Pinches S A, Gribble S M, Beechey R B, Ellis A, Shaw J M, Shirazi-Beechey S P
Department of Biochemistry, University of Wales, Aberystwyth, Dyfed, U.K.
Biochem J. 1993 Sep 1;294 ( Pt 2)(Pt 2):529-34. doi: 10.1042/bj2940529.
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.
从猪和正常人结肠细胞的基底外侧结构域分离出膜囊泡。哇巴因敏感的钾激活磷酸酶作为基底外侧膜标志物，其活性在这些膜囊泡中比原始匀浆富集了13倍。这些膜与抗（钠/钾）ATP酶抗体和RLA I类主要组织相容性抗原发生交叉反应，这两者都是基底外侧膜的已知指标。通过标志物酶分析和蛋白质印迹法（使用抗绒毛蛋白抗体）显示，其他细胞器和腔面膜的污染可忽略不计。这些囊泡以细胞松弛素B抑制的、不依赖钠的方式转运D - 葡萄糖，Km为28.1±0.8 mM，Vmax为每毫克蛋白质3.1±0.4 nmol/s。该转运受到2 - 脱氧 - D - 葡萄糖和3 - O - 甲基 - D - 葡萄糖的抑制，但不受L - 葡萄糖或甲基 - α - D - 葡萄糖的抑制。用人肝GLUT 2 C末端抗体探测结肠细胞基底外侧膜，在52 kDa处产生交叉反应。这些特性表明人和猪结肠细胞的基底外侧膜上存在GLUT 2同工型。
