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An immunological investigation of F(1)-ATPase
Abstract
Stable hybridoma cell lines producing monoclonal antibodies (Mabs) against beef heart mitochondrial F$\sb1$-ATPase that had been modified with BzATP (benzophenone ATP) were established. Four of these Mabs (A11, C1, F6, and G5) were shown to bind to BzATP modified F$\sb1$-ATPase, unmodified F$\sb1$-ATPase, submitochondrial particle-bound F$\sb1$-ATPase, and F$\sb1$ from nine other sources. The antibodies were shown to have no effect on the hydrolytic activity of soluble bovine F$\sb1$-ATPase. They were shown only to bind the intact enzyme. Competitive binding studies were performed with the four Mabs and using unmodified F$\sb1$, BzATP modified F$\sb1$, submitochondrial particles, rat liver F$\sb1$ and spinach chloroplast F$\sb1$ as antigens. It was shown that binding of Mab A11 to unmodified F$\sb1$ induced conformational changes to occur in subunits other than the one to which the Mab bound, allowing more of the competing antibody to bind. Depending on the antigen employed, and other experimental conditions, the other Mabs (C1, F6, and G5) were shown to exhibit this same enhanced binding effect. These results show effectors other than small molecular weight molecules effect specific subunit-subunit interactions in F$\sb1$. Circular dichroism (CD) studies of F$\sb1$ and F$\sb1$ to which effectors were bound, demonstrates that the binding of ADP and MgCl$\sb2$ to F$\sb1$ induced changes in the secondary structure of F$\sb1$. The covalent binding of BzATP to F$\sb1$ did not produce significant detectable changes in the secondary structure of the enzyme. All four Mabs induced changes in secondary structure of the enzyme upon binding. Each Mab induced different changes in the secondary structure and the magnitude of the changes were dependent upon the nature of the enzyme before Mab binding (i.e. if it had been modified with ADP/Mg or BzATP). It has therefore been concluded that the Mabs produced are not only specific for a particular epitope but also sensitive to the conformation of F$\sb1$. These antibodies produce conformational changes to occur in F$\sb1$ upon binding. These changes and magnitude thereof have been shown to be dependent upon the Mab chosen, the environment of the enzyme, the presence or absence of effectors, and the origin of the enzyme.
Subject Area
Biochemistry|Immunology
Recommended Citation
Krakowski, Letitia Ann, "An immunological investigation of F(1)-ATPase" (1988). ETD collection for University of Nebraska-Lincoln. AAI8904494.
https://digitalcommons.unl.edu/dissertations/AAI8904494