Preparation and characterization of basolateral membrane vesicles from pig and human colonocytes: the mechanism of glucose transport.
Membrane vesicles were isolated from the basolateral domains of pig and normal human colonocytes. The activity of the ouabain-sensitive K(+)-activated phosphatase, the basolateral membrane marker, was enriched 13-fold in these membrane vesicles over the original homogenate. The membranes displayed cross-reactions with antibodies to the (Na+/K+)ATPase and the RLA class I major histocompatibility antigen, both known indicators of the basolateral membrane. There was negligible contamination by other organelles and the luminal membrane, as revealed by marker-enzyme analysis and Western blotting, using an antibody to villin. The vesicles transported D-glucose in a cytochalasin B-inhibitable Na(+)-independent manner, with a Km of 28.1 +/- 0.8 mM and Vmax. of 3.1 +/- 0.4 nmol/s per mg of protein. The transport was inhibited by 2-deoxy-D-glucose and 3-O-methyl-D-glucose, but not by L-glucose or methyl-alpha-D-glucose. Probing the colonocyte basolateral membranes with an antibody against the C-terminus of the human liver GLUT 2 produced a cross-reaction at 52 kDa. These properties indicate the presence of a GLUT 2 isoform on the basolateral membranes of human and pig colonocytes.
Department of Biochemistry, University of Wales, Aberystwyth, Dyfed, U.K.
S A
SA
Pinches
S M
SM
Gribble
R B
RB
Beechey
A
A
Ellis
J M
JM
Shaw
S P
SP
Shirazi-Beechey
D019325:3-O-Methylglucose; D000469:Alkaline Phosphatase / Q000032:analysis; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D003106:Colon / Q000648:ultrastructure*; D003571:Cytochalasin B / Q000494:pharmacology; D003847:Deoxyglucose / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism*; D051273:Glucose Transporter Type 2; D015395:Histocompatibility Antigens Class I / Q000032:analysis; D006801:Humans; D008757:Methylglucosides / Q000494:pharmacology; D009004:Monosaccharide Transport Proteins / Q000032:analysis; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D012964:Sodium / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis; D013552:Swine
D051273:Glucose Transporter Type 2; D015395:Histocompatibility Antigens Class I; D008757:Methylglucosides; D009004:Monosaccharide Transport Proteins; D019325:3-O-Methylglucose; D003571:Cytochalasin B; D010042:Ouabain; D003847:Deoxyglucose; D012964:Sodium; D000469:Alkaline Phosphatase; D000254:Sodium-Potassium-Exchanging ATPase; D005947:Glucose; D011188:Potassium