Mechanism of energy coupling to entry and exit of neutral and branched chain amino acids in membrane vesicles of Streptococcus cremoris.
The energetics of neutral and branched chain amino acid transport by membrane vesicles from Streptococcus cremoris have been studied with a novel model system in which beef heart mitochondrial cytochrome c oxidase functions as a proton-motive force (delta p) generating system. In the presence of reduced cytochrome c, a large delta p was generated with a maximum value at pH 6.0. Apparent H+/amino acid stoichiometries (napp) have been determined at external pH values between 5.5 and 8.0 from the steady state levels of accumulation and the delta p. For L-leucine napp (0.8) was nearly independent of the pH. For L-alanine and L-serine napp decreased from 0.9-1.0 at pH 5.5 to 0-0.2 at pH 8.0. The napp for the different amino acids decreased with increasing external amino acid concentration. At pH 6.0, first order rate constants for amino acid exit (kex) under steady state conditions for L-leucine, L-alanine, and L-serine were 1.1-1.3, 0.084, and 0.053 min-1, respectively. From the pH dependence of kex it is concluded that amino acid exit in steady state is the sum of two processes, pH-dependent carrier-mediated amino acid exit and pH-independent passive diffusion (external leak). The first order rate constant for passive diffusion increased with increasing hydrophobicity of the side chain of the amino acids. As a result of these processes the kinetic steady state attained is less than the amino acid accumulation ratio predicted by thermodynamic equilibrium. The napp determined from the steady state accumulation represents, therefore, a lower limit. It is concluded that the mechanistic stoichiometry (n) for L-leucine, L-alanine, and L-serine transport most likely equals 1.
D026905:Amino Acid Transport Systems; D000596:Amino Acids / Q000378:metabolism*; D000597:Amino Acids, Branched-Chain / Q000378:metabolism; D000818:Animals; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism*; D003576:Electron Transport Complex IV / Q000378:metabolism; D004734:Energy Metabolism; D006863:Hydrogen-Ion Concentration; D008929:Mitochondria, Heart / Q000201:enzymology; D011522:Protons; D013291:Streptococcus / Q000378:metabolism*; D013816:Thermodynamics
D026905:Amino Acid Transport Systems; D000596:Amino Acids; D000597:Amino Acids, Branched-Chain; D002352:Carrier Proteins; D011522:Protons; D003576:Electron Transport Complex IV