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

Rifat Pamukcu, Allen M. Spiegel, Eugene B. Chang

Research output: Contribution to journalArticle

1 Scopus citations

Abstract

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: αGs, αG1(1 or 2), αG1(3), αGo, αGz 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 αGs, αG1(1 or 2), and αG1(3), but failed to identify αGo or αGz 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.

Original languageEnglish (US)
Pages (from-to)689-696
Number of pages8
JournalThe Journal of Laboratory and Clinical Medicine
Volume121
Issue number5
StatePublished - May 1993
Externally publishedYes

ASJC Scopus subject areas

  • Pathology and Forensic Medicine

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