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Proton accumulation and ATPase activity in Golgi apparatus–enriched vesicles from rat liver
Author(s) -
Yeh HorngI,
Van Rossum G. D. V.
Publication year - 1991
Publication title -
hepatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.488
H-Index - 361
eISSN - 1527-3350
pISSN - 0270-9139
DOI - 10.1002/hep.1840130321
Subject(s) - nigericin , valinomycin , chemistry , atpase , acridine orange , monensin , proton transport , vesicle , biochemistry , gramicidin , chloride , enzyme , membrane , apoptosis , organic chemistry
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 NO 3 − markedly inhibited H + uptake. Nitrate competitively inhibited both the chloride‐dependent ATPase (apparent K i 6 mmol/L) and proton uptake (apparent K i 2 mmol/L). Nitrate and SCN − also inhabited uptake of 36 Cl. 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 NH 4 + and choline reduced proton uptake without affecting ATPase. The ATPase and H + uptake were supported equally well by Mg 2+ or Mn 2+ . The ATPase was competitively inhibited by 4‐acetamido‐4′‐isothiocyano‐stilbene‐2,2′‐disulfonic acid (apparent K i 39 μmol/L). Other agents inhibiting both H + uptake and ATPase were N ‐ethylmaleimide, N , N ′‐dicyclohexylcarbodiimide, chlorpromazine, diethylstilbestrol, Zn 2+ , Co 2+ and Cu 2+ . In the Cl − medium, accumulated protons were released by ionophores at the relative rates, monensin = nigericin > valinomycin > 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. (H EPATOLOGY 1991;13:523–533.)