Properties of soluble and bound protein kinases isolated from swine kidney.

Abstract

Several protein kinases which catalyze the phosphorylation of glyeogen synthetase, histones, protamine and proteins present in isolated plasma membranes, microsomes and nuclei have been extensively purified from swine kidney homogenates. About 60% of the protein kinase activity in kidney was recovered in the soluble 100000 × g supernatant fraction following differential centrifugation. The remaining 40% of the activity was found to be associated with the plasma membrane, nuclear and microsomal subcellular fractions. The activity of the protein kinases which were bound to the particulate structures was not enhanced by adenosine 3′:5′ ‐mono‐phosphate (cyclic AMP) when kidney glycogen synthetase or histone was used as the protein substrate, however the rate of phosphorylation of these substrates by the soluble enzymes was increased 50 to 100% by the addition of 0.5 μM cyclic AMP. The bound protein kinase activity was dissociated from the particulate structures by treatment with 0.7 M NaCl. The soluble and bound protein kinase were purified by a procedure involving treatment with alumina Cγ, adsorption to calcium phosphate gel, and chromatography on diethylaminoethylcellulose. Both protein kinase preparations were separated into two fractions by the last procedure and their elution profiles were very similar. The apparent Km for ATP in the presence of 10 mM MgCl2 varied from 5 to 9 μM When either glycogen synthetase or histone was the substrate. All four preparations showed a very broad specificity for protein substrates. They phosphorylated histones, protamine, casein and glycogen synthetases. The molecular weight of the four kidney protein kinase preparations calculated from data obtained by sucrose‐density centrifugation and chromatography on Sephadex G‐200 was 170000 ± 10000. Upon polyacrylamide‐gel electrophoresis in 1% sodium dodecylsulfate all of the samples showed two major components.The kinetic properties of the bound form of protein kinase was examined with a sonicated preparation of kidney plasma membranes. The molecular weight of the active fragment was greater than 1000000. Proteins present in the membrane were labeled with 32P when the preparation was incubated with magnesium ion and [γ‐32P] ATP. This preparation also phosphorylated glycogen synthetase and histone. However, the apparent Km of the bound form of the enzyme for ATP was 2.8 mM when glycogen synthetase was used as the protein substrate.