Reconstitution into liposomes of the B degrees -like glutamine-neutral amino acid transporter from renal cell plasma membrane.
Na+ dependent [3H]glutamine uptake was found in liposomes reconstituted with solubilized rat kidney brush border in the presence of intraliposomal K+. The reconstituted system was optimised with respect to the critical parameters of the cyclic detergent removal procedure, i.e., the detergent used for the solubilization, the protein concentration, the detergent/phospholipid ratio and the number of passages through a single Amberlite column. Time dependent [3H]glutamine accumulation in proteoliposomes occurred only in the presence of external Na+ and internal K+. The transporter showed low if there is any tolerance towards the substitution of Na+ or K+ for other cations. Valinomycin strongly stimulated the transport indicating that it is electrogenic. Intraliposomal glutamine had no effect. From the dependence of the transport rate on the Na+ concentration cooperativity index close to 1 was derived, indicating that 1 Na+ should be involved in the cotransport with glutamine. The electrogenicity of the transport originated from the Na+ transport. Optimal rate of 0.1 mM [3H]glutamine uptake was found in the presence of 50 mM intraliposomal K-gluconate. At higher K-gluconate concentrations the transport rate decreased. The activity of the reconstituted transporter was pH dependent with optimal function in the range pH 6.5-7.0. [3H]glutamine (and [3H]leucine) uptake was inhibited by all the neutral but not by the positively or negatively charged amino acids. The sulfhydryl reagents HgCl2, mersalyl, p-hydroxymercuribenzoate and the substrate analogue 2-aminobicyclo[2,2,1]heptane-2-carboxylate strongly inhibited the transporter, whereas the amino acid analogue alpha-(methylamino)isobutyrate had no effect. The inhibition by mersalyl was protected by the presence of the substrate. On the basis of the Na+ dependence, the electrogenic transport mode and the specificity towards the amino acids, the reconstituted transporter was classified as B degrees-like.
Department of Cell Biology, University of Calabria, Via P. Bucci 4c, 87036 Arcavacata di Rende, Italy.
Francesca
F
Oppedisano
Cesare
C
Indiveri
D026905:Amino Acid Transport Systems / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism*; D007668:Kidney / Q000166:cytology* / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry*; D026901:Membrane Transport Proteins / Q000378:metabolism*; D008871:Microvilli / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D012964:Sodium / Q000378:metabolism; D013379:Substrate Specificity
D026905:Amino Acid Transport Systems; D008081:Liposomes; D026901:Membrane Transport Proteins; D005973:Glutamine; D012964:Sodium