PURIFICATION AND CHARACTERIZATION OF RABBIT PANCREAS PROTEIN-KINASE-C

被引：8

作者：

EDERVEEN, AGH ^{[1
]}

VANEMSTDEVRIES, SE ^{[1
]}

BURGERS, LHC ^{[1
]}

DEPONT, JJHHM ^{[1
]}

机构：

[1] CATHOLIC UNIV NIJMEGEN,DEPT BIOCHEM,POB 9101,6500 HB NIJMEGEN,NETHERLANDS

来源：

PANCREAS | 1992年 / 7卷 / 01期

关键词：

PROTEIN KINASE-C; SIGNAL TRANSDUCTION; PHORBOL ESTER;

D O I：

10.1097/00006676-199201000-00007

中图分类号：

R57 [消化系及腹部疾病];

学科分类号：

摘要：

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 K(a) values of 3.1-mu-g/ml and 247-mu-M for phosphatidylserine and Ca2+, respectively. When dioctanoylglycerol or the phorbol ester 12-O-tetradecanoyl-phorbol 13 acetate (TPA) was present, the K(a) value for Ca2+ decreased to 10 and 18-mu-M, 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 K(a) 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 K(m) value for ATP and for histone-H-1 was 8.5-mu-M and 20.4-mu-g/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.

引用

页码：34 / 44

页数：11

共 39 条

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