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Several protein kinases which catalyze the phosphorylation of glycogen 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 s3mthetase 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. [ABSTRACT FROM AUTHOR]

The intracellular distribution of thymidine phosphorylating kinases has been studied ill rat liver at different times after partial hepatectomy. After conventional fractionation in 0.25 M sucrose, thymidine kinase is present in all fractions, the highest proportion being found, however, in the supernatant; phosphorylation of thymidine to TDP and TTP is detected only in the supernatant fraction from 24 h onwards after hepatectomy. Nuclei prepared in 2.2 M sucrose or in non-aqueous media have the same level of thymidine kinase activity and can never phosphorylate thymidine to TDP and TTP. In the nuclear fractions, the absolute activity of thymidine kinase increases from 16 to 46 h after hepatectomy but represents a decreasing proportion of the activity of the homogenate. At the same time, the proportion of kinase activity rises markedly in the supernatant fraction with a maximum of 83% in 40 h regenerating livers. The implications of these results are discussed. [ABSTRACT FROM AUTHOR]

Disc polyacrylamide-gel electrophoresis analyses have been carried out on the dT kinase activities of “cytosol” (high-speed supernatant) and mitochondrial fractions of parental HeLa S3 and enzyme-deficient HeLa(BU25) cells. The HeLa S3 cytosol fraction contained a very active dT kinase molecular form with a mobility relative to the tracking dye (Rx) of about 0.25. Conversion of HeLa S3 cells to dT-kinase-deficient HeLa(BU25) resulted in the loss of most of the cytosol dT kinase activity. The mitochondrial fraction of HeLa(BU25) cells did not contain the 0.25 Rx activity, but did shown 2 more rapidly migrating molecular forms (Rx of 0.4 and 0.6–0.7). The mitochondrial fraction of parental HeLa S3 cells also exhibited the 0.4 and 0.6 to 0.7 Rx dT kinase activities, and addition, contained the 0.25 Rx cytosol-like enzyme. The distinctive dT kinase molecular forms observed in the mitochondrial fraction account for most of the residual enzyme activity of mutant HeLa(BU25) cells. [ABSTRACT FROM AUTHOR]

Glass electrode measurements of the pH of anaerobic suspensions of rat fiver mitochondria during the hydrolysis of ATP to ADP and P1 by the oligomycin-sensitive ATPase show that almost 2H+ ions are translocated outwards through the M phase of the cristae membrane per ATP molecule hydrolysed. Adenylate kinase activity is minimised by the presence of EDTA, and valinomycin must be added to the normal mitochondrial suspensions to collapse the membrane potential and permit the outwardly translocated H+ ions to diffuse practically quantitatively to the glass electrode system. Classical inhibitor studies of the overall ATPase reaction catalysed by the mitochondria indicate that the specific ATP/ADP porter is not rate-limiting under the conditions of these experiments; and it is also shown that the overall process of ATP entry and ADP + Pi exit through the porter systems in the M phase is not coupled to net proton translocation. It is concluded that the ATPase system responsible for or coupled to proton translocation has a proton translocation quotient (→ H+/P) of 2, corresponding to that of the type II ATPase of the chemiosmotic hypothesis. [ABSTRACT FROM AUTHOR]

Conversion of uridine to UTP is enhanced in lymphocytes under the influence of phytohemagglutinin concomitant with the induction of uridine kinase. The uridine kinase activity in the induced cells decreases if the cells are treated with antibodies against phytohemagglutinin. The phosphorylation of uridine may be the limiting step in the incorporation of uridine into RNA. Uridine incorporation can thus not be taken as a direct measure of RNA synthesis. [ABSTRACT FROM AUTHOR]