Histidyl residues at the active site of the Na/succinate cotransporter in rabbit renal brush borders

Abstract

Mono-, dicarboxylic acid-, and D-glucose transport were measured in brush border vesicles from renal cortex after treatment with reagents known to modify terminal amino, lysyl, epsilon-amino, guanidino, serine/threonine, histidyl, tyrosyl, tryptophanyl and carboxylic residues. All three sodium-coupled co-transport systems proved to possess sulfhydryl (and maybe tryptophanyl sulfhydryl, disulfide, thioether and tyrosyl) residues but not at the substrate site or at the allosteric cavity for the Na co-ion. Histidyl groups seem to be located in the active site of the dicarboxylic transporter in that the simultaneous presence of Na and succinate protects the transporter against the histidyl specific reagent diethylpyrocarbonate. Lithium, which specifically competes for sodium sites in the dicarboxylic acid transporter, substantially blocked the protective effect of Na and succinate. Hydroxylamine specifically reversed the covalent binding of diethylpyrocarbonate to the succinate binding site. The pH dependence of the Na/succinate cotransport is consistent with an involvement of histidyl and sulfhydryl residues. We conclude that a histidyl residue is at, or is close to, the active site of the dicarboxylate transporter in renal brush border membranes.

Electrophysiology of succinate transport across rabbit renal brush border membranes.

“…Hg2+ also inhibits the succinate potential, but this effect is irreversible, and it is due to a reduction in the AAbmax* p-chloromercuribenzene sulphonic acid produces a similar effect on succinate transport (Bindslev & Wright, 1984), and both effects are probably due to interactions with an essential sulphydryl group on the transporter.…”

“…Thus the affinity of the sites for Li+ appears to be about sixfold higher than the affinity for Na+. Although the identities of the Na+ sites are unknown, there is good evidence that a histidyl residue is at, or close to, the succinate binding site (Bindslev & Wright, 1984).…”

Inhibition of vincristine binding to plasma membrane vesicles from daunorubicin-resistant Ehrlich ascites cells by multidrug resistance modulators

“…Cells were maintained as ascitic tumours as described (Skovsgaard, 1978b Pldsma membrane vesicle preparation Plasma membrane vesicles were prepared from EHR2/ DNR + as previously described (Sehested et al, 1989). Briefly, after swelling and disruption of the cells by polytronation in a hypotonic buffer, differential centrifugation (Bindslev & Wright, 1984) was followed by separation on a discontinous Ficoll gradient under ultracentrifugation (Spitzer et al, 1983). Plasma membranes, which were enriched >20-fold over homogenate, were stored at -80°C till use.…”

“…J. Cancer (1989 (Bindslev & Wright, 1984;Sehested et al, 1989). Experiments were performed with either simultaneous incubation of drug tracer with a modulator or with 30 min preincubation at 4°C for each modulator before incubation with drug tracer.…”

Na+(Li+)/Branched-chain amino acid cotransport inPseudomonas aeruginosa

“…It is considered that low activities at pH 9 may be attributed to either inactivation of the transport systems or reduction of a driving force generated by intact cells, but that a difference in pH dependence of the two transport activities between pH 5 and 7 should be due to the property of the respective systems. The big difference in the leucine transport activity between pH 5 and 7 could be interpreted as titration of histidine with a pKa around 6.0 involved in the cotransport pathway (Bindslev & Wright, 1984). The possibility of involvement of histidine remains to be investigated.…”