Reconstitution into liposomes of the glutamine/amino acid transporter from renal cell plasma membrane: functional characterization, kinetics and activation by nucleotides.
The glutamine/amino acid transporter was solubilized from rat renal apical plasma membrane (brush-border membrane) with C12E8 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 HgCl2, 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 Km 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.
Oppedisano
Francesca
F
Department of Cell Biology, University of Calabria, Via P.Bucci 4c 87036 Arcavacata di Rende, Italy.
Pochini
Lorena
L
Galluccio
Michele
M
Cavarelli
Mariangela
M
Indiveri
Cesare
C
D000595:Amino Acid Sequence; D026962:Amino Acid Transport System ASC / Q000378:metabolism*; D026905:Amino Acid Transport Systems / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism*; D011756:Diphosphates / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005973:Glutamine / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry*; D008871:Microvilli / Q000378:metabolism; D015778:Minor Histocompatibility Antigens; D008969:Molecular Sequence Data; D009705:Nucleosides / Q000494:pharmacology; D009711:Nucleotides / Q000737:chemistry / Q000494:pharmacology*; D051381:Rats; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D013379:Substrate Specificity
D026962:Amino Acid Transport System ASC; D026905:Amino Acid Transport Systems; D011756:Diphosphates; D008081:Liposomes; D015778:Minor Histocompatibility Antigens; D009705:Nucleosides; D009711:Nucleotides; C498849:Slc1a5 protein, rat; D005973:Glutamine