A characterization of the nucleotide uptake by chromaffin granules of bovine adrenal medulla

Abstract

Chromaffin granules isolated from bovine adrenal gland were incubated with 3H-labelled nucleotides and [14C]noradrenaline to study the uptake of these substances. [3H]ATP, [3H]ADP and [3H]AMP are taken up by these organelles by the same temperature-dependent mechanism. The apparent Km for ATP and ADP is 1.4mm, and for AMP it is 2.9mm. The uptake of ATP has a flat pH optimum, whereas the catecholamine uptake increases with more alkaline pH. Atractyloside and carboxyatractyloside are competitive and specific inhibitors of nucleotide uptake, whereas reserpine inhibits only that for catecholamines. Mg2+ ions activate uptake of both catecholamine and nucleotides, whereas EDTA and N-ethylmaleimide inhibit these processes. Nucleotide and catecholamine uptakes are inhibited by uncouplers of oxidative phosphorylation and by two ATP analogues. NH4+ ions and nigericin in the presence of KCl inhibit only catecholamine uptake. It is concluded that nucleotide uptake, as proposed previously for catecholamine uptake, depends on an electrochemical proton gradient produced by a proton-translocating adenosine triphosphatase localized in the membrane of chromaffin granules. Furthermore, as suggested by the effect of NH4+ and nigericin, catecholamine uptake apparently depends on the chemical part of this gradient, whereas the results for nucleotide uptake are consistent with its dependence on the electrical component.