Characterization of phosphate:hexose 6-phosphate antiport in membrane vesicles of Streptococcus lactis

Membrane vesicles of Streptococcus lactis were used to characterize a novel anion exchange involving phosphate and sugar 6-phosphates. For vesicles loaded with 50 mM phosphate at pH 7, homologous phosphate:phosphate exchange had a maximal rate of 130 nmol/min/mg of protein and a K(t) of 0.21 mM external phosphate; among phosphate analogues tested, only arsenate replaced phosphate. Heterologous exchange was studied by 2-deoxyglucose 6-phosphate entry into phosphate-loaded vesicles; this reaction had a maximal velocity of 31 nmol/min/mg of protein and a K(t) of 26 μM external substrate. Sugar phosphate moved intact during this exchange, since its entry led to internal ³²Pi without transfer of ³²P to sugar phosphate. Inhibitions of phosphate exchange suggested that the preferred sugar phosphate substrates were (K(iapp)): glucose, 2-deoxyglucose, and mannose 6-phosphates (approximately 20 μM) > fructose 6-phosphate (150 μM) > glucosamine 6-phosphate (420 μM) > α-methylglucoside 6-phosphate (740 μM). Stoichiometry for phosphate:2-deoxyglucose-6-phosphate antiport was 2:1 at pH 7, and since initial rates of exchange were unaffected by charge carrying ionophores (gramicidin, valinomycin, a protonophore), this unequal stoichiometry indicated the electroneutral exchange of two monovalent phosphates for a single divalent sugar phophate.