AJP - Gastrointestinal and Liver Physiology, Vol 265, Issue 1 56-G62, Copyright © 1993 by American Physiological Society
Timed photoaffinity labeling and characterization of bile acid binding and transport proteins in rat ileum
M. C. Lin, E. Mullady and F. A. Wilson
Department of Metabolic Diseases, Bristol-Myers Squibb Research Institute, Princeton, New Jersey 08543.
Rat ileal enterocytes were radiolabeled by flash photolysis with a
photolabile derivative of taurocholate (7,7-azo-[3H]TC) and subjected to
sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Maximal labeling
of the bile acid binding proteins (BABPs) was achieved between 15 and 90 s.
When enterocytes were pulsed with 7,7-azo-[3H]TC for 2 min, and then 0.5 mM
TC was added to chase the radiolabel, the radioactivity in the BABPs was
displaced by 50% after 2 min. The 99-kDa brush-border membrane (BBM)
protein had the highest initial labeling rate, followed by 43-kDa actin,
35- and 14-kDa cytosolic proteins, 54-kDa basolateral membrane (BLM)
protein, 59-kDa BLM-associated protein, and 20-kDa microsomal protein. When
a mixed microsomal and cytosolic fraction was photolabeled with
7,7-azo-[3H]TC and then separated, the 20-kDa microsomal protein was
labeled. However, if the microsomal fraction alone was photolabeled, the
20-kDa protein was not labeled, suggesting this protein required a
cytosolic cofactor for labeling. Using Triton X-114 phase separation and
EDTA extraction, the BABPs were separated into amphiphilic integral
membrane proteins (99- and 54-kDa proteins) and hydrophilic proteins (14-,
35-, 43-, and 59-kDa proteins). From these data, a model is proposed for
transcellular bile acid transport in rat ileal enterocytes.
