Activation and inhibition of phosphorylase kinase by monospecific antibodies against preparatively isolated alpha, beta and gamma subunits.
Homogeneous alpha and beta subunits were isolated for the first time in preparative amounts in the presence of sodium dodecyl sulfate. Analysis by analytical polyacrylamide electrophoresis, sedimentation velocity, and immunoprecipitation with monospecific antibodies indicated homogeneity. The apparent molecular masses of the purified subunits as determined electrophoretically in the presence of dodecyl sulfate are: alpha = 140.2 +/- 2.1 kDa and beta = 123 +/- 1.8 kDa. Amino acid analyses show that per 100 mol amino acid the alpha-subunit has a higher serine content (Ser alpha/Ser beta = 1.32, Ser alpha/Ser gamma = 1.42) and a lower aspartic acid/asparagine (Asx) content (AsX alpha/Asx beta = 0.76, Asx alpha/Asx gamma = 0.90) than the beta and gamma subunits. Monospecific antibodies against the purified alpha, beta and gamma subunits were produced in sheep [J. Immunol. Methods (1984) 70, 193-209] and their action on the catalytic activity of non-activated phosphorylase kinase assayed. It can be shown that certain antibody fractions of anti-alpha, anti-beta and anti-gamma inhibit the Ca2+-dependent and Ca2+-independent activity at pH 6.8 as well as at pH 8.2. Other antibody fractions against the beta and gamma subunits however activate the Ca2+-dependent activity at pH 6.8 threefold to fourfold, although they inhibit the activity at pH 8.2. These antibodies lead to a ca. five fold increase in the pH 6.8/8.2 activity ratio. Activating anti-beta can even overcome the inhibitory action of anti-alpha at pH 6.8. A kinetic analysis shows that inhibition is the result of a mixed type mechanism whereas activation is due to a fivefold to tenfold increase in V for phosphorylase b. The results illustrate the importance of possibly large, concerted conformational changes of phosphorylase kinase. It appears that activation or inhibition can be triggered by the antibody binding to conformational determinants of a single subunit type leading to a structural alteration of the holoenzyme.
D000596:Amino Acids / Q000032:analysis; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D002118:Calcium / Q000378:metabolism; D055598:Chemical Phenomena; D002621:Chemistry; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007120:Immunochemistry; D005779:Immunodiffusion; D008970:Molecular Weight; D010446:Peptide Fragments / Q000276:immunology; D010764:Phosphorylase Kinase / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000378:metabolism; D014461:Ultracentrifugation
D000596:Amino Acids; D000906:Antibodies; D010446:Peptide Fragments; D010764:Phosphorylase Kinase; D002118:Calcium