Biochemical and immunochemical localization of GTP-binding proteins in the rat ileal enterocyte

This study characterizes the distribution of various guanosine triphosphate-binding proteins (G proteins) in rat intestinal epithelial membranes. Enriched basolateral membranes were prepared from isolated enterocytes through differential density centrifugation; apical membranes were prepared with a chaotropic agent. Enrichment and purity of the membrane fractions were assessed by various biochemical markers. G proteins were identified by sodium dodecyl sulfate-polyacrylamlde gel electrophoresis after adenosine diphosphate ribosylation in the presence of pertussis or cholera toxins. Western blotting was performed with the use of highly specific antibodies against the following subunits: αG_s, αG_{1(1 or 2)}, αG₁₍₃₎, αG_o, αG_z and β subunits. Adenosine dlphosphate ribosylatton catalyzed by cholera toxins revealed two major substrates of molecular weights 47 and 43 kd in only the crude and basolateral fractions. The reaction catalyzed by pertussis toxin revealed a 41 kd substrate in the crude and basolateral fractions and a 40 kd substrate in the apical fraction. Immunoblotting confirmed the presence of αG_s, αG_{1(1 or 2)}, and αG₁₍₃₎, but failed to identify αG_o or αG_z subunits in the basolateral fraction; none of these subunits were identified in the apical fraction. Beta subunits were identified in both apical and basolateral fractions. These findings suggest selective sorting of the G proteins to regional domains in the plasma membrane of intestinal epithelial cells. The presence of previously unidentified G proteins is also suggested.