STUDIES ON PHOSPHATE TRANSPORT IN ESCHERICHIA-COLI .2. EFFECTS OF METABOLIC-INHIBITORS AND DIVALENT-CATIONS

A study was made of the effects of a variety of metabolic inhibitors and divalent cations (Ni2+ and Mn2+), normally after 5 min exposure, on the biphasic uptake of Pi exhibited by phosphate-deprived cells of E. coli, strains AB3311 (Reeves met-) and CBT302 (a (Ca2+ + Mg2+)-ATPase-deficient mutant). In AB3311 cells cyanide (1-10 mM) produced comparable reductions in phosphate uptake to anaerobiosis, but in both instances significant uptake was maintained. Examination of intracellular Pi concentrations showed that, despite these inhibitions, Pi is still concentrated 130 times compared to 394 times under aerobic conditions. Arsenate (100 .mu.M) and iodoacetate (100 .mu.M pre-exposed 15 min) both abolished anaerobic-supported uptake. Under aerobic conditions the former eliminated primary uptake while the latter reduced both phases of uptake 60%. The uncouplers, dinitrophenol (100-1000 .mu.M) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP) (50 .mu.M), produced very significant, but not complete, inhibitions of both phases of uptake. Inhibitions by iodoacetate and dinitrophenol were additive while dithiothreitol protected against the effects of 50-250 .mu.M CCCP. N,N''-Dicyclohexylcarbodiimide (DCCD), the potent inhibitor of membrane-bound (Ca2+ + Mg2+)-ATPase, at 10-3 M caused significant inhibitions of aerobic- (about 60%) and anaerobic- (about 80%) supported uptakes, suggesting some obligatory requirement for this ATPase. CBT302 cells, like AB3311, supported Pi transport aerobically and anaerobically. CCCP (50 .mu.M) reduced the primary uptake similarly to AB3311 cells, but the secondary uptake was less affected. DCCD (10-5-10-3 M), as expected, showed no effects in contrast to AB3311 cells. In AB3311 cells Ni2+ (10 mM) caused significant but different reductions of secondary (70%) and primary (33%) phases of phosphate uptake. Mn2+ (10 mM) showed a greater differential effect with the primary uptake being minimally affected and the secondary uptake being abolished (97%). Partial relief of these inhibitions by Mg2+ (10 mM) suggested that these ions compete with Mg2+ transport. High voltage electrophoresis studies showed that Ni2+ cause intensification in the labeling from 32Pi (i.e., during Pi uptake) of hexose phosphates and a reduction in the labeling of complex molecules left at the origin. With Mn2+, labeling of fructose 1,6-diphosphate was reduced, the triose phosphate area was intensified and an unknown area (X) was intensely labeled. When Mn2+ was combined with anaerobiosis, phosphate uptake, though diminished in rate, exceeded after 16 min the plateau level of uptake under aerobic conditions with Mn2+ present.