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Temperature-dependence of activation and inhibition of rat-brain adenosine triphosphatase activated by sodium and potassium ions.

ABSTRACT: 1. The adenosine-triphosphatase activity of rat-brain microsomes was measured between 0 degrees and 37 degrees . The stimulatory effect of Na(+) plus K(+) on the Mg(2+)-dependent adenosine-triphosphatase activity decreased sharply with decreasing temperature and became negligible at 0 degrees . An Arrhenius plot drawn from the experimental data showed two discontinuities: one at about 6 degrees and the other at about 20 degrees . 2. The increment in activity induced by Na(+) plus K(+) was more sensitive to oligomycin at lower than at higher temperatures, but the opposite was observed for ouabain. The action of oligomycin showed a biphasic character, since below a certain concentration it caused slight activation of Na(+)-plus-K(+)-activated adenosine triphosphatase. 3. Where oligomycin increased the activity of the enzyme, it also enhanced the accumulation of an acid-precipitable phosphorylated compound formed through the transfer of the gamma-phosphate group of [(32)P]ATP to the enzyme system. Stimulatory concentrations of oligomycin did not interfere with K(+)-mediated dephosphorylation of the intermediate, though high concentrations of oligomycin counteracted the effect of K(+). 4. The temperature profile of K(+)-stimulated microsomal phosphatase qualitatively resembled that of microsomal adenosine triphosphatase.

SUBMITTER: Gruener N

PROVIDER: S-EPMC1265212 | biostudies-other | 1966 Sep

REPOSITORIES: biostudies-other

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Project description:1. The Na(+)-plus-K(+)-stimulated adenosine triphosphatase [(Na(+),K(+))-ATPase] of microsomal preparations from ox brain was inactivated or diminished in activity by exposure to 2-8m-urea. Similar concentrations of urea diminished the turbidity of the suspensions. 2. Low concentrations (about 2.5mm) of NaATP with the urea gave partial or complete protection of the ATPase, without altering the concomitant change in turbidity. Some protection of the (Na(+),K(+))-ATPase was afforded by tris ATP, but the greatest protection was found with NaATP and in its presence the change in (Na(+),K(+))-ATPase with 3m-urea included a phase in which activity was enhanced by 40%. 3. The protective effect was specific to NaATP: KATP, NaADP, NaAMP and sodium pyrophosphate were without protective effect and in some cases they augmented the action of urea. 4. The turbidity of cerebral microsomal suspensions was diminished also by ultrasonic irradiation; NaATP did not alter this change. After ultrasonic treatment up to 55% of the protein and of the ATPase activity were no longer deposited by centrifugal forces of 4.5x10(6)g-min. 5. Ultrasonic treatment and centrifugation could be carried out with little or no loss of ATPase and ammonium sulphate flocculation of the supernatant then afforded in the first material precipitated a three- to five-fold enrichment of (Na(+),K(+))-ATPase activity. 6. Sodium borohydride and dimethyl sulphoxide also diminished the turbidity of the microsomal fraction but enrichment of the ATPase was not effected by these reagents; ten other compounds were without action on the ATPase. 7. Acetyl phosphate was hydrolysed by the microsomal preparation and this activity was increased by added K(+). Acetyl-phosphatase activity persisted in the ultrasonically treated and ammonium sulphate-fractionated preparations, which were more exacting in their requirements for K(+). 8. The findings are discussed in relation to the mechanism of the (Na(+),K(+))-ATPase.

Project description:1. Adenosine triphosphatase activities of dispersions prepared from bovine cerebral cortex that had been frozen, were greater than those of dispersions prepared from fresh tissue. The subcellular distribution of components of the dispersion was not altered by freezing the tissue and a microsomal fraction enriched in Na(+)+K(+)-stimulated adenosine triphosphatase activity was prepared. 2. The bovine cerebral microsomes were further treated with a 2m-sodium iodide reagent to obtain a particulate preparation with minimal Na(+)+K(+)-independent adenosine triphosphatase activity. Na(+)+K(+)-stimulated activity was increased by the sodium iodide treatment and this preparation was shown to be enriched in lipid constituents. 3. Density-gradient centrifugation of the sodium iodide treated preparation gave three main subfractions each containing approximately equal amounts of phospholipid and protein. Further exposure of the sodium iodide-treated preparation to the 2m-sodium iodide reagent altered the distribution of protein and phospholipid among the fractions obtained by density-gradient centrifugation. Dissociation of phospholipids from protein in the sodium iodide-treated preparation was brought about also by high concentrations of arginine. Concentrated solutions of arginine and sodium thiocyanate brought about dissociation of phospholipids from protein of the microsomal preparation. 4. Many amino acids were found to inhibit Na(+)+K(+)-stimulated adenosine triphosphatase activity when present in high concentrations. The inhibition was complex but resulted, in part at least, from diminished affinity for ATP and Na(+) in the presence of the amino acids. 5. A non-ionic detergent, Lubrol W, solubilized up to 40% of the enzyme activity of the sodium iodide-treated preparation together with 30% of the protein and phospholipid in the preparation. Protein was released from the sodium iodide-treated preparation by pancreatic elastase but Na(+)+K(+)-stimulated adenosine triphosphatase activity of the residue was diminished. Ultrasonic treatment of the sodium iodide-treated preparation failed to release a significant proportion of Na(+)+K(+)-stimulated adenosine triphosphatase activity into a form not deposited by ultracentrifugation.

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Temperature-dependence of activation and inhibition of rat-brain adenosine triphosphatase activated by sodium and potassium ions.

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