Dataset Information

A role for aminopeptidase N in Na(+)-dependent amino acid transport in bovine renal brush-border membranes.

ABSTRACT: A monoclonal antibody FD19 which removes reconstitutable Na(+)-dependent amino acid transport activity from solubilized bovine renal brush-border membrane vesicles was found to react specifically with the enzyme aminopeptidase N. Cleavage of aminopeptidase N from the membranes with papain inhibited Na(+)-dependent amino acid transport activity without affecting that of alpha-methyl D-glucoside. Removal of aminopeptidase substantially increased the Km values for the Na(+)-dependent transport of alanine, glutamine, leucine and phenylalanine without affecting the Vmax. Both Na(+)-dependent amino acid transport and aminopeptidase activity in intact vesicles were competitively inhibited by amino acids with very similar specificity. These results suggest that the amino acid-binding sites of aminopeptidase N and the transporter interact in some way to increase the Km of the transport process for its substrates. However, independent direct inactivation of the transport system by papain cannot be ruled out.

SUBMITTER: Plakidou-Dymock S

PROVIDER: S-EPMC1132382 | biostudies-other | 1993 Feb

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:Phosphate uptake by rat renal brush-border membrane vesicles was studied under experimental conditions where transmembrane electrical potential (delta psi) could be manipulated. Experiments were performed under initial rate conditions to avoid complications associated with the dissipation of ion gradients. First, phosphate uptake was shown to be strongly affected by the nature of Na+ co-anions, the highest rates of uptake being observed with 100 mM-NaSCN (1.010 +/- 0.086 pmol/5 s per micrograms of protein) and the lowest with 50 mM-Na2SO4 (0.331 +/- 0.046 pmol/5 s per micrograms of protein). Anion substitution studies showed that potency of the effect of the co-anions was in the order thiocyanate greater than nitrate greater than chloride greater than isethionate greater than gluconate greater than sulphate, which correlates with the known permeability of the membrane to these anions and thus to the generation of transmembrane electrical potentials of decreasing magnitude (inside negative). The stimulation by ion-diffusion-induced potential was observed from pH 6.5 to 8.5, indicating that the transport of both monovalent and divalent phosphate was affected. In addition, inside-negative membrane potentials were generated by valinomycin-induced diffusion of K+ from K+-loaded vesicles and showed a 57% stimulation of phosphate uptake, at pH 7.5. Similar experiments with H+-loaded vesicles, in the presence of carbonyl cyanide m-chlorophenylhydrazone gave a 50% stimulation compared with controls. Inside-positive membrane potentials were also induced by reversal of the K+ gradient (outside greater than inside) in the presence of valinomycin and gave 58% inhibition of phosphate uptake. The membrane-potential dependency of phosphate uptake was finally analysed under thermodynamic equilibrium, and a stimulation by inside-negative potential was observed. The transport of phosphate was thus driven against a concentration gradient by a membrane potential, implicating the net transfer of a positive charge during the translocation process. These results indicate a major contribution of electrical potential to phosphate uptake in renal brush-border membranes.

Dataset Information

A role for aminopeptidase N in Na(+)-dependent amino acid transport in bovine renal brush-border membranes.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets