Functional separation of dipeptide transport and hydrolysis in kidney brush border membrane vesicles.

Abstract

Dipeptides serve as substrates for both transport and hydrolytic processes. This has caused uncertainty as to whether different systems mediate both processes, and if so, whether they can be functionally separated. To investigate these problems, we determined the effects of a series of compounds previously characterized as aminopeptidase (AP) inhibitors on transport and hydrolytic activities of the kidney brush border membrane. The substrate used for assaying transport activity was Gly-Gln whereas Leu-, Arg-, and Glu-nitroanilides, as well as Gly-Tyr and Ala-Tyr, were used in assaying hydrolytic activity. The AP inhibitors, arphamenines A and B and bestatin, strongly inhibited transport by the oligopeptide/H+ symporter (EC50 values of 15 to 67 microM). The mechanism of inhibition appeared to be competition for the binding site of the symporter. In contrast, leucinethiol, leuhistin, and amastatin had little or no effect on dipeptide transport (EC50 values of 4 to more than 50 mM). Arphamenine and leucinethiol, in concentrations as high as 100 microM, were found to be either ineffective or weak inhibitors of membrane-associated hydrolysis. In contrast, amastatin and leuhistin, in concentrations as low as 20 microM, almost completely inhibited dipeptide hydrolysis. These results show that dipeptide hydrolysis can be selectively suppressed by either amastatin or leuhistin and dipeptide transport by arphamenine. Furthermore, the results provide new insight into the structural features of substrates that are recognized at the binding site of the oligopeptide/H+ symporter.