Na+-gradient-dependent transport of L-proline and analysis of its carrier system in brush-border membrane vesicles of the guinea-pig ileum.
Transport of L-proline was studied with membrane vesicles prepared from the brush borders of the guinea-pig ileum. The presence of an Na+ gradient from outside to inside of the vesicles stimulated L-proline uptake. Accumulation of amino acid in the vesicles reached a maximum 30 s after incubation, then decreased due to efflux and finally equilibrated at a level nearly identical to that shown in the absence of an Na+ gradient in 30 min. The peak level of the uptake was 3.5-times greater than the final equilibrium level. The equilibrium level of L-proline uptake decreased with increasing medium osmolarity. Extrapolation to infinite medium osmolarity, that is, under the condition of zero intravesicular space, showed no uptake, indicating transport of L-proline into membrane vesicles. The initial rate of uptake for 15 s was enhanced with increasing concentrations of Na+ in the external medium. A small part of the L-proline transport occurred by simple diffusion in addition to Na+-gradient-dependent transport. When L-proline concentrations were varied and transport due to diffusion was subtracted, the initial rate of uptake dependent on Na+ gradient (out greater than in) obeyed Michaelis-Menten kinetics with Km and V values of 0.67 mM and 2.73 nmol/15 s per mg protein, respectively. Evidence was obtained which indicates that L-cysteine is a substract specific for transport through system ASC (alanine-, serine-, and cysteine-preferring) and that transport in the presence of an Li+ gradient (out > in) also takes palce by the ASC system. The uptake of L-proline in the presence of an Na+ gradient (out > in) was inhibited 90% by a large excess of alpha-(methylamino)-isobutyrate, the model substrate specific for the A system (alanine-preferring). This indicates than 90% of Na+-gradient-dependent L-proline uptake is supported by the A system. The remaining 10% of L-proline uptake was found to be catalyzed by the ASC system, since L-proline uptake equivalent to this alpha-(methylamino)-isobutyrate-uninhibited part was demonstrated in the presence of Li+ gradient.
D000621:Aminoisobutyric Acids / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism*; D003545:Cysteine / Q000494:pharmacology; D006168:Guinea Pigs; D007082:Ileum / Q000378:metabolism*; D007700:Kinetics; D008094:Lithium / Q000494:pharmacology; D008871:Microvilli / Q000378:metabolism*; D009994:Osmolar Concentration; D011392:Proline / Q000378:metabolism*; D012964:Sodium / Q000494:pharmacology*
D000621:Aminoisobutyric Acids; C017911:2-(methylamino)isobutyric acid; D011392:Proline; D008094:Lithium; D012964:Sodium; D003545:Cysteine