Activated states of phosphorylase kinase as detected by the chemical cross-linker 1,5-difluoro-2,4-dinitrobenzene.
When phosphorylase kinase from rabbit skeletal muscle was activated by phosphorylation and then cross-linked with 1,5-difluoro-2,4-dinitrobenzene at pH 6.8, dimers of beta subunits were formed that were not observed during cross-linking of nonphosphorylated enzyme under the same conditions. The ability to form these dimers was due to phosphorylation of the beta subunit because when enzyme phosphorylated in the alpha and beta subunits was incubated with a protein phosphatase relatively specific for the beta subunit (Ganapathi, M.K., Silberman, S.R., Paris, H., and Lee, E.Y.C. (1981) J. Biol. Chem. 256, 3213-3217), the ability to form the cross-linked beta dimers was lost. Significant amounts of two complexes also judged to be dimers of beta subunits were observed when nonphosphorylated phosphorylase kinase was cross-linked after preincubation with Ca2+ plus Mg2+ ions, after proteolysis by chymotrypsin, or when it was cross-linked at pH 8.2, three conditions known to stimulate the activity of the nonphosphorylated enzyme. From these results, we conclude that 1,5-difluoro-2,4-dinitrobenzene can serve as a structural probe for activated states of phosphorylase kinase. The activation is associated with a conformational change in which two beta subunits either move closer together or have a reactive group on one, or both, of them unmasked. Our results suggest that the diverse mechanisms listed above for stimulating phosphorylase kinase activity cause a common conformational change to occur.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D004139:Dinitrofluorobenzene / Q000031:analogs & derivatives / Q000494:pharmacology*; D004789:Enzyme Activation; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D009578:Nitrobenzenes / Q000494:pharmacology*; D010764:Phosphorylase Kinase / Q000378:metabolism*; D010766:Phosphorylation; D011817:Rabbits
D046911:Macromolecular Substances; D009578:Nitrobenzenes; D004139:Dinitrofluorobenzene; D010764:Phosphorylase Kinase; C002041:1,5-difluoro-2,4-dinitrobenzene; D008274:Magnesium; D002118:Calcium