Proline uptake by an isolated cytoplasmic membrane preparation of Escherichia coli.

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Abstract
Isolated membrane preparations from E. coli W6, a proline auxotroph, catalyze energy-dependent concentrative uptake of proline. Membranes from E. coli W157, a mutant deficient in proline uptake and exchange capacity, do not catalyze concentrative proline uptake. Membranes prepared from penicillin-induced sphero-plasts by osmotic shock and treatment with ethylene diamine tetraacetate (EDTA), could be separated from trace amounts of intact cells and spheroplasts by sucrose density sedimentation. Homogeneity of the preparations was established by electron and phase contrast microscopy, viability assays, disk gel electrophoresis and enzymatic assay. The isolated membranes contained less than 5% as much deoxyribonucleic acid (DNA) or ribonucleic-acid (RNA) and 15% as much protein as sphero-plasts; their proline uptake capacity is 50% or greater than spheroplasts. More than 80% of the proline fixed by membranes was recovered as proline by chromatography. Proline uptake is O2 -dependent and is stimulated by glucose; it is inhibited by dinitrophenyl (DNP), carbonyl cyanide p-trifluoromethoxyphenylhydrazone, iodoacetamide, amytal and hydroxyproline but not by other amino-acids or by chloromycetin. Fixed proline exchanges with external proline and hydroxyproline but not with other amino-acids.