A study of 3':5'-cyclic mononucleotide-dependent protein kinase from canine prostate glands.
1. An adenosine 3':5'-cyclic monophosphate (cyclic AMP)-dependent protein kinase, located predominantly in the cytosol, was studied in canine prostate. 2. The enzyme exhibited cyclic AMP-binding activity, and could be isolated by chromatography on diethylaminoethyl cellulose. 3. The enzyme was maximally stimulated (fourfold) by 1mum-cyclic AMP, and half-maximal activation of the enzyme was observed in presence of 50nm-cyclic AMP. 4. Equilibrium studies at pH5.0 indicated the presence of one major class of binding site for cyclic AMP, with an association constant of approx. 10(8)m(-1). 5. Stimulation of the enzyme was also observed with the 3':5'-cyclic monophosphate derivatives of cytidine, inosine, guanosine and uridine as well as with dibutyryl cyclic AMP, but higher concentrations of these cyclic nucleotides were required to provide the same degree of activation as that seen with cyclic AMP. 6. Comparing alpha-casein, protamine and different histone subfractions as substrates, highest cyclic AMP stimulation was demonstrated with histones. 7. Although maximum velocity of the enzyme was enhanced approximately fivefold in presence of cyclic AMP, kinetic studies indicated that the apparent K(m) for histone (0.5mg/ml) remained the same whether determined in the presence or absence of the cyclic nucleotide. 8. In addition, cyclic AMP did not significantly change the apparent K(m) for ATP (1.2x10(-5)m). 9. The purified enzyme showed an absolute requirement for bivalent metal ion. Substitution of Mn(2+) for Mg(2+) decreased basal protein kinase activity as well as the stimulation noted with cyclic AMP. Similarly, the basal activity was lowered when Mg(2+) was replaced by Ca(2+) and cyclic AMP produced only little stimulation of the prostatic enzyme.
D000255:Adenosine Triphosphate; D000818:Animals; D001665:Binding Sites; D003994:Bucladesine / Q000378:metabolism; D002118:Calcium; D002848:Chromatography, DEAE-Cellulose; D000242:Cyclic AMP / Q000378:metabolism*; D006152:Cyclic GMP / Q000378:metabolism; D007289:Cyclic IMP / Q000378:metabolism; D003597:Cytosine Nucleotides / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D004285:Dogs; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008274:Magnesium; D008297:Male; D008345:Manganese; D011467:Prostate / Q000201:enzymology*; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D014500:Uracil Nucleotides / Q000378:metabolism
D003597:Cytosine Nucleotides; D014500:Uracil Nucleotides; D007289:Cyclic IMP; D008345:Manganese; D003994:Bucladesine; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D011494:Protein Kinases; D006152:Cyclic GMP; D008274:Magnesium; D002118:Calcium