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-(3)H] 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-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).
Mandal
Anita
A
Department of Biology, University of North Florida, 4567 St. John's Bluff Road (S), Jacksonville, FL 32224, USA. amandal@unf.edu
Verri
Tiziano
T
Mandal
Prabir K
PK
Storelli
Carlo
C
Ahearn
Gregory A
GA
D000818:Animals; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D005260:Female; D005947:Glucose / Q000493:pharmacokinetics*; D008099:Liver; D008757:Methylglucosides / Q000494:pharmacology; D008845:Microinjections; D009004:Monosaccharide Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D008121:Nephropidae; D009865:Oocytes / Q000502:physiology; D010179:Pancreas; D010695:Phlorhizin / Q000494:pharmacology; D012333:RNA, Messenger / Q000494:pharmacology; D012964:Sodium / Q000493:pharmacokinetics*; D014982:Xenopus laevis
D008757:Methylglucosides; D009004:Monosaccharide Transport Proteins; D012333:RNA, Messenger; C027020:methylglucoside; D012964:Sodium; D010695:Phlorhizin; D005947:Glucose