In vivo and in vitro incorporation of endogenous nucleotides by the energy-transducing ATPase of Streptococcus faecalis.

The soluble ATPase isolated from Streptococcus faecalis membranes containing tightly bound endogenous nucleotides do not exchange in the presence of ATP and Mg+2 added during the purification of the enzyme. In this paper the stoichiometry of endogenous nucleotides in the soluble ATPase obtained from (a) growing cells, (b) nongrowing glycolyzing cells, and (c) isolated cell membranes has been defined. The time course of incorporation was also studied in nongrowing, glycolyzing cells and isolated cell membranes. In all cases, 1-2 mol of nucleotide was bound per mol of enzyme. Maximal incorporation required approximately 1 h at 38 degrees C. Incorporation of cytoplasmic nucleotide into the enzyme occurred by a process of slow exchange for bound nucleotide. N,N'-dicyclohexylcarbodiimide, which inhibits the membrane-bound ATPase and prevents generation of the protonmotive force, had no effect on incorporation of endogenous nucleotides in glycolyzing cells. Treatment of glycolyzing cells with gramicidin D plus K+, which dissipates the protonmotive force but has no effect on ATPase activity, did not inhibit incorporation of nucleotide. These results support the view that the slow exchange-incorporation of endogenous nucleotide(s) is independent of ATP hydrolysis and a protonmotive force. An in vitro system for the study of nucleotide binding at endogenous sites is described.