The activation of phosphatase activity of the Ca2+-ATPase from human red cell membranes by calmodulin,ATP and partial proteolysis |
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| Institution: | 1. College of Food Science and Nutritional Engineering, China Agricultural University, Beijing Advanced Innovation Center for Food Nutrition and Human Health, National Engineering Research Centre for Fruit and Vegetable Processing, Key Lab of Fruit and Vegetable Processing, Ministry of Agriculture, Beijing Key Laboratory for Food Nonthermal Processing, Beijing 100083, China;2. Institute of Quality Standard & Testing Technology for Agro-Products, Chinese Academy of Agricultural Sciences, Key Laboratory of Agro-food Safety and Quality, Ministry of Agriculture, Beijing 100081, China;3. Institute of Agro-products Storage and Processing, Xinjiang Academy of Agricultural Sciences, Urumqi 830091, China;1. Department of Food Science and Engineering, Institute of Food Safety and Nutrition, Guangdong Engineering Technology Center of Food Safety Molecular Rapid Detection, Jinan University, Guangdong, China;2. School of Chemical Engineering and Light Industry, Guangdong University of Technology, Guangzhou 510006, PR China;1. Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, China;2. Fish Nutrition and Safety Production University Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, China;3. Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Chengdu 611130, China;4. Animal Nutrition Institute, Sichuan Academy of Animal Science, Chengdu 610066, China;5. Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China |
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| Abstract: | (1) Depending on the assay conditions, the ability of the Ca2+-ATPase from intact human red cell membranes to catalyze the hydrolysis of p-nitrophenylphosphate is elicited by either calmodulin or ATP. The response of the phosphatase activity to p-nitrophenylphosphate, ATP, Mg2+ and K+ is the same for the activities elicited by ATP or by calmodulin, suggesting that a single process is responsible for both activities. (2) In media with calmodulin, high-affinity activation is followed by high-affinity inhibition of the phosphatase by Ca2+ so that the activity becomes negligible above 30 μM Ca2+. Under these conditions, addition of ATP leads to a large decrease in the apparent affinity for inhibition by Ca2+. (3) In membranes submitted to partial proteolysis with trypsin, neither calmodulin nor Ca2+ are needed and phosphatase activity is maximal in media without Ca2+. This is the first report of an activity sustained by the Ca2+-ATPase of red cell membranes in the absence of Ca2+. Under these conditions, however, ATP still protects against high-affinity inhibition by Ca2+. These results strongly suggest that during activation by calmodulin, Ca2+ is needed only to form the calmodulin-Ca2+ complex which is the effective cofactor. (4) Protection by ATP of the inhibitory effects of Ca2+ and the induction of phosphatase activity by ATP + Ca2+ suggests that activation of the phosphatase by Ca2+ in media with ATP requires the combination of the cation at sites in the ATPase. (5) Results can be rationalized assuming that E2, the conformer of the Ca2+-ATPase, is endowed with phosphatase activity. Under this assumption, either the calmodulin-Ca2+ complex or partial proteolysis would elicit phosphatase activity by displacing the equilibrium between E1 and E2 towards E2. On the other hand, ATP + Ca2+ would elicit the activity by establishing through a phosphorylation-dephosphorylation cycle a steady-state in which E2 predominates over other conformers of the ATPase. |
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