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EXAMINATION OF THE PHYSICAL PROPERTIES OF F(1)-ATPASES (PROTEASE, ADENYLATE KINASE, NEUTRAL SALT)
Abstract
The kinetic and immunochemical properties of corn mitochondrial ATPase (F(,1)) were examined. The enzyme was found to exhibit extremely nonlinear kinetics in which the rate of hydrolysis of a particular nucleoside triphosphate (adenosine 5'-triphospate, inosine 5'-triphosphate, or guanosine 5'-triphosphate) was maximized by incubation of the enzyme with that compound prior to the initiation of hydrolysis by addition of divalent metal ions. A plausible mechanism for such effects was suggested, based on the differential binding of nucleotides to noncatalytic, regulatory sites rapidly prior to and slowly during catalysis, with the enzyme being most active when the protein, and consequently the subunit interactions, were maximally symmetric. The instability and low ATPase activity of the corn mitochondrial preparations, as well as other factors, led to the study of the better characterized beef heart enzyme. A characterization of purity and physical properties of beef heart F(,1) preparations revealed a number of problems. As commonly prepared the enzyme was found to be highly pure by denaturing gel electrophoresis. However, size exclusion chromatographic analysis showed the preparations to contain anywhere from 2 to greater than 50% dissociated enzyme. The preparations were also found to be contaminated to varying degrees by the F(,1) inhibitor protein, and an adenylate kinase-like activity. In addition, a protease capable of cleaving the alpha and delta subunits of F(,1), as well as the inhibitor protein were found to co-purify with the enzyme. The proteolytic activity was examined and means were found to diminish its effects. The process of dissociation was examined both in order to characterize under what conditions the enzyme was unstable and in order to understand what types of forces hold the subunits together.
Subject Area
Biochemistry
Recommended Citation
PARTRIDGE, BRUCE EDWARD, "EXAMINATION OF THE PHYSICAL PROPERTIES OF F(1)-ATPASES (PROTEASE, ADENYLATE KINASE, NEUTRAL SALT)" (1986). ETD collection for University of Nebraska-Lincoln. AAI8706241.
https://digitalcommons.unl.edu/dissertations/AAI8706241