Regulation of purine utilization in bacteria. VII. Involvement of membrane-associated nucleoside phosphorylase in the uptake and the base-mediated loss of the ribose moiety of nucleosides by Salmonella typhimurium membrane vesicles

Abstract

Although uridine and adenosine are converted by membrane-associated nucleoside phosphorylases to ribose-1-phosphate (ribose-1-P) and the corresponding bases (uracil and adenine), only ribose-1-P is accumulated within S. typhimurium LT2 membrane vesicles. No uptake is observed when the vesicles are incubated with the bases or nucleosides labeled in their base moieties. The vesicles lack a transport system for ribose-1-P, since excess ribose-1-P does not inhibit the uptake of the ribose moiety of uridine. There is no exchange with preaccumulated ribose-1-P. Uridine, rather than ribose-1-P, serves as the initially transported substrate. The uptake of the ribose portion of uridine is coupled to electron transport, and the levels to which ribose-1-P are accumulated are reduced by adding various bases to the reaction mixtures. The bases appear to inhibit the uridine phosphorylase reaction and/or cause an efflux of ribose-1-P from the vesicles. This loss of ribose-1-P reflects the accumulation of nucleosides in the external medium after being synthesized within the membranes. Synthesis of the nucleosides from intravesicular ribose-1-P and exogenous bases proceeds even though the bases are not accumulated by the vesicles. Ribose-1-P cannot significantly inhibit uridine phosphorylase activity unless the membranes are disrupted. The membrane-associated nucleoside phosphorylases may have a transmembranal orientation with their base and ribose-1-P binding sites on opposite sides of the membranes. Such an asymmetric arrangement of these enzymes may facilitate the uptake of the ribosyl moiety of nucleosides by a group translocation mechanism. Nucleosides are cleaved during the membrane transport process, with the resultant bases delivered to the external environment while ribose-1-P is shunted to the intravesicular space.