It has been proposed that cells contain a calcium-dependent protein, "calcatin", that specifically inhibits the calcium- ATPase. Since the calcium ATPase is the enzymatic activity underlying the calcium pump, calcatin could affect the ability of the cell to control intracellular calcium. This laboratory has identified and purified a protein that may be calcatin. The proposed research is directed toward verifying that this protein is, indeed, responsible for the observed inhibition of the calcium-ATPase, characterizing the protein, and assessing its potential role in regulating the calcium-ATPase. The specific aims of the project are to: 1) complete the purificaiton of the protein identified as calcatin; 2) determine whether the purified protein is calcatin, the proposed inhibitor; 3) raise antibodies to calcatin (to inhibit its activity and to use in affinity purification for the large scale production of calcatin); 4) characterize the biochemical and physical properties of calcatin; 5) determine the mechanism by which calcatin inhibits the calcium-ATPase; 6) determine whether calcatin inhibits the calcium pump. This research may lead to the identification of a calcium- dependent protein, "calcatin", that regulates the calcium-ATPase or "calcium pump". This would be of major physiological significance, because the calcium-ATPase is one of the most important mechanisms controlling intracellular calcium. The calcium-ATPase of the human red cell is an ideal model system in which to study the effects of calcatin, because the enzyme has been purified and its interaction with other modifiers (calcium, ATP, and calmodulin) has already been extensively characterized. Ultimately it should be possible to study the interaction of the regulatory protein, calcatin, with the purified calcium-ATPase at the molecular level.
