Characterization of the cyclic AMP-dependent protein kinase from rat pancreas, further purification of the catalytic subunit, substrate specificity, effect of the pancreatic heat stable inhibitor.
In order to investigate the sequence of events triggered by cyclic AMP and cyclic GMP in exocrine pancreatic cells, the identification of the various protein kinases possibly present in this tissue is of major interest. Further analysis of the two cyclic AMP-dependent protein kinases previously reported [11] suggests that KI is a degraded form of KII. It is therefore likely that a single holoenzyme is present in exocrine cells. In addition no protein kinase, specifically stimulated by cyclic GMP, has been detected in any fraction obtained in the course of purification of the cyclic AMP-dependent protein kinase. A faster and more efficient method than the one previously described [11] allows the purification (5000 times) of the protein kinase catalytic subunit. Analysis of the subunit by sodium dodecyl sulphate polyacrylamide gel electrophoresis indicates a molecular weight of 40 000 +/- 1 000. The enzyme phosphorylates specifically histone H2B (Vm = 236 min(-1), Km = 1.15 10(-5) M) and to a lesser extent H2A, H5 and H1 (Vm = 55--77 min(-1), Km 5--25 10(-5) M). Histones H3 and H4 are not phosphorylated. The effect of the heat stable inhibitor, extracted from rat pancreas, on the phosphorylation of H2B has been investigated. The inhibition is of the non competitive type with respect to ATP. The inhibition at various histone concentrations cannot be described by the Michaelis-Menten equation.
D000818:Animals; D002848:Chromatography, DEAE-Cellulose / Q000379:methods; D000242:Cyclic AMP / Q000378:metabolism*; D066298:In Vitro Techniques; D008970:Molecular Weight; D010179:Pancreas / Q000201:enzymology*; D010184:Pancreatic Extracts / Q000494:pharmacology; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity
D010184:Pancreatic Extracts; D047428:Protein Kinase Inhibitors; D000242:Cyclic AMP; D011494:Protein Kinases