Abstract
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.
Highlights
Using fresh enzyme and enzyme has two sites/chain (13, 14). These sites oneach heptapeptide as substrate, Site 1 occupation occurred cAMP isozyme were shown to be differentwithrespect to at lower concentrations of cGMP than did Site 2 occu- cAMP dissociation rate and cyclic nucleotide analog specificpation, and was associated withan increase in protein ity (15, 16)
Correlate better with total cGMP binding than with subunit (10, 17). This finding habseen reinvestigated in light binding to eitherof the two sites, and the activatbioyn cGMP exhibited positive cooperativity( n= 1.57).It is suggested that both intrachain sites are involvedin protein kinase activation
Stoichiometry of cGMP binding membrane filtration of protein-bound 3H-labeled cyclic nu- For Preparations 1, 2, and 3, the cGMP kinase was eluted from cleotide was previously carried out using an 8-ml potassium the affinity column with [3H]cGMP. l x and ly were different fracphosphate buffer rinse step. the rinsewas appropriate for the R subunit and produced lower blanks, as can be seen in Fig. 3, it was inappropriate for the cG kinase
Summary
CCKinase Purification-All steps were performed at 4 “C.Through the (NH,),SO, precipitation step thebeef lung cG kinase was purified from approximately 10 kg of tissue according to the procedure described earlier (20). The binding reaction mixture contained 50 mM potassium phosphate The counts were corrected for 38% quenching due to thepaper, and for 5-15% due to [3H]H20contamination of [3H]cGMP.The latterwas determined by chromatographing each reaction mixture on a Sephadex G-25 column (0.9 X 50 cm) equilibrated in 50 mM NH4HC03 (pH 7.8).
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