Abstract
We and others have shown previously the existence of high and low affinity systems for oligopeptide transport in kidney brush border membrane vesicles (BBMV). In the present study we investigated the relationship between the structure of substrates and their affinity for interaction with the high-affinity oligopeptide/H+ transporter in kidney BBMV. Based on competition experiments using [3H]Gly-Gln as a probe we determined the Ki values for more than 60 selected peptides. For a high-affinity interaction with the carrier site the following structural features of substrates are required: (a) both a free amino and carboxyl terminus; (b) the amino group and peptide bond nitrogen located in the alpha-position; (c) a trans peptide bond rather than the cis configuration; (d) L-alpha-amino acid isomers in both COOH and NH2 termini, although D-isomers of hydrophobic amino acids are acceptable in the NH2 terminus; and (e) a backbone of less than 3 amino acid residues. A striking finding of the present study is that, for peptides satisfying these minimal structural requirements, the primary determinant of affinity is hydrophobicity. The fact that there is a highly significant (p less than 0.001) correlation between Ki and hydrophobicity allows the prediction of the affinity for any di- or tripeptide composed of alpha-amino acids in the L-form.
Highlights
From the Clinical Nutrition Research Unit and DeDartment of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213
In the present study we investigated the relationship between the structure of substrates and theiraffinity for interaction with the high-affinity oligopeptide/H+transporter in kidney brush border membrane vesicles (BBMV)
Hydrolysis ofGly-Gln by BBMV-In a previous study (1) we showed that under the incubation conditions used for the assay of dipeptide transport, the hydrolysis of 100 p~ Glyof uptake) was subtracted from the transportdata
Summary
0 1992 by The American Society for Biochemistry and Molecular Biology, Inc. Vol 267, No 14, Issue of May 15, pp. 9565-9573,1992 Printed in U.S.A. As shown previously (I), under the conditions of our transport assay Gly-Gln uptake intoBBMV preloaded with a buffer pH 8.3 and incubated in a buffer pH 6.0 (final incubation pH 6.7) is linear up to 5 s and displays an overshoot of eight times the equilibrium values (0.962 f 0.092 pmol/mg protein a t 5 s versus 0.111 f 0.002 pmol/mg much lower concentration of Gly-Gln (0.1 p ~ )T.herefore, it is possible that hydrolysis could be a significant problem under the incubation conditions of the present study To investigate this problem, the following experiment was perprotein at 30 min). Effect of Free Amino Acids on Dipeptide Uptake- Gly-Gln was not appreciably hydrolyzed in the medium under radioactive tracer intothe BBMV represents the influx of the the conditions of our transport assay, other oligopeptides parent dipeptide, the amount of [3H]Gly-Gln(0.1 p ~hy)drolyzed in serving as inhibitors might behydrolyzed. This appeared to be a corresponding a-amino dipeptide configurations (Table 11)
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