title: Proton accumulation and ATPase activity in Golgi apparatus-enriched vesicles from rat liver.
abstract: 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.
authors.affiliation: Department of Pharmacology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
authors.forename: H I
authors.initials: HI
authors.lastname: Yeh
authors.forename: G D
authors.initials: GD
authors.lastname: van Rossum
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D002712:Chlorides / Q000494:pharmacology; D006056:Golgi Apparatus / Q000187:drug effects / Q000201:enzymology*; D007477:Ions; D008099:Liver / Q000378:metabolism*; D008297:Male; D009566:Nitrates / Q000494:pharmacology; D009711:Nucleotides / Q000494:pharmacology; D011522:Protons; D051381:Rats; D011919:Rats, Inbred Strains
chemical_list: D002712:Chlorides; D007477:Ions; D009566:Nitrates; D009711:Nucleotides; D011522:Protons; D000251:Adenosine Triphosphatases