H+ ATPase of chromaffin granules. Kinetics, regulation, and stoichiometry.
The chromaffin granule ATPase mediates an inwardly directed transport of H+ against concentration gradients, thereby forming and maintaining an electrochemical transmembrane H+ gradient. The kinetics of this ATPase, its activity modulation by changes in electrochemical H+ gradients, and the stoichiometry between H+ transport and ATP hydrolysis were studied in intact bovine chromaffin granules, resealed chromaffin granule ghosts, and highly purified fragmented chromaffin granule membranes. In fragmented membranes the H+ ATPase has a KM for ATP of 69 microM, a maximum of activity at pH 7.3, and a Vmax of 111 nmol/min/mg of protein at 20 degrees C. Trimethyl tin inhibits the ATPase at much lower concentrations than dicyclohexylcarbodiimide, whereas oligomycin, reserpine, and other inhibitors were without effect. In intact chromaffin granules, the ATPase activity was stimulated up to 300% by collapsing the H+ transmembrane gradients. H+/ATP stoichiometry was measured in resealed chromaffin ghosts devoid of ATP and catecholamines under conditions where no net pH changes occur upon ATP hydrolysis. After addition of ATP, the rates of H+ accumulation in the ghosts and ATP hydrolysis were both linear for about 60-100 s, and the ratio of H+ to ATP was 1.71. These data indicate that the H+ ATPase of chromaffin granules has both kinetic similarities and dissimilarities with other known H+ ATPases. The regulation by changes in H+ gradients and the fixed H+/ATP ratio of this ATPase is further evidence of its primary role in establishing electrogenic H+ translocation and H+ gradients in chromaffin granules.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000641:Ammonia / Q000494:pharmacology; D000818:Animals; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002837:Chromaffin Granules / Q000201:enzymology*; D002838:Chromaffin System / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D006180:Proton-Translocating ATPases; D013861:Thiocyanates / Q000494:pharmacology
D013861:Thiocyanates; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D000641:Ammonia; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases