AJP - Gastrointestinal and Liver Physiology, Vol 240, Issue 4 317-G323, Copyright © 1981 by American Physiological Society
Gastric oxyntic cell tubulin: characterization and possible significance
H. E. Stewart and D. K. Kasbekar
Colchicine-binding activity in the homogenate fractions of the whole
gastric epithelium and oxynein tissue has been studied to determine if
tubulin is present in the bullfrog stomach. Most of the specific binding
activity is found in the 100,000-g soluble fraction and is enriched in the
oxynein tissue. Binding is proportional to protein concentration, linear up
to 30 min, and half saturable at 16 microM colchicine concentration.
Scatchard and direct linear plot analysis yields the following values for
binding constants: Kd = 12 microM, Ka = 0.08 X 10(6) M-1, maximal binding
capacity (Bmax) = 0.19 X 10(-6) M, and n = 84 pmol/mg protein. Fiftyfold
excess lumicolchicine does not affect colchicine binding. The binding
activity has been partially purified by the temperature-sensitive cycling
technique. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic
analysis of the product shows a major band corresponding to tubulin in the
55,000-dalton region. Tubulin in isolated oxyntic cells can be visualized
by indirect immunofluorescence. These data and the observed increase in
colchicine-binding activity in the burimamide- over the histamine-treated
tissue, possibly due to changes in the levels of tubulin polymerization,
suggest a likely involvement of the cytoskeleton in gastric secretion.
