Properties of adenosine 3',5'-monophosphate-dependent protein kinases isolated from bovine epididymal spermatozoa.

Three protein kinases were separated from bovine epididymal spermatozoa by using DEAE-Sephadex ion exchange chromatography and Sephadex G-100 gel filtration. The molecular weights of these three kinases were estimated to be 120,000, 78,000 and 56,000 by the use of Sephadex G-100 gel filtration. The activity of all three kinases was greatly enhanced by cyclic adenosine 3’,5’-monophosphate (cyclic AMP). In the presence of 1OF M cyclic AMP, a catalytic subunit of molecular weight 30,000 to 35,000 was separated from each of the three protein kinases. Under these same conditions, cyclic AMP-binding proteins were located in fractions corresponding to molecular weights of 78,000, 35,000 to 40,000, and 17,000 to 18,000; the latter may result from proteolysis for it was not observed in fresh preparations. The cyclic AMP-binding protein of molecular weight 78,000 combined with the isolated catalytic subunits of all three protein kinases. The apparent K, for casein (approximately 3 mg per ml) appeared to be neither different for the three kinases nor affected by the addition of cyclic AMP. The apparent Km for ATP varied from 3.5 to 8.7 pM, but it appeared to be the same for all three protein kinases and also appeared to be unaffected by cyclic AMP. The isolated catalytic subunit of the kinase of molecular weight 120,000 exhibited a K, for ATP of 5.5 pM, which agreed with the K, for the intact kinase. Maximum kinase activity was at about pH 6.5 with casein as substrate and at about pH 7.5 with histone as substrate for all three kinases. The isolated catalytic subunit of the 120,000 molecular weight kinase showed identical responses to pH. Cyclic AMP activated all three kinases half-maximally in the lop7 M range, whereas cyclic GMP activated the kinases half-maximally in the lo-” M range. %Methylthiocyclic AMP appeared to be more potent than cyclic AMP in activating the kinases.