μm CaM. Carboxymethylation of PbK with [3H]iodoacetic acid was carried out for 20 min at 30 °C. Final concentrations in the reaction were: 1.73 μm PbK protomers, 50 mmHepes, pH 6.8 or 8.2, 1.0 mm EDTA, and 2.5 mm[3H]iodoacetic acid (28.6 Ci/mol, American Radiochemicals). Concentrations of Ca2+ and CaM identical to those used in the reductive methylation were also used, where indicated, in the alkylation reaction. Methylation and carboxymethylation of the kinase subunits were quenched by dilution of an aliquot of the assay mixture into an equivalent volume of SDS buffer (0.125 m Tris, pH 6.8, 20% glycerol, 5% β-mercaptoethanol, 4% SDS), followed by brief mixing. After heating at 80 °C for 10 min, the samples were run on SDS-polyacrylamide gradient (4–20%) gels (35Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207165) Google Scholar) and stained with Coomassie Blue. All gels were destained in 40% methanol, 10% acetic acid (2 h) and 7% acetic acid, 4% methanol (15 h). The integrated optical density of the protein bands was determined on a BioImage whole band analyzer. Each band was then excised, solubilized, and decolorized by heating in 250 μl of 30% H2O2 for 2 h at 80 °C. The samples and blanks, which contained equivalent amounts of polyacrylamide and H2O2, were diluted with 7 ml of Ecoscint scintillation mixture (ICN), and the3H content was determined. Standard conditions for cross-linking were developed by optimizing time and cross-linker concentration to allow formation of sufficient amounts of intermediate sized complexes of interest for convenient quantification, while the amounts formed were still capable of increasing or decreasing in a linear manner in response to effectors. With o-phenylenedimaleimide (o-PDM, 17.3 μm),m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS, 8.6 μm), and 1,1′-(methylenedi-4,1-phenylene)bismaleimide (mdPDM, 17.3 μm), the cross-linking was carried out at 30 °C for 2 min with the indicated final concentrations of cross-linkers. In the case of the photocross-linkerN-5-azido-2-nitrobenzoyloxysuccinimide (ANB·NOS, 86.0 μm), it was first incubated with PbK for 30 min in the dark, and cross-linking was then initiated by irradiation with UV light (360 nm) for 1 min at 4 °C. Besides cross-linkers, the final concentrations of the other components in the reactions were: 1.73 μm PbK αβγδ protomer, 50 mm Hepes, pH 8.2, and 1.0 mm EDTA. These same conditions were used to test the effects of Ca2+ (1.25 mmCaCl2, i.e. 250 μm in excess of chelator), Ca2+/CaM (1.25 mm/1.73 μm), and Ca2+/BtCaM (1.25 mm/1.73 μm), except for the experiment shown in Fig. 2, where CaM was used at the indicated concentrations. Cross-linking was quenched with SDS buffer, and the subunits were resolved by SDS-PAGE as described above. As was observed previously with PDM (31Nadeau O.W. Sacks D.B. Carlson G.M. J. Biol. Chem. 1997; 272: 26196-26201Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar), cross-linking of PbK by MBS and ANB·NOS was intramolecular, as judged by coelution of the indicated conjugates with native enzyme on Sepharose 6B (data not shown); mdPDM was not evaluated in this aspect. Subunit composition of cross-linked species was analyzed by Western blotting using subunit-specific mAbs as described previously (29Nadeau O.W. Carlson G.M. J. Biol. Chem. 1994; 269: 29670-29676Abstract Full Text PDF PubMed Google Scholar, 31Nadeau O.W. Sacks D.B. Carlson G.M. J. Biol. Chem. 1997; 272: 26196-26201Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The extent of cross-linking for individual bands was determined using transmissive and reflective densitometry to measure bands stained with Coomassie Blue in SDS-PAGE and alkaline phosphatase in Western blots, respectively. The electroblotting conditions used to achieve quantitative transfer of the various conjugates onto nitrocellulose resulted in a relatively low recovery of monomeric CaM, presumably because of its low molecular weight. For the detection of BtCaM, streptavidin-alkaline phosphatase (Southern Biotechnology) was exposed to blots and assayed with an alkaline phosphatase kit from Bio-Rad, following the supplier's suggested protocol. Apparent molecular masses of cross-linked species were determined from comparison with the migration of commercial protein standards (29–250 kDa) on 4–20% linear gradient PAGE (31Nadeau O.W. Sacks D.B. Carlson G.M. J. Biol. Chem. 1997; 272: 26196-26201Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 34Jentoft N. Dearborn D.G. Methods Enzymol. 1983; 91: 570-579Crossref PubMed Scopus (174) Google Scholar). Also, the migration of known αβ dimer, prepared by cross-linking PbK with transglutaminase (29Nadeau O.W. Carlson G.M. J. Biol. Chem. 1994; 269: 29670-29676Abstract Full Text PDF PubMed Google Scholar), was used in identifying αβ dimers formed by the chemical cross-linkers and as the maximum molecular mass standard (no attempt was made to estimate the masses of oligomers with apparent molecular masses greater than that of the αβ dimer). The assays at pH 6.8 for the phosphorylase conversion activity of PbK, with and without cross-linking, were performed exactly as described in the previous report (31Nadeau O.W. Sacks D.B. Carlson G.M. J. Biol. Chem. 1997; 272: 26196-26201Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). To screen for perturbation of the catalytic γ subunit induced by the binding of Ca2+ to endogenous CaM (δ) or by the binding of exogenous CaM/Ca2+ (δ′) to the (αβγδ)4holoenzyme, PbK was incubated with radioactive, general chemical modifiers as conformational probes either alone (control), with Ca2+, or with δ′/Ca2+ (equimolar to αβγδ protomers), and the incorporation of label into the γ subunit was followed. Carboxymethylation by iodoacetate ([3H]ICH2CO2−), which is selective for thiols, and reductive methylation by formaldehyde ([3H]CH2O), which is selective for amines, were used for the modifications, which were performed at both pH 6.8, where the nonactivated enyzme has little activity, and pH 8.2, where it is nearly fully active. At either pH, there was a linear carboxymethylation of the γ subunit for 20 min (data not shown), which was enhanced by Ca2+ and δ′/Ca2+, respectively, by 2.1 × and 2.8 × at pH 6.8 and by 1.8 × and 2.4 × at pH 8.2 (Fig.1 A). Similarly, under conditions where reductive methylation of the γ subunit increased linearly with time, Ca2+ enhanced its modification by 1.5 × at pH 6.8 and by 2.0 × at pH 8.2; however, for this particular conformational probe, δ′/Ca2+ had little effect over that of Ca2+ alone at either pH (Fig.1 B). These data suggest that regardless of the activity state of the enzyme as defined by pH, Ca2+ increases the accessibility of at least one thiol group and multiple amine groups on the γ subunit (Fig. 1), which is consistent with the fact that catalytic activity is Ca2+-dependent at both pH values. Chemical cross-linkers with different chemistries and spans were evaluated for their ability to detect changes in subunit interactions (in addition to those described above) that were induced by Ca2+ and δ′/Ca2+. Because the effects of δ′/Ca2+ seemed to be overlaid on those of Ca2+ alone (Fig. 1), our initial screening was with δ′/Ca2+, where the excess Ca2+ would simultaneously saturate the δ subunit. Cross-linking of control enzyme by o-PDM, MBS, ANB·NOS, and mdPDM (Fig.2) resulted in the formation of low and intermediate molecular mass cross-linked species of known subunit composition, of αβ dimers, and of high molecular mass oligomers containing all four subunits, but in indeterminate amounts (denoted asa and b in Figs. 2, 3 and 6). As before (29Nadeau O.W. Carlson G.M. J. Biol. Chem. 1994; 269: 29670-29676Abstract Full Text PDF PubMed Google Scholar, 31Nadeau O.W. Sacks D.B. Carlson G.M. J. Biol. Chem. 1997; 272: 26196-26201Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar), the subunit composition and stoichiometry of cross-linked conjugates were determined by their masses and cross-reactivities against subunit-specific mAbs. The δ′/Ca2+ promoted significant changes in the rates of subunit cross-linking of PbK by all of the cross-linkers. With o-PDM and mdPDM, the cross-linking of α, β, and γ was increased (i.e. their rates of disappearance increased), with this effect more pronounced at higher concentrations of δ′ and with the longer cross-linker (Fig. 2,B and E). In contrast, with ANB·NOS or MBS, δ′/Ca2+ protected the β subunit from cross-linking, with higher concentrations of δ′ being more effective (Fig. 2,C and D).Figure 6Effect of Ca2+ and δ′/Ca2+ on the cross-linking of autophosphorylated phosphorylase kinase. Under the standard cross-linking conditions, autophosphorylated PbK (control, lane 1) was cross-linked with o-PDM (panel A) or MBS (panel B) in the absence of effectors (lane 2) or in the presence of Ca2+ (lane 3) or δ′/Ca2+(lane 4) or BtCaM/Ca2+ (lane 5), subjected to SDS-PAGE, and stained for protein to determine the extent of cross-linking or electroblotted onto nitrocellulose and probed with the anti-CaM mAb or with streptavidin-alkaline phosphatase (Avidin-AP).View Large Image Figure ViewerDownload Hi-res image Download (PPT) In addition to altering the rates of cross-linking, δ′/Ca2+ also changed the patterns of subunit cross-linking, with new cross-linked species formed that contained CaM: αγδδ with o-PDM and βCaM with MBS (Fig. 2, B and C, and Figs. 3 and4). In all such complexes, the CaM could, of course, represent either δ or δ′; in many cases, as is described in a later section, we were able to distinguish between the two alternatives by utilizing derivatized CaM (BtCaM) as δ′. As a result, whenever possible throughout this report, conjugates are specifically denoted as containing either δ or δ′ (e.g. αγδδ above); if δ could be present with δ′ or if the two are alternatively present in a conjugate (depending on cross-linking conditions), then the conjugate is simply denoted as containing CaM (e.g. βCaM above). In figures, for the labeling of a given band that might contain δ under one condition (one gel lane) but not another (a different gel lane), the “CaM” nomenclature is also used. The complication regarding the presence in conjugates of δversus δ′ is relevant, of course, only for cross-linking carried out in the presence of δ′/Ca2+, not Ca2+ alone, where only the δ subunit is present. Because cross-linking by o-PDM and MBS demonstrated the most significant changes in response to δ′/Ca2+, we focused primarily on these cross-linkers as probes for targeting interactions in the holoenzyme mediated by δ and δ′. To establish reference data against which to determine the effects of Ca2+ and δ′/Ca2+ on subunit interactions, we characterized as fully as possible the cross-linking of nonactivated, control PbK by o-PDM and MBS. For o-PDM (4.8 Å cross-linking span), the conditions for cross-linking were as described previously (31Nadeau O.W. Sacks D.B. Carlson G.M. J. Biol. Chem. 1997; 272: 26196-26201Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar), and correspondingly, the cross-linked species formed by a 10-fold molar excess of the cross-linker over protomers were as before, namely predominant doublets of αβ dimers and βγγ trimers, plus small amounts of an αγγ trimer and a doublet with the mass of an αδ dimer, but which cross-reacted only with anti-α mAb (Fig. 3, lane 2). As was discussed previously (31Nadeau O.W. Sacks D.B. Carlson G.M. J. Biol. Chem. 1997; 272: 26196-26201Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 36Fitzgerald T.J. Carlson G.M. J. Biol. Chem. 1984; 259: 3266-3274Abstract Full Text PDF PubMed Google Scholar), the presence of doublets most likely results from intramolecular cross-linking within the large α and β subunits. In addition to the cross-linked complexes, the degradation product of α (αfrag) that commonly occurs in small amounts in purified preparations of the enzyme (14Cohen P. Eur. J. Biochem. 1973; 34: 1-14Crossref PubMed Scopus (508) Google Scholar) also showed the ability to cross-react with more than one mAb; its predominant interaction was, as expected, with the anti-α mAb, but it also showed highly variable cross-reactivity with the anti-CaM mAb (Fig. 3, lane 2). Because of this cross-reactivity, any species that, based on mass and cross-reactivity with the different mAbs, could have possibly contained the αfrag was eliminated from further analysis. The variability in the cross-reactivity may be related to epitope presentation in the blotting process itself because in all cases there was no cross-reactivity by the anti-CaM mAb with the intact α subunit, only with the αfrag. When nonactivated, control enzyme was cross-linked with MBS (9.9 Å cross-linking span), the majority of the cross-linked complexes contained the β subunit (Fig. 4, lane 2). The predominant species formed were a βγγ trimer (5.0% error) and an αβ dimer (2.3% error) and in smaller amounts, a ββ dimer (masstheor = 250 kDa; 3.0% error) and an only partially classified conjugate termed βX225 that contained β and δ and migrated with a mass of 225 kDa, slightly slower than βγγ. The mass and cross-reactivity of this last complex do not correspond to any straightforward combination of subunits. Several other complexes containing the β subunit that were present in only small amounts had masses that most closely corresponded to a βδ dimer (masstheor = 142 kDa) but did not cross-react with the anti-CaM" @default.
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• W1995314021 title "The Structural Effects of Endogenous and Exogenous Ca2+/Calmodulin on Phosphorylase Kinase" @default.
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