Glycine Uptake in Escherichia coli: II. GLYCINE UPTAKE, EXCHANGE, AND METABOLISM BY AN ISOLATED MEMBRANE PREPARATION

Abstract

Abstract In this paper, glycine uptake by isolated membrane preparations of Escherichia coli W (W+) and the d-serine-resistant mutant (WS) is examined in detail. Glycine uptake by W+ membrane preparations is dependent on pH and on the presence of Mg++ and an energy source. In W+ membranes, glycine is first taken up into an exchangeable pool, from which it is subsequently removed as the incubation proceeds. The exchangeable pool is built up within 30 sec and remains constant with time, although glycine uptake by the membranes continues. Glycine uptake into the exchangeable pool of W+ membranes is independent of temperature. Uptake and exchange in W+ membranes are specific for glycine, dl-alanine, dl-serine, and dl-threonine. Membrane preparations from the WS mutant are unable to catalyze either the uptake of glycine or the exchange of glycine between the incubation medium and the pool. Glycine uptake by W+ membranes is inhibited 50 to 70% by anaerobiosis, incubation at 0°, or incubation in the presence of 2,4-dinitrophenol, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, NaN3, KAsO4, KCN, iodoacetamide, and p-chloromercuriphenylsulfonate. No inhibition is oberved with NaF, sodium Amytal, hydroxyquinoline N-oxide, antimycin A, Chloromycetin, or ouabain. Although glycine uptake at 30 min is inhibited by the conditions listed above, there is no diminution of glycine uptake when measured at 30 sec. Under none of the conditions studied is the uptake (at either 30 sec or 30 min) decreased to the values found with WS membranes at 37° or 0°. W+ membranes incubated at 37° convert about 94% of the glycine carbon taken up into compounds that have the properties of phosphatidylethanolamine and phosphatidylserine. Under all of the conditions studied, the concentration of glycine in the intramembranal pool approximates that of the incubation medium. Incubation at 0° or in the presence of dinitrophenol or iodoacetamide inhibits the conversion of glycine to phospholipids, and the amount of glycine in the intramembranal pool is increased above that found for uninhibited (control) preparations. WS membranes convert significant amounts of glycine to phospholipids, but the concentration of glycine in the intramembranal pool is only 10 to 20% of that in the external pool. When the external glycine concentration is raised so that the rate of glycine uptake by WS membranes is comparable to the rate exhibited by W+ membranes, the WS membranes convert as much glycine to phospholipids as do W+ membranes, but the concentration of glycine in the intramembranal pool remains only 10 to 20% of that in the external pool. Hydroxylamine almost quantitatively inhibits the conversion of glycine to phospholipids, and in the presence of this inhibitor the WS membranes manifest the same defect.