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).
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 IC50 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 IC50 value for Ala-Lys but did decrease the IC50 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 IC50 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.
Brandsch
M
M
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta 30912, USA.
Brandsch
C
C
Ganapathy
M E
ME
Chew
C S
CS
Ganapathy
V
V
Leibach
F H
FH
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D018938:Caco-2 Cells / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D002506:Cephalexin / Q000494:pharmacology; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004151:Dipeptides / Q000378:metabolism / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006639:Histidine / Q000502:physiology*; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism; D007700:Kinetics; D000074011:Peptide Transporter 1; D011522:Protons; D051381:Rats; D027981:Symporters
D002352:Carrier Proteins; D004151:Dipeptides; D000074011:Peptide Transporter 1; D011522:Protons; C082186:SLC15A1 protein, human; C489940:Slc15a1 protein, rat; D027981:Symporters; C093694:hydrogen-coupled oligopeptide transporter PepT2; C038047:alanylaspartic acid; C004194:glycylsarcosine; D006639:Histidine; D004047:Diethyl Pyrocarbonate; D002506:Cephalexin