Proton accumulation and ATPase activity in Golgi apparatus-enriched vesicles from rat liver

We have studied the mechanism by which liver Golgi apparatus maintains the acidity of its contents, using a subcellular fraction from rat liver highly enriched in Golgi marker enzymes. Proton accumulation (measured by quenching of acridine-orange fluorescence) and anion-dependent ATPase were characterized and compared. Maximal ATPase and proton accumulation required ATP; GTP and other nucleotides gave 10% to 30% of maximal activity. Among anions, Cl- and Br- approximately doubled the activities; others were much less effective. Half-maximal increase of ATPase and H+ uptake required 55 mmol/L and 27 mmol/L Cl-, respectively. In predominantly chloride media, SCN- and NO3- markedly inhibited H+ uptake. Nitrate competitively inhibited both the chloride-dependent ATPase (apparent Ki 6 mmol/L) and proton uptake (apparent Ki 2 mmol/L). Nitrate and SCN- also inhibited uptake of 36Cl. Replacing K+ with Na+ had no effect on the initial rate of proton uptake but somewhat reduced the steady state attained. Replacement of K+ with NH4+ and choline reduced proton uptake without affecting ATPase. The ATPase and H+ uptake were supported equally well by Mg2+ or Mn2+. The ATPase was competitively inhibited by 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonic acid (apparent Ki 39 mumol/L). Other agents inhibiting both H+ uptake and ATPase were N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide, chlorpromazine, diethylstilbestrol, Zn2+, Co2+ and Cu2+. In the Cl- medium, accumulated protons were released by ionophores at the relative rates, monensin = nigericin greater than valinomycin greater than carbonyl cyanide mchlorophenylhydrazone; the last of these also reduced ATPase activity. In the absence of Cl-, monensin and valinomycin both stimulated the ATPase. These results show a close association between ATPase activity and acidification of liver Golgi vesicles. They support a role for Cl- that depends on its uptake as a counter ion for H+ and suggest that it may also stimulate proton transport by a more direct effect on a component of the transport system.