Studies of two different intrachain cGMP-binding sites of cGMP-dependent protein kinase.
The binding of [3H]cGMP to purified beef lung cGMP-dependent protein kinase (cG kinase) was examined using two methods of membrane filtration which avoided loss of bound [3H]cGMP. The enzyme bound 1.6-2.0 mol of [3H]cGMP/mol of monomer. If the kinase was saturated with [3H]cGMP and then excess unlabeled cGMP was added, [3H]cGMP dissociated from the enzyme as two approximately equal components (Sites 1 and 2). When 8-bromo-cGMP or cIMP was added to the [3H]cGMP-binding reaction at a concentration sufficient to competitively inhibit binding by greater than 50%, the relative amount of the slower or faster component, respectively, of [3H]cGMP dissociation decreased during the cGMP chase. The data indicated that the cG kinase, like its cAMP-dependent protein kinase homologue, possesses two highly conserved intrachain cyclic nucleotide-binding sites which have different dissociation rates and analog specificity. The Ka of the kinase for cGMP was about 20-fold lower using histone instead of heptapeptide as substrate. Aging of the enzyme caused conversion to a higher Ka form of the kinase and an apparent increase in the Site 1 cGMP dissociation rate. Using fresh enzyme and heptapeptide as substrate, Site 1 occupation occurred at lower concentrations of cGMP than did Site 2 occupation, and was associated with an increase in protein kinase activity. However, kinase activity appeared to correlate better with total cGMP binding than with binding to either of the two sites, and the activation by cGMP exhibited positive cooperativity (n = 1.57). It is suggested that both intrachain sites are involved in protein kinase activation. E2 + 4 cGMP in equilibrium E2 . cGMP4 The cG kinase could be photoaffinity-labeled using 8-azido-[32P]cAMP. When the labeled cG kinase was trypsin-treated followed by sodium dodecyl sulfate-slab gel electrophoresis, a single major peptide of approximate Mr = 12,000 was resolved.
D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D001386:Azides; D001665:Binding Sites; D002417:Cattle; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism*; D004789:Enzyme Activation; D007700:Kinetics; D008168:Lung / Q000201:enzymology; D010777:Photochemistry; D011494:Protein Kinases / Q000378:metabolism*
D000345:Affinity Labels; D001386:Azides; C017042:8-azidoadenosine-3',5'-monophosphate; D000242:Cyclic AMP; D011494:Protein Kinases; D006152:Cyclic GMP