Tightness and orientation of vesicles from guinea-pig kidney estimated from reactions of adenosine triphosphatase dependent on sodium and potassium ions.
In order to study the "sidedness" of the ligands of the Na+, K+-ATPase in the phosphorylation from [32P]ATP, tight vesicles were prepared from guinea pig kidney and partially purified by a two-stage sucrose and Ficoll gradient centrifugation procedure. These vesicles were derived presumably from plasma membrane fragments resealed after the initial disruption of the cells during homogenization. Tightness of the vesicles was estimated according to activation by the nonionic detergent, Triton X-100. Treatment with Triton X-100 increased both the activity of the Na+, K+-ATPase and its Na+-dependent phosphorylation from [32P]ATP at least three-fold. Activation of both functions also appeared when the vesicles were shocked osmotically. These results suggest that the preparation contains a major population of tight normal vesicles (approximately 75%) in which the phosphorylation site faces the intravesicular solution. In the response to ouabain breakdown of the phosphoenzyme was inhibited in vesicles treated with Triton X-100 but not in intact ones as if ouabain could not get to its binding site. Correspondingly in phosphorylation from ATP pretreatment with ouabain in the presence of inorganic phosphate produced less inhibition in intact vesicles than in those disrupted with Triton X-100 beforehand. These data suggest the presence of an everted vesicle fraction in the preparation (approximately 20%). Apparently only a small fraction of the vesicles was leaky. In the everted vesicles the action of K+ on the phosphoenzyme was slow. In order to accelerate the dephosphorylation in intact vesicles as effectively as in disrupted ones, K+ had to be added before the start of phosphorylation. This supports the view that K+ was acting from the side of the membrane opposite to that where the gamma-phosphoryl group was accepted from ATP.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D006168:Guinea Pigs; D007668:Kidney / Q000201:enzymology* / Q000648:ultrastructure; D009940:Organoids / Q000201:enzymology*; D009994:Osmolar Concentration; D010042:Ouabain / Q000494:pharmacology; D010710:Phosphates / Q000494:pharmacology; D011092:Polyethylene Glycols / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology*; D012964:Sodium / Q000494:pharmacology*
D010710:Phosphates; D011092:Polyethylene Glycols; D010042:Ouabain; D000255:Adenosine Triphosphate; D012964:Sodium; D000251:Adenosine Triphosphatases; D011188:Potassium