BINDING-PROTEINS FOR CYCLIC AND LINEAR OLIGOPEPTIDES IN PLASMA-MEMBRANES AND THE CYTOSOL OF RAT HEPATOCYTES

Using a cyclolinopeptide A analogue, the hydrophobic cyclic peptide c( -Ala-Lys-Pro-Phe-Phe-Ala-Lys-Pro-Phe-Phe-), termed CDP (cyclodecapepti de), as ligand in affinity chromatography, hepatocellular peptide bind ing proteins were isolated from the integral part of plasma membranes and the cytosol. The sequence of the isolated protein with MW of 50 kD a from the integral part of the plasma membrane fraction was identical to cytochrome P450 II C13 and cytochrome P450 II C22, whereas the seq uence of the 54 kDa protein was identical to 3-hydroxyandrogen-UDP-glu curonosyltransferase. These proteins have also been described as bindi ng proteins for bile acids. As shown in earlier studies, bile acids an d CDP also compete for uptake into hepatocytes. In the cytosol, a furt her known bile acid binding protein, the glutathione-S-transferase (G- S-T) subunit Yb1, was isolated and sequenced as binding protein for CD P and also for a further cyclopeptide, the somatostatin analogue OO8, and a linear peptide with renin-inhibiting activity, EMD 55068. As sho wn in uptake studies using isolated basolateral plasma membrane vesicl es, G-S-T was able to increase the uptake of EMD 51921, a linear pepti de with renin-inhibiting potency, into the vesicles when the latter we re preloaded with G-S-T. The binding of the substrate to the outside o f the preloaded vesicles was not different than binding to unloaded ve sicles. The maximal transport rate of the carrier-mediated/facilitated diffusion and the rate of permeation, however, were doubled in the pr esence of G-S-T, pointing to the involvement of intracellular binding proteins such as G-S-T in the unloading of the carrier protein and in the reduction of the free substrate concentration. (C) 1997 Elsevier S cience Inc.