Progressive myocardial dysfunction during acute and/or chronic disease is likely to involve defects in the regulation of Ca2+ uptake and release by the cardiac sarcoplasmic reticulum. In order to understand the mechanisms underlying these processes it is critical to develop a clear idea as to how they may be regulated. To this end, the studies outlined in this proposal would use immunolocalization to examine the interactions of the sarcoplasmic reticulum Ca2+ pump protein and two putative regulatory polypeptides. The sarcoplasmic reticulum actively accumulates Ca2+ from the cytoplasm through the activity of an intrinsic Ca2+-ATPase. In skeletal sarcoplasmic reticulum there is evidence that the coupling of ATP hydrolysis to Ca2+ transport is influenced by a 53-Kda glycoprotein. In cardiac sarcoplasmic reticulum it is well documented that phosphorylation of a low molecular mass, intrinsic proteolipid, enhances the rate of Ca2+ uptake. Electron microscopic studies with monoclonal and polyclonal antibodies, as well as biotinylated oligopeptides, would be performed using thin section, fracture label and deep etch procedures to localize: 1) the Ca2+-ATPase in native, isolated skeletal and cardiac sarcoplasmic reticulum membranes and in reconstituted proteoliposomes; 2) phospholamban in native sarcoplasmic reticulum and proteopliposomes; 3) the 53-Kda glycoprotein in native and vanadate crystallized skeletal and cardiac sarcoplasmic reticulum membranes. Ultimately, since the regulatory nature of the 53-Kda glycoprotein and phospholamban may depend on a direct interaction with the Ca2+-ATPase, all of these proteins would be co-localized in native sarcoplasmic reticulum membranes. The Ca2+-ATPase and phospholamban would also be co-localized in reconstituted proteoliposomes.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL034779-05
Application #
3348108
Study Section
Cardiovascular and Pulmonary Research A Study Section (CVA)
Project Start
1987-02-05
Project End
1994-03-31
Budget Start
1992-04-01
Budget End
1993-03-31
Support Year
5
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Cincinnati
Department
Type
Schools of Medicine
DUNS #
City
Cincinnati
State
OH
Country
United States
Zip Code
45221