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

Abstract

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 α- d-[2- 3H] 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-α- d-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).