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KMID : 0358619950290020269
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Korean Journal of Physiology
1995 Volume.29 No. 2 p.269 ~ p.277
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Inositol 1,4,5-Trisphosphate-induced Increase in Activity in the Microsomes of Tracheal Epithelial Cells
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Cho Hyoung-Jin
Park Sung-Shin
Kim Young-Kee
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Abstract
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Membrane vesicles were prepared by differential centrifugation from epithelial cells of porcine trachea. Total activity of microsomal ATPases was measured spectrophotometrically by a coupled enzyme assay. The steady-state activity of the enzyme was nmol/min mg protein. Thapsigargin, a specific antagonist of intracellular , inhibited about 50% of the activity, leaving protein (n=6), indicating that the is one of the major microsomal ATPases. The microsomes used in this study appeared to be tight-sealed vesicles since they showed saturation in uptake experiments. Inositol 1,4,5-trisphosphate , an agonist of -sensitive release channel (, receptor), and Ca-ionophore A23187 induced releases of 20% and 50% of stored , respectively. The addition of also increased the microsomal ATPase activity from nmol/min mg protein to nmol/min . mg protein in the intact vesicles. Similar increase in the activity was observed by making microsomes leaky (uncoupling) using the Ca-ionophore A23187. ; effects were blocked by either thapsigargin or heparin suggesting that: 1) the increase in ATPase activity is mediated by microsomal , and 2) dissipation of gradient across the microsomal membrane is responsible for the effect. In order to test the dependence of the activity on the activity of the activity of ATPases was monitored in various concentrations of free using buffers. The -dependent biphasic change is the well-known character of receptor but not of microsomal in non-excitable cells; however, the activity of microsomal ATPase appeared biphasic and a maxim진 activity of protein was obtained in the solution containing 100 nM free . Below or above this concentration, the activity of ATPases was lower. These results strongly support a positive correlation of microsomal to the receptors in epithelial microsomes.
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KEYWORD
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Porcine tracheal epithelium, $InsP_{3}\, receptor$, Coupling, uptake and release
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