CHARACTERIZATION OF THE RABBIT INTESTINAL FRUCTOSE TRANSPORTER (GLUT5)

Recent studies suggest that the jejunal/kidney-type facilitative gluco se transporter (GLUT5) functions as a high-affinity D-fructose transpo rter. However, its precise role in the small intestine is not clear. I n an attempt to identify the fructose transporter in the small intesti ne, we measured fructose uptake in Xenopus oocytes expressing jejunal mRNA from five species (rat, mouse, rabbit, hamster and guinea-pig). O nly jejunal mRNA from the rabbit significantly increased fructose upta ke. We also cloned a rabbit GLUT5 cDNA from a jejunal library. The pre dicted amino acid sequence of the 487-residue rabbit GLUT5 showed 72.3 and 67.1% identity with human and rat GLUT5 respectively. Northern-bl ot analysis revealed GLUT5 transcripts in rabbit duodenum, jejunum and , to a lesser extent, kidney. After separation of rabbit jejunal mRNA on a sucrose density gradient, the fractions that conferred D-fructose transport activity in oocytes also hybridized with rabbit GLUT5 cDNA. Hybrid depletion of jejunal mRNA with a GLUT5 antisense oligonucleoti de markedly inhibited the mRNA-induced fructose uptake in oocytes. Imm unoblot analysis indicated that GLUT5 (49 kDa) is located in the brush -border membrane of rabbit intestinal epithelial cells. Xenopus oocyte s injected with rabbit GLUT5 cRNA exhibited fructose uptake activity w ith a K-m of 11 mM for D-fructose. D-Fructose transport by GLUT5 was s ignificantly inhibited by D-glucose and D-galactose. D-Fructose uptake in brush-border membrane vesicles shows a K-m similar to that of GLUT 5, but was not inhibited by D-glucose or D-galactose. Finally, cytocha lasin B photolabelled a 49 kDa protein in rabbit brush-border-membrane preparations that was immuno-precipitated by antibodies to GLUT5. Our results suggest that GLUT5 functions as a fructose transporter in rab bit small intestine. However, biochemical properties of fructose trans port in Xenopus oocytes injected with GLUT5 cRNA differed from those i n rabbit jejunal vesicles.