Expression of Na+/D-glucose cotransport in Xenopus laevis oocytes by injection of poly(A)+ RNA isolated from lobster (Homarus americanus) hepatopancreas

Xenopus laevis oocytes were used for expression and characterization of lobster (Homarus americanus) hepatopancreas Na+-dependent D-glucose transport activity. Poly(A)(+) RNA from the whole hepatopancreatic tissue was injected and transport activity was assayed by alpha-D-[2-H-3] glucose. Injection of lobster hepatopancreatic poly(A)(+) RNA resulted in a dose (1-20 ng) and time (1-5 days) dependent increase of Na+-dependent D-glucose uptake. Kinetics of Na+-dependent glucose transport was a hyperbolic function (K-m = 0.47 +/- 0.04 mM) of external D-glucose concentration and a sigmoidal function (K-Na =68.32 +/- 1.57 mM; Hill coefficient = 2.22 +/- 0.09) of external Na+ concentration. In addition, Na+-dependent D-glucose uptake was significantly inhibited by both (0.1-0.5 mM) phloridzin and (0.1-0.5 mM) methyl-alpha-glucopyranoside. After size fractionation through a sucrose density gradient, poly(A)(+) RNA fractions with an average length of 2-4 kb induced a twofold increase in Na+-dependent phloridzin-inhibited D-glucose uptake as compared to total poly(A) RNA-induced uptake. The results of this study provide the functional basis to screen lobster hepatopancreatic cDNA libraries for clones encoding putative and still not known crustacean SGLT-type Na+/glucose co-transporter(s). (C) 2003 Elsevier Science Inc. All rights reserved.