Effect of diethylpyrocarbonate on pH-driven monoamine uptake by chromaffin granule ghosts.

Chromaffin granules, the catecholamine storage organelles of adrenal medulla, accumulate monoamines by an active process which involves 2 different entities: an electrogenic ATP-dependent H+ctranslocase which generates a proton electrochemical gradient ApH+ (interior acid and positive) [l-3] and a specific monoamine carrier which mediates the coupling between Ac(H+ and substrate accumulation [4-61. Kinetics and pharmacological properties of the latter have been described [7,8], but little is known about its biochemistry. Diethylpyrocarbonate , a histidine specific reagent, has been shown to affect lactose/H+ symport in Escherichia coli membranes by directly reacting with the lac carrier [9]. A similar reaction was reported for proline and D-lactate transport [9]. We investigated the possibility that DEPC might also react with the monoamine carrier of chromaffm granule membranes. Monoamine/proton antiport was assayed independently of the H’-translocase by imposing a pH-gradient (interior acid) on chromaffm granule ghosts [5,8,10]. gifts of Dr A. Stempel (Hoffman-La Roche) and Dr B. S. Cooperman, respectively. [3H]Dihydrotetrabenazine (12 Ci/mmol) was prepared as in [ 111. S-Hydroxy [G-3H]tryptamine creatinine sulfate (18 Ci/mmol), l-[7,8-3H]Noradrenaline (12 Cijmmol), [7-14C] tyramine hydrochloride (50 mCi/mmol) were from Amersham, [ 14C] methylamine hydrochloride (38 mCi/mmol) was from CEA.