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 an d normal human colonocytes. The activity of the ouabain-sensitive K+-a ctivated phosphatase, the basolateral membrane marker, was enriched 13 -fold in these membrane vesicles over the original homogenate. The mem branes displayed cross-reactions with antibodies to the (Na+/K+)ATPase and the RLA class I major histocompatability antigen, both known indi cators of the basolateral membrane. There was negligible contamination by other organelles and the luminal membrane, as revealed by marker-e nzyme analysis and Western blotting, using an antibody to villin. The vesicles transported D-glucose in a cytochalasin B-inhibitable Na+-ind ependent manner, with a K(m) of 28.1 +/- 0.8 mM and V(max.) 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-alp ha-D-glucose. Probing the colonocyte basolateral membranes with an ant ibody against the C-terminus of the human liver GLUT 2 produced a cros s-reaction at 52 kDa. These properties indicate the presence of a GLUT 2 isoform on the basolateral membranes of human and pig colonocytes.