Influence of proton and essential histidyl residues on the transport kinetics of the H +/peptide cotransport systems in intestine (PEPT 1) and kidney (PEPT 2)

Abstract

The mechanism by which H + alters the kinetics of the H +-coupled peptide transporters PEPT 1 and PEPT 2 was investigated in two different cell lines which differentially express these transporters, namely Caco-2 cells (PEPT 1) and SKPT cells (PEPT 2). The effects of H + on the affinity and the maximal velocity of Gly-Sar uptake were analyzed in these cells under identical conditions. In both cells, H + influenced only the maximal velocity of uptake and not the apparent affinity. The effects of H + on the IC 50 values (i.e., concentration necessary to cause 50% inhibition) of the cationic dipeptide Ala-Lys and the anionic dipeptide Ala-Asp for inhibition of Gly-Sar uptake were also investigated. H + did not change the IC 50 value for Ala-Lys but did decrease the IC 50 value for Ala-Asp considerably. The influence of diethylpyrocarbonate (DEP) on the kinetic parameters of PEPT 1 and PEPT 2 was then studied. Histidyl residues are the most likely amino acid residues involved in H + binding and translocation in H +-coupled transport systems and DEP is known to chemically modify histidyl residues and block their function. DEP treatment altered the maximal velocity of Gly-Sar uptake but had no effect on its K t (Michaelis-Menten constant) or the IC 50 values of Ala-Lys or Ala-Asp for the inhibition of Gly-Sar uptake. It is concluded that H + stimulates PEPT 1 and PEPT 2 primarily by increasing the maximal velocity of the transporters with no detectable influence on the substrate affinity.