Reconstitution into liposomes of the glutamine/amino acid transporter from renal cell plasma membrane: Functional characterization, kinetics and activation by nucleotides

Francesca Oppedisano, Lorena Pochini, Michele Galluccio, Mariangela Cavarelli, Cesare Indiveri

Research output: Contribution to journalArticle

Abstract

The glutamine/amino acid transporter was solubilized from rat renal apical plasma membrane (brush-border membrane) with C 12E 8 and reconstituted into liposomes by removing the detergent from mixed micelles by hydrophobic chromatography on Amberlite XAD-4. The reconstitution was optimised with respect to the protein concentration, the detergent/phospholipid ratio and the number of passages through a single Amberlite column. The reconstituted glutamine/amino acid transporter catalysed a first-order antiport reaction stimulated by external, not internal, Na +. Optimal activity was found at pH 7.0. The sulfhydryl reagents HgCl 2, mersalyl and p-hydroxymercuribenzoate and the amino acids alanine, serine, threonine, cysteine, asparagine, methionine and valine strongly inhibited the transport, whereas the amino acid analogue methylaminoisobutyrate had no effect. Glutamine, alanine, serine, asparagine, threonine were efficiently translocated from outside to inside and from inside to outside the proteoliposomes as well. Cysteine and valine were translocated preferentially from outside to inside. The K m for glutamine on the external and internal side of the transporter was 0.47 and 11 mM, respectively; the values were not influenced by the type of the counter substrate. The transporter is functionally asymmetrical and it is unidirectionally inserted into the proteoliposomal membrane with an orientation corresponding to that of the native membrane. By a bisubstrate kinetic analysis of the glutamine antiport, a random simultaneous mechanism was found. The glutamine antiport was strongly stimulated by internal nucleoside triphosphates and, to a lower extent, by pyrophoshate. The reconstituted glutamine/amino acid transporter functionally corresponds to the ASCT2 protein.

Original languageEnglish
Pages (from-to)122-131
Number of pages10
JournalBiochimica et Biophysica Acta - Biomembranes
Volume1667
Issue number2
DOIs
Publication statusPublished - Dec 15 2004

Keywords

  • Amino acid
  • ASCT2
  • Glutamine
  • Liposome
  • Plasma membrane
  • Reconstitution
  • Transport

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Biophysics

Fingerprint Dive into the research topics of 'Reconstitution into liposomes of the glutamine/amino acid transporter from renal cell plasma membrane: Functional characterization, kinetics and activation by nucleotides'. Together they form a unique fingerprint.

  • Cite this