The membrane bound enzyme Na+,K+ ATPase is responsible for the active transport of Na+ and K+ across cell membranes and is, therefore, an essential component of all higher animal cells. It is also the putative receptor site for the cardiac glycosides which are specific inhibitors of its ATPase activity. This enzyme is of special interest because of its role in the maintenance of the electrophysiological properties of heart muscle cells. The goal of this project is to determine the membrane organization, structure, and function of the Na+, K+ ATPase and, thereby, aid in the elucidation of the mechanism of the inotropic effect of the cardiac glycosides. I plan to use antibodies directed against the """"""""native"""""""" enzyme and its components in order to determine structure-function relationships. Studies using mouse monoclonal antibodies will investigate specific antibody-enzyme interactions in order to understand the mechanism(s) of antibody effects on enzyme function and the interactions between the different ligand binding sites of the enzyme. In addition, antibodies (polyclonal and monoclonal) with selective specificities towards the holoenzyme, the catalytic, glycoprotein subunit, and specific haptens will be used to determine the influence of the holoenzyme conformation on its antigenic properties and to identify, localize, and isolate functional regions of the enzyme containing the cardiac glycoside receptor, the phosphorylation site, and cation binding sites. This project will also provide the tools and develop the methodologies needed to study the biosynthesis and processing of the catalytic and glycoprotein subunits and to identify the cDNA or chromosomal clones of the catalytic subunit of the Na+, K+ ATPase.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL032214-02
Application #
3343547
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1984-09-01
Project End
1987-08-31
Budget Start
1985-09-01
Budget End
1986-08-31
Support Year
2
Fiscal Year
1985
Total Cost
Indirect Cost
Name
University of Cincinnati
Department
Type
Schools of Medicine
DUNS #
City
Cincinnati
State
OH
Country
United States
Zip Code
45221
Amler, E; Abbott, A; Malak, H et al. (1996) The carbohydrate moieties of the beta-subunit of Na+, K(+)-ATPase: their lateral motions and proximity to the cardiac glycoside site. Biophys J 70:182-93
Abbott, A; Ball Jr, W J (1993) The epitope for the inhibitory antibody M7-PB-E9 contains Ser-646 and Asp-652 of the sheep Na+,K(+)-ATPase alpha-subunit. Biochemistry 32:3511-8
Abbott, A; Ball Jr, W J (1992) The inhibitory monoclonal antibody M7-PB-E9 stabilizes E2 conformational states of Na+,K(+)-ATPase. Biochemistry 31:11236-43
Sun, Y; Ball Jr, W J (1992) Determination of Na(+)-K(+)-ATPase alpha- and beta-isoforms and kinetic properties in mammalian liver. Am J Physiol 262:C1491-9
Amler, E; Abbott, A; Ball Jr, W J (1992) Structural dynamics and oligomeric interactions of Na+,K(+)-ATPase as monitored using fluorescence energy transfer. Biophys J 61:553-68
Ball Jr, W J; Abbott, A; Sun, Y et al. (1992) Monoclonal antibodies and the identification of functional regions of the Na+,K(+)-ATPase. Ann N Y Acad Sci 671:436-9
Incerpi, S; Jefferson, J R; Wood, W G et al. (1992) Na pump and plasma membrane structure in L-cell fibroblasts expressing rat liver fatty acid binding protein. Arch Biochem Biophys 298:35-42
Abbott, A J; Amler, E; Ball Jr, W J (1991) Immunochemical and spectroscopic characterization of two fluorescein 5'-isothiocyanate labeling sites on Na+,K(+)-ATPase. Biochemistry 30:1692-701
Cuppoletti, J; Abbott, A J (1990) Interaction of melittin with the (Na+ + K+)ATPase: evidence for a melittin-induced conformational change. Arch Biochem Biophys 283:249-57
Ting-Beall, H P; Beall, H C; Hastings, D F et al. (1990) Identification of monoclonal antibody binding domains of Na+,K(+)-ATPase by immunoelectron microscopy. FEBS Lett 265:121-5

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