Identification and conformational changes of the intestinal proline carrier.
Fluorescein isothiocyanate (FITC) was used to selectively label the rabbit intestinal brush-border imino carrier, identify the binding protein on SDS-polyacrylamide gel electrophoresis, and monitor the effect of ions on fluorescein quenching. FITC inhibits Na+-dependent L-proline transport irreversibly, but transport is protected by physiological concentrations of Na+ and L-proline. About 1 nmol of FITC/mg of protein binds specifically to the transporter, which was identified by SDS-polyacrylamide gel electrophoresis as a 100 +/- 5-kDa peptide. Na+ produced a specific, saturable quench in the fluorescence of FITC bound to the proline carrier. Both transport and FITC quenching are inhibited by n-acetylimidazole, and membranes are protected from acetylation by Na+. We conclude that Na+ binds to the proline carrier (100-kDa peptide) to produce a change in conformation that results in an increase in the affinity of the carrier for proline.
D026921:Amino Acid Transport Systems, Neutral; D000818:Animals; D002352:Carrier Proteins / Q000032:analysis / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007413:Intestinal Mucosa / Q000378:metabolism*; D007700:Kinetics; D008871:Microvilli / Q000378:metabolism*; D008970:Molecular Weight; D011392:Proline / Q000378:metabolism*; D011487:Protein Conformation; D011817:Rabbits; D013861:Thiocyanates
D026921:Amino Acid Transport Systems, Neutral; D002352:Carrier Proteins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D013861:Thiocyanates; C041832:proline transport protein; D011392:Proline; D016650:Fluorescein-5-isothiocyanate