Relation of ATPases in rat renal brush-border membranes to ATP-driven H+ secretion (original) (raw)

1989, The Journal of Membrane Biology

ATPase, (Na ~ + K+)-and Ca2LATPases, and alkaline phosphatase, sealed brush-border membrane vesicles hydrolyse externally added ATP demonstrating the existence of ATPases at the outside of the membrane ("ecto-ATPases"), These ATPases accept several nucleotides, are stimulated by Ca-'~ and Mg ~, and are inhibited by N,N'-dicyclohexylcarbodiimide (DCCD), but not by N-ethylmaleimide (NEM). They occur in both brushborder and basolateral membranes, Opening of brush-border membrane vesicles with Triton X-100 exposes ATPases located at the inside (cytosolic side) of the membrane. These detergentexposed ATPases prefer ATP, are activated by Mg TM and Mn 2+, but not by Ca 2+, and are inhibited by DCCD as well as by NEM. They are present in brush-border, but not in basolateral membranes. As measured by an intravesicularly trapped pH indicator, ATP-loaded brush-border membrane vesicles extrude protons by a DCCD-and NEM-sensitive pump. ATP-driven H ~ secretion is electrogenic and requires either exit of a permeant anion (CI) or entry of a cation, e.g., Na ~ via electrogenic Na~/ D-glucose and Na+/L-phenylalanine uptake. [n the presence of Na t , ATP-driven H + &flux is stimulated by blocking the Na*/H ' exchanger with amiloride. These data prove the coexistence of Na+-coupled substrate transporters, Na+/H + exchanger, and an ATP-driven H" pump in brush-border membrane vesicles. Similar location and inhibitor sensitivity reveal the identity of ATPdriven H + pumps with (a part of) the DCCD-and NEM-sensitive ATPases at the cytosolic side of the brush-border membrane.