title: Reconstitution of Escherichia coli membrane vesicles with D-amino acid dehydrogenase.
abstract: When purified D-amino acid dehydrogenase [Olsiewski, P. J., Kaczorowski, G. J., & Walsh, C. T. (1980) J. Biol. Chem. 255, 4487] is incubated with right-side-out membrane vesicles from Escherichia coli, the enzyme binds to the membrane in a time- and concentration-dependent manner. As a result, the vesicles acquire the ability to oxidize D-alanine and catalyze D-alanine-dependent active transport. Similarly, incubation of D-amino acid dehydrogenase with inside-out vesicles results in binding of enzyme and D-alanine oxidase activity. Antibody inhibition studies indicate that the enzyme is bound exclusively to the inner cytoplasmic surface of the membrane in native vesicles (i.e., membrane vesicles prepared from cells induced for D-amino acid dehydrogenase). In contrast, similar studies with reconstituted vesicles demonstrate that enzyme binds to the surface exposed to the medium regardless of the orientation of the membrane. Thus, enzyme bound to right-side-out vesicles is located on the opposite side of the membrane from where it is normally found. Remarkably, in the presence of D-alanine, reconstituted right-side-out and inside-out vesicles generate electrochemical proton gradients of similar magnitude but opposite polarity, indicating that enzyme bound to either surface of the membrane is physiologically functional. The results suggest that vectorial proton translocation via the respiratory chain occurs at a point distal to the site where electrons enter the respiratory chain from the primary dehydrogenase, a conclusion that is inconsistent with the notion that the dehydrogenase forms part of a proton-translocating loop.
mesh_terms: D000409:Alanine / Q000378:metabolism; D000906:Antibodies; D000936:Antigen-Antibody Complex; D001692:Biological Transport; D001693:Biological Transport, Active; D002462:Cell Membrane / Q000201:enzymology* / Q000502:physiology; D003605:D-Amino-Acid Oxidase / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D005033:Ethylmaleimide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D010101:Oxygen Consumption
chemical_list: D000906:Antibodies; D000936:Antigen-Antibody Complex; D003605:D-Amino-Acid Oxidase; D005033:Ethylmaleimide; D000409:Alanine