Open AccessSolubilization and Partial Purification of Amino Acid-Specific Components of the D-Lactate Dehyrogenase-Coupled Amino Acid-Transport Systems
Author(s) -
Adrienne S. Gordon,
Frank J. Lombardi,
H. Ronald Kaback
Publication year - 1972
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.69.2.358
Subject(s) - chemistry , proline , amino acid , biochemistry , vesicle , sephadex , dithiothreitol , proline dehydrogenase , glycine , molecular mass , membrane , enzyme
A protein-containing fraction has been solubilized fromE. 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 byp -chloromercuribenzoate andN -ethylmaleimide, and the inhibition observed withp -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.