A high-affinity in plasma membranes of rat ascites hepatoma AH109A cells |
| |
Authors: | Takafumi Iwasa Yasushi Iwasa Rajabather Krishnaraj |
| |
Institution: | 1. Department of Pharmacology, Kumamoto University Medical School, Kumamoto 860 Japan;2. Department of Biochemistry, University of Health Sciences/The Chicago Medical School, 3333 Green Bay Road, North Chicago, IL 60064 U.S.A. |
| |
Abstract: | The activity of calcium-stimulated and magnesium-dependent adenosinetriphosphatase which possesses a high affinity for free calcium (high-affinity , EC 3.6.1.3) has been detected in rat ascites hepatoma AH109A cell plasma membranes. The high-affinity had an apparent half saturation constant of for free calcium, a maximum reaction velocity of ATP hydrolyzed/mg protein per min, and a Hill number of 0.8. Maximum activity was obtained at 0.2 μM free calcium. The high-affinity was absolutely dependent on 3–10 mM magnesium and the pH optimum was within physiological range (pH 7.2–7.5). Among the nucleoside trisphosphates tested, ATP was the best substrate, with an apparent of 30 μM. The distribution pattern of this enzyme in the subcellular fractions of the ascites hepatoma cell homogenate (as shown by the linear sucrose density gradient ultracentrifugation method) was similar to that of the known plasma membrane marker enzyme alkaline phosphatase (EC 3.1.3.1), indicating that the ATPase was located in the plasma membrane. Various agents, such as K+, Na+, ouabain, KCN, dicyclohexylcarbodiimide and NaN3, had no significant effect on the activity of high-affinity . Orthovanadate inhibited this enzyme activity with an apparent half-maximal inhibition constant of 40 μM. The high-affinity was neither inhibited by trifluoperazine, a calmodulin-antagonist, nor stimulated by bovine brain calmodulin, whether the plasma membranes were prepared with or without ethylene glycol . Since the kinetic properties of the high-affinity showed a close resemblance to those of erythrocyte plasma membrane , the high-affinity of rat ascites hepatoma cell plasma membrane is proposed to be a calcium-pumping ATPase of these cells. |
| |
Keywords: | Nucleoside specificity (Rat ascites hepatoma) EGTA DCCD dicyclohexylcarbodiimide Pipes To whom correspondence should be addressed at the University of Health Sciences/The Chicago Medical School |
本文献已被 ScienceDirect 等数据库收录! |
|