STUDIES OF 2 DIFFERENT INTRACHAIN CGMP-BINDING SITES OF CGMP-DEPENDENT PROTEIN-KINASE

  • 1 January 1983
    • journal article
    • research article
    • Vol. 258  (18) , 1391-1397
Abstract
The binding of [3H]cGMP to purified beef lung cGMP-dependent protein kinase (cG kinase) was examined using 2 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 2 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 > 50%, the relative amount of the slower or faster component, respectively, of [3H]cGMP dissociation decreased during the cGMP chase. The cG kinase, like its cAMP-dependent protein kinase homologue, probably possesses 2 highly conserved intrachain cyclic nucleotide-binding sites which have different dissociation rates and analog specificity. The Ka of the kinase for cGMP was aobut 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 2 sites, and the activation by cGMP exhibited positive cooperativity (n = 1.57). Both intrachain sites may be involved in protein kinase activation. E2 + 4 cGMP .dblarw. E2 .cntdot. 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 peptides of approximate MW = 12,000 was resolved.

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