Uptake of glycine from L-alanylglycine into renal brush border vesicles.

Isolated renal brush border microvilli vesicles were employed to study the uptake of radiolabel from L-Ala. [3H]Gly and D-Ala.[3H]Gly as well as to determine the presence of dipeptidase activity. Microvilli vesicles were prepared from porcine kidney cortex by differential centrifugation through hypotonic Tris buffer containing Mg2+. The microvilli vesicles transiently accumulated radiolabel from L-Ala. [3H]Gly to higher levels than were initially present in the incubation medium (overshoot phenomenon). This accumulation was dependent on the presence of an inward-directed (extravesicular greater than intravesicular) Na+ gradient and was osmotically sensitive and linear with respect to microvilli protein concentration. Analysis of intravesicular contents revealed that all 3H uptake from L-Ala. [3H]Gly appeared as free glycine. Hydrolysis studies demonstrated the rate of L-Ala.[3H]Gly hydrolysis to free alanine and [3H[glycine by the microvilli to be greatly in excess of their rate of radiolabel uptake from this dipeptide. In addition, the uptake profiles and kinetic constants for vesicular uptake of radiolabel from L-Ala.[3H]Gly and free glycine were demonstrated to be identical when measured by double-labeling techniques in the same experiments. These results indicate that L-Ala.[3H]Gly is hydrolyzed at the external surface of the microvilli with the [3H]glycine released being transported into the vesicles by a Na+ gradient-dependent system identical to that employed for free glycine. Microvilli vesicle uptake of radiolabel from D-Ala.[3H]Gly exhibited no Na+ dependent "over-shoot" effect. D-Ala.[3H]Gly was completely resistant to microvilli-catalyzed hydrolysis. Analysis of the microvilli for renal dipeptidase, an enzyme with hydrolytic activity against a wide range of L-dipeptides, revealed this enzyme to be enriched in the microvilli vesicles to a degree equivalent to that observed for marker enzymes for renal microvilli. Renal dipeptidase catalyzed hydrolysis of L-Ala.Gly but not D-Ala.Gly, as was the case with microvilli-catalyzed hydrolysis of the dipeptides. With its location in the renal brush border microvilli and its hydrolytic action against L-dipeptides, renal dipeptidase my act at the luminal surface of the proximal tubule cell to hydrolyze L-dipeptides present in the glomerular filtrate, with the resultant free amino acids transported across the brush border microvilli by Na+ gradient-dependent processes.