title: Differential affinity cross-linking of phosphorylase kinase conformers by the geometric isomers of phenylenedimaleimide.
abstract: Phosphorylase b kinase (PbK) from skeletal muscle is a highly regulated oligomer consisting of four copies of four distinct subunits (alphabetagamma)delta4. The gamma subunit is catalytic, and the remaining subunits are regulatory. To characterize effector-induced changes in the quaternary structure of the enzyme, we utilized the ortho-, meta, and para-isomers of phenylenedimaleimide (PDM), which in addition to having different geometries, also vary 2.5-fold in their cross-linking spans. Even at concentrations equivalent to the alphabetagammadelta protomers of PbK, all three isomers caused specific, rapid, and extensive cross-linking of the holoenzyme to form primarily alphabeta dimers, plus smaller amounts of betagammagamma and alphagammagamma trimers. The formation of these three conjugates was nearly totally inhibited by a 10-fold molar excess over PDM of N-(o- and p-tolyl)succinimide, which are chemically inert structural analogs of PDM. This inhibition suggests that PbK has binding sites for PDM and that PDM acts as an affinity cross-linker in binding to these sites prior to forming cross-linked conjugates. The largest effect on cross-linking in progressing from o- to p-PDM was on the alphagammagamma trimer, which is preferentially formed by the p-isomer. Activation of the enzyme by either phosphorylation or the allosteric activators ADP and GDP resulted in large increases in the amount of alphagammagamma formed, small increases in betagammagamma, and little change in alphabeta. When cross-linked in the presence of the reversibly activating nucleoside diphosphates, PbK remained activated after their removal, indicating that cross-linking had locked it in the active conformation. Our results provide direct evidence for perturbations in the interactions of the catalytic gamma subunit with the regulatory alpha and beta subunits upon activation of PbK.
authors.affiliation: Department of Biochemistry, College of Medicine, the University of Tennessee, Memphis, Tennessee 38163, USA.
authors.forename: O W
authors.initials: OW
authors.lastname: Nadeau
authors.forename: D B
authors.initials: DB
authors.lastname: Sacks
authors.forename: G M
authors.initials: GM
authors.lastname: Carlson
mesh_terms: D000345:Affinity Labels; D000818:Animals; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004789:Enzyme Activation; D007700:Kinetics; D008301:Maleimides / Q000737:chemistry*; D008970:Molecular Weight; D010764:Phosphorylase Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D011817:Rabbits
chemical_list: D000345:Affinity Labels; D003432:Cross-Linking Reagents; D008301:Maleimides; D010764:Phosphorylase Kinase