The goal of this project is to determine the structure, organization and function of the membrane bound Na+, K+- ATPase. This enzyme is essential for the active regulation of and maintenance of Na+ and K+ levels within the cell. It is also the pharmacological receptor for cardiac glycosides which are used to treat some heart diseases. These studies are designed to help elucidate the molecular mechanisms of cardiac glycoside inhibition of enzyme activity and how this inhibition is linked to the drug's inotropic effects on heart muscle contraction. In this project we will continue with studies of the mechanisms of monoclonal antibody effects on enzyme function in order to determine how they inhibit enzyme activity and alter the normal interactions between the regulatory ligands which bind to the catalytic, or alpha subunit of Na+, K+-ATPase. This is accomplished by studying the Na+,K+-ATPase, p- nitrophenylphosphatase, and acetylphosphatase activities, and the partial reactions of phosphoenzyme intermediate formation and dephosphorylation. Ligand-induced conformational changes in the enzyme are monitored using fluorescent probe-labeled enzyme. These studies will provide new information about cardiac glycoside action. In addition we will use synthetic peptides which have the amino acid sequences of various regions of the enzyme to identify antigenic sites of the enzyme. Polyclonal antibodies raised to these peptides and holoenzyme-directed antibodies which bind to these peptides will be used to locate functional regions such as the cardiac glycoside, the ATP and cation binding sites of the enzyme. Our collection of holoenzyme- and synthetic peptide-directed antibodies will also be used to probe the tertiary structure of the enzyme. Finally, we will use various immunochemical techniques to determine both the organization and possible functional role of beta, the glycoprotein subunit of Na+, K+ -ATPase. We will then determine the mechanism(s) of its effects on enzyme catalytic activity.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL032214-07
Application #
3343551
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1984-09-01
Project End
1992-08-31
Budget Start
1990-09-01
Budget End
1991-08-31
Support Year
7
Fiscal Year
1990
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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