Studies of two different intrachain cGMP-binding sites of cGMP-dependent protein kinase.

摘要: The binding of [3H]cGMP to purified beef lung cGMP-dependent protein kinase (cG kinase) was examined using two methods membrane filtration which avoided loss bound [3H]cGMP. enzyme 1.6-2.0 mol [3H]cGMP/mol monomer. If the saturated with and then excess unlabeled cGMP added, dissociated from as approximately equal components (Sites 1 2). When 8-bromo-cGMP or cIMP added [3H]cGMP-binding reaction at a concentration sufficient competitively inhibit by greater than 50%, relative amount slower faster component, respectively, dissociation decreased during chase. data indicated that cG kinase, like its cAMP-dependent homologue, possesses highly conserved intrachain cyclic nucleotide-binding sites have different rates analog specificity. Ka for about 20-fold lower histone instead heptapeptide substrate. Aging caused conversion higher form an apparent increase in Site rate. Using fresh substrate, occupation occurred concentrations did 2 occupation, associated activity. However, activity appeared correlate better total either sites, activation exhibited positive cooperativity (n = 1.57). It is suggested both are involved activation. E2 + 4 equilibrium . cGMP4 could be photoaffinity-labeled 8-azido-[32P]cAMP. labeled trypsin-treated followed sodium dodecyl sulfate-slab gel electrophoresis, single major peptide approximate Mr 12,000 resolved.