Purification and characterization of rabbit pancreas protein kinase C.
Protein kinase C was purified 6,900-fold from rabbit pancreas with a total yield of 15% by a procedure involving ammonium sulfate fractionation, diethyl aminoethyl ion exchange chromatography, hydroxylapatite chromatography, and finally protamine-agarose affinity chromatography. After these purification steps the protein kinase C preparation contained two major protein bands as judged by silver staining after SDS-polyacrylamide gel electrophoresis: 80 and 69-kDa bands. Monoclonal antibodies directed against bovine brain protein kinase C (alpha- and beta-subtype) recognized only the 80-kDa band. On the other hand, both the 80 and 69-kDa proteins were recognized by a polyclonal monospecific antibody directed against rat brain protein kinase C. Analysis of rabbit pancreas protein kinase C subtypes by means of hydroxylapatite chromatography showed the presence of the III (alpha) subtype as the major subtype. The enzyme depended absolutely on the presence of both phosphatidylserine and Ca2+ for its activity, with apparent Ka values of 3.1 micrograms/ml and 247 microM for phosphatidylserine and Ca2+, respectively. When dioctanoylglycerol or the phorbol ester 12-O-tetradecanoyl-phorbol 13 acetate (TPA) was present, the Ka value for Ca2+ decreased to 10 and 18 microM, respectively. In the presence of the phorbol ester, pancreatic protein kinase C could be activated without added Ca2+. The enzyme also required Mg2+ for its activity. The Ka value was 3.6 mM and maximal activity was reached at 10 mM Mg2+. Pancreatic protein kinase C activity showed a broad pH dependence, with optimal activity at pH 6.75. The Km value for ATP and for histone-H1 was 8.5 microM and 20.4 micrograms/ml, respectively. The present study shows that the kinetic properties of protein kinase C purified from rabbit pancreas closely resemble those found in other tissues.
Ederveen
A G
AG
Department of Biochemistry, University of Nijmegen, The Netherlands.
Van Emst-De Vries
S E
SE
Burgers
L H
LH
De Pont
J J
JJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002118:Calcium / Q000502:physiology; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D008274:Magnesium / Q000502:physiology; D010179:Pancreas / Q000201:enzymology*; D011493:Protein Kinase C / Q000276:immunology / Q000302:isolation & purification* / Q000493:pharmacokinetics; D011817:Rabbits; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D011493:Protein Kinase C; D008274:Magnesium; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium