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Beef liver mitochondrial amine oxidase: purification and studies on some physical and chemical properties
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|Title:||Beef liver mitochondrial amine oxidase: purification and studies on some physical and chemical properties|
|Abstract:||Beef (steer) liver mitochondrial amine oxidase was prepared according to the method reported earlier (Adv. Pharmacol., 6, Part A, 43, 1968). In addition to the usual preparation with high activity, (component 2, specific activity of 8,000) another component of the enzyme (component 1) with lower activity (specific activity 3,000) was isolated (Biochem. Biophys. Res. Commun., submitted). Studies were made on some physical and chemical properties of these two components. The amine oxidase components were bright yellow in color; they were thermolabile, and unstable at room temperature. The rate of inactivation of component 2 was faster than that of component 1. The optimum pH for activity was found to be 9.2. Both the components were non-competitively inhibited by p-chloromercuribenzoate. Metal chelators like cuprizone, 8-hydroxyquinoloine, o-phenanthroline inhibited the enzyme components. Ammonia or aldehyde reagents did not have significant effects on the activity. Both the components had almost the same substrate specificity. The molecular weights of the enzyme component 1 was found to be 400,000 by the gel filtration technique, 396,000 ± 10,000 on the basis of Stoke's radius, sedimentation coefficient, and partial specific volume, and 425,000 ±10,000 on the basis of sedimentation diffusion method. These values for component 2 were 1,300,000, 1,195,000, and 1,355,000, respectively. The sedimentation coefficients of component 1 and component 2 were 14.4 ± 0.3 and 20.6, respectively. Metal analyses of the enzyme yielded 1 gram atom of copper per 400,000 grams or 3 gram atoms of the metal per mole of component 2. Other metals, such as cobalt, iron, manganese, and molybdenum were examined and found to be either absent or insignificant (J. Biol. Chem., 241, 2774, 1966). Both the components of the mitochondrial enzyme were found to be flavoproteins. This was amply proved (1) from their riboflavin content as determined microbiologically, and spectrophotometrically (Biochem. Biophys. Res. Commun., 23, 324, 1966), (2) from a steady increase of riboflavin during purification processes, and (3) from the spectrum of flavo-peptide obtained from pronase digest of the enzyme. Besides, the prosthetic group was found to contain ribose (Biochem. Biophys. Res. Commun., 29, 562, 1967), adenine and phosphorus in integral values suggesting that the "flavin prosthetic" group was a flavin adenine dinucleotide of unknown structure. Accordingly, component 1 contained 4 and component 2 contained 12 FAD or FAD-like substance per mole, respectively. Examination of the sulfhydryl groups revealed that components 1 and 2 of the enzyme contained 28 and 86 titratable sulfhydryl residues, respectively in their molecules, and that they were not directly involved in enzyme catalysis. In addition, the enzyme was found to contain 24 and 106 moles of phospholipid in components 1 and 2, respectively. Finally, it appeared that the high molecular weight component was the native form from which the small molecular component arose during the purification of the enzyme, although no interconversions were observed with the purified enzyme preparations.|
Thesis (Ph. D.)--University of Hawaii, 1968.
Bibliography: leaves -162.
xiii, 162 l illus., tables
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|Appears in Collections:||Ph.D. - Biomedical Sciences (Biochemistry)|
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