Solubilization and partial purification of amino acid-specific components of the D-lactate dehydrogenase-coupled amino acid-transport systems (E. coli-cell membranes-sephadex-detergent-solubilized-vesicles).
A protein-containing fraction has been solubilized from E. coli ML 308-225 membrane vesicles that has many of the properties of the amino acid "carrier proteins" of the D-lactate dehydrogenase-coupled amino acid-transport systems. Membrane vesicles were partially solubilized with the nonionic detergent Brij 36-T, and the solubilized material was fractionated by Sephadex G-100 chromatography in the presence of the same detergent. Three fractions possess binding activity for proline: one of relatively low molecular weight with a high specific activity, and two of higher molecular weight with low specific activities. The higher molecular weight fractions exhibit D-lactate dehydrogenase activity; however, there is no corresponding activity associated with the low molecular weight fraction. Moreover, proline-binding activity is highly specific as it is not inhibited by structurally-unrelated amino acids. In addition to proline, the low molecular weight fraction exhibits binding activities for serine, glycine, lysine, and tyrosine. The proline-binding activity of the low molecular weight fraction is not inhibited by electron transfer or by general metabolic inhibitors that inhibit the accumulation of proline by intact membrane vesicles. In contrast, binding activity is inhibited by p-chloromercuribenzoate and N-ethylmaleimide, and the inhibition observed with p-chloromercuribenzoate is reversed by the addition of dithiothreitol. The effect of these sulfhydryl reagents on binding corresponds to the effect of these compounds on proline transport by intact membrane vesicles.
D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D002850:Chromatography, Gel; D002855:Chromatography, Thin Layer; D004926:Escherichia coli / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D009243:NAD; D010088:Oxidoreductases / Q000378:metabolism; D010759:Phosphorus Isotopes; D011392:Proline / Q000378:metabolism; D011485:Protein Binding; D012995:Solubility; D013385:Succinate Dehydrogenase / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000494:pharmacology
D001426:Bacterial Proteins; D002352:Carrier Proteins; D010759:Phosphorus Isotopes; D013438:Sulfhydryl Compounds; D009243:NAD; D011392:Proline; D010088:Oxidoreductases; D007770:L-Lactate Dehydrogenase; D013385:Succinate Dehydrogenase