Phosphorylase kinase conformers. Detection by proteases.
A variety of proteases have been evaluated as potential structural and conformational probes of nonphosphorylated and phosphorylated phosphorylase kinase. In general, the enzyme's alpha subunit is rapidly degraded, followed in most cases by hydrolysis of the beta subunit; the gamma subunit is resistant to most proteases. Trypsin clearly distinguishes between the nonactivated and activated conformers of phosphorylase kinase, in that the beta subunit in phosphorylated enzyme, as opposed to nonphosphorylated enzyme, is markedly protected from tryptic attack. In contrast, only a small difference in the rates of proteolysis of the alpha subunit in phosphorylated and nonphosphorylated enzyme is seen, even when a protease is used that is highly selective for the alpha subunit, such as chymotrypsin or endoproteinase Arg C. Incubation of nonphosphorylated phosphorylase kinase with either Mg2+ or Ca2+, which are activating cations, also protects the beta subunit from tryptic hydrolysis, whereas Mn2+, which inhibits the kinase activity, has little effect on proteolysis. The allosteric activator ADP also causes the beta subunit to become refractory to trypsin and mimics the effects of phosphorylation. Similar effector-induced conformational changes in the beta subunit are also observed with enzyme in which the alpha subunit has previously been selectively destroyed. These data indicate that activation of phosphorylase kinase by dissimilar mechanisms is associated with a conformational change in the enzyme's beta subunit that is detectable by trypsin and confirm earlier studies from this laboratory employing a chemical cross-linker as a conformational probe for activated and nonactivated conformers of the enzyme (Fitzgerald, T. J., and Carlson, G. M. (1984) J. Biol. Chem. 259, 3266-3274).
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000494:Allosteric Regulation; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002413:Cations, Divalent; D002918:Chymotrypsin / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005994:Glycerophosphates / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D010447:Peptide Hydrolases / Q000378:metabolism*; D010764:Phosphorylase Kinase / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation / Q000187:drug effects; D011817:Rabbits; D014357:Trypsin / Q000378:metabolism
D002413:Cations, Divalent; D005994:Glycerophosphates; D000244:Adenosine Diphosphate; D010764:Phosphorylase Kinase; D010447:Peptide Hydrolases; D002918:Chymotrypsin; D014357:Trypsin; D008274:Magnesium; D002118:Calcium; C031463:beta-glycerophosphoric acid