Requirement of Poiycations for the Enzymatic Activity of a New Protein Kinase-Substrate Complex from Morris Hepatoma 3924A
From isotonic extracts of fast-growing Morris hepatoma 3924A, a soluble protein kinase was purified about 25-fold by diethylaminoethyl cellulose column chromatography followed by gel filtration on a Sephadex G-200 column and isoelectrofocusing electrophoresis. The enzyme preparation phosphorylated Itself and did not require any added substrate. The reaction was greatly stimulated by H1 histone as well as by polylysine. Polyarginine and other histone fractions were less effective. Spermine and spermidine were slightly active. Acid hydrolysis of the phosphorylated product resulted in the identification of phosphoserine and phosphothreonine in a ratio of about 1.2:1. Analysis of sodium dodecyl sulfate :polyacrylamide gel electrophoresis revealed that histones were not phosphorylated, but endogenous proteins with approximate molecular weights of around 7 x 10 4 were labeled. The enzyme did not show homogeneous size distribution upon gel filtration on a Sephadex column, but the major fraction corresponded to an approximate molecular weight of 2.6 x 10 5. Nevertheless, the enzyme showed a single isoelectric point of 4.9. Km for adenosine triphosphate was about 4 x 10 5 m. Apparent K a was 8 βg/ml for polylysine and 16 Mg/ml for whole histone. A pH optimum was 7.5 to 8.5. In the absence of polylysine, the enzyme showed a biphasic concentration curve for Mg 2+ with an optimum concentration of 50 to 100 ms, but in the presence of polylysine the curve was normalized and the optimum concentration was 3 to 100 ms. Subcellular fractionation with organic solvents revealed that the enzyme did not represent a leakage of nuclear phosphoproteins into the cytoplasm during isolation procedures. The enzyme was not stimulated or inhibited by cyclic nucleotides and is clearly distinguished from cyclic nucleotide-dependent protein kinases. © 1978, American Association for Cancer Research. All rights reserved.
Criss, Wayne E.; Yamamoto, Masahiro; Takai, Yoshimi; Nishizuka, Yasutomi; and Morris, Harold P., "Requirement of Poiycations for the Enzymatic Activity of a New Protein Kinase-Substrate Complex from Morris Hepatoma 3924A" (1978). Howard University Cancer Center Faculty Publications. 332.