Abstract
Two protein kinases (MPK1 and MPK2) were isolated from bovine heart mitochondria. After the solubilization of submitochondrial particles with cholate, these protein kinases were purified by ammonium sulfate precipitation, Sepharose 6B gel filtration, and affinity chromatography on phosvitin-Sepharose. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the final preparation of MPK1 contained two major polypeptide bands (40,000 and 36,000 daltons). MPK2 contained one major polypeptide of 34,000 daltons. Under nondenaturing conditions, the molecular weights of MPK1 and MPK2 were estimated to be approximately 250,000 and 70,000-90,000, respectively. MPK1 had a pH optimum at 9.0 and MPK2 at 7.5. Both enzymes required Mg2+ for activity and responded poorly to Mn2+ or Ca2+. Both had similar apparent Km values for ATP and were not affected by either cyclic AMP or Ca2+-calmodulin. MKP1 phosphorylated threonine residues and MPK2 serine residues of casein. With beta casein as substrate, MPK1 was more active than MPK2, whereas with alpha casein, MPK2 was more active than MPK1.
Highlights
Purification of MPK, a n d MPK2-As can be seen from Table I, solubilization of submitochondrial membranes with cholate increased, over 2-fold, the protein kinase activity measured with casein as substrate
This suggests that the exogenous substrate is not accessible to theprotein kinases in the membrane
The proteins were rapidly precipitated with ammonium sulfate to avoid prolonged contact of the enzyme with high concentra
Summary
From the Section of Biochemistry, Molecular a n d Cell Biology, Division of Biological Sciences, Cornel University, Zthaca, New York 14853. Two protein kinases (MPK1 and MPKzw) ere isolated dehydrogenase kinase [13], since it is membranous and acts from bovine heart mitochondria. Dodecyl sulfate-polyacrylamide gel electrophoresthise, In this paper we describe the purification of two protein final preparation of MPKl contained two major poly- kinases from bovine heart mitochondria. They exhibit differpeptide bands (40,000 and 36,000 daltons). MPKz con- ences in substrate specificity, pH optimum, and amino acid tained one major polypeptide of 34,000 daltons.
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