Transport and membrane binding of the glutamine analogue 6-diazo-5-oxo-L-norleucine (DON) in Xenopus laevis oocytes

Abstract

We have examined transport and membrane binding of 6-diazo-5-oxo-L-norleucine (DON, a photoactive diazo-analogue of glutamine) and their relationships to glutamine transport in Xenopus laevis oocytes. DON uptake was stereospecific and saturable (Vmax of 0.44 pmol/oocyte.min and a Km of 0.065 mM). DON uptake was largely Na+ dependent (80% at 50 microM DON) and inhibited (greater than 75%) by glutamine and arginine (substrates of the System B0,+ transporter) at 1 mM. Glutamine and DON show mutual competitive inhibition of Na(+)-dependent transport. Preincubation of oocytes in medium containing 0.1 mM DON for 24 or 48 hr depressed the Vmax for System B0,+ transport (as measured by Na(+)-dependent glutamine uptake), this effect was highly specific (neither D-DON nor the System B0,+ substrates glutamine and D-alanine showed any independent effect) and required Na+ ions. Glutamine (1 mM in preincubation medium) protected transport from inhibition by DON. The possibility that specific inactivation of System B0,+ by DON reflects attachment of DON to the transporter was tested by examining the binding of [14C]DON to Xenopus oocyte membranes. Oocytes incubated in 100 mM NaCl in the presence of [14C]DON for up to 48 hr showed 2.4-fold higher 14C-binding to membranes than oocytes incubated in choline chloride. Na(+)-dependent DON binding (31 +/- 11 fmol/micrograms membrane protein) was suppressed by external glutamine, arginine or alanine and was largely confined to a membrane protein fraction of 48-65 kDa (as assessed by SDS-polyacrylamide gel electrophoresis). The present studies indicate that DON and glutamine uptake in oocytes are both mediated by System B0,+ and demonstrate the DON binding to a particular membrane protein fraction is associated with inactivation of the transporter, offering the prospect of using [14C]DON as a covalent label for the transport protein in order to facilitate its isolation and subsequent biochemical characterization.