Dataset Information

Lipid requirement of the membrane sodium-plus-potassium ion-dependent adenosine triphosphatase system.

ABSTRACT: The dependence of the (Na-++K-+)-dependent ATPase (adenosine triphosphatase) (EC 3.6.1.3) on lipid has been examined in a number of different ways, with the use of various preparations from kidney tissue. The main findings were as follows. (1) The ATPase activities of the preparations examined were closely correlated with their total phospholipid content. (2) Extraction of the ATPase with deoxycholate or Lubrol W, combined with suitable salt-fractionation and washing procedures, removed phospholipid, cholesterol and enzymic activity in parallel; but activity was completely lost before all lipid had been removed. (3) The loss of activity could not be attributed to inhibition by residual detergent. (4) No selective removal of any particular phospholipid class by detergent could be detected. (5) Consistent reactivation of the Lubrol-extracted enzymes was obtained by adding dispersions of exogenous phospholipid, but only some, bearing a net negative charge, such as phosphatidylserine and phosphatidylglycerol, were effective. (6) The degree of reactivation was correlated with the amount of residual activity remaining after lipid depletion. (7) Partial purification of the ATPase, giving a 50-fold increase in specific activity, was not accompanied by selective enhancement of any particular class of phospholipid. We conclude that although the ATPase is dependent on phospholipid, only the reactivation results provide evidence for specificity.

SUBMITTER: Wheeler KP

PROVIDER: S-EPMC1165362 | biostudies-other | 1975 Mar

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

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.

Dataset Information

Lipid requirement of the membrane sodium-plus-potassium ion-dependent adenosine triphosphatase system.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets