Methodological and biological studies of the amino acid oxidases.

HOLME, David J. (1978). Methodological and biological studies of the amino acid oxidases. Masters, Sheffield Hallam University (United Kingdom)..

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Abstract

Methods are described in which the liberation of ammonia from amino acid substrates by the D- and L-amino acid oxidases is coupled to the NALH dependent reductive amination of 2-oxoglutarate, the reaction being catalysed by exogenous glutamate dehydrogenase L-glutamate: NAL(P) oxidoreductase (deaminating), EC 1.4.1.3.). The inhibition of L-amino acid oxidase (L-amino acid: oxygen oxidoreductase (deaminating), EC 1.4.3.3.) by the ALP needed to activate and stabilise glutamate dehydrogenase was relieved by FAL. The Michaelis constant (Km) for the enzyme was 3.3 mmol 1[-1] with D-alanine as the substrate which, when used in the assay at a concentration of. 17 mmol 1[-1], permitted 84% of the maximum velocity. Neither FAD nor FMN were required in the L-amino acid oxidase (L-amino acid: oxygen oxidoreductase (deaminating) EC 1 .4.3.2.) assay which utilized L-leucine as substrate (Km 0.6 mmol 1 ) at a concentration of 3.3 mmol 1[-1]. This concentration of substrate was low enough to avoid any significant effect of substrate inhibition and yet permitted 85% of the maximum velocity. The oxidation of NALH was monitored both as a fall in absorbance at 340 nm and by the increase in fluorescence due to NAD[+] in alkaline solution (excitation maximum 365 nm, emission maximum 455 nm). The former provided the basis for a kinetic spectrophotometric assay which was sensitive and precise and lent itself to valid kinetic studies of the enzymes. The fluorescence due to NAL+ in a solution of 6 mol 1[-1] NaOH was stabilised by the presence of 10 mmol 1[-1] imidazole and formed the basis of a sensitive, fixed time assay. A study of human tissues demonstrated the presence of significant concentrations of L-amino acid oxidase in kidney and liver with lower concentrations in samples of brain tissue. L-amino acid oxidase could only be detected in kidney and liver and no other tissues investigated showed any amino acid oxidase activity. The specificities of human, hog and snake venom amino acid oxidases were shown to be significantly different.

Item Type: Thesis (Masters)
Additional Information: Thesis (M.Phil.)--Sheffield Hallam University (United Kingdom), 1978.
Research Institute, Centre or Group: Sheffield Hallam Doctoral Theses
Depositing User: EPrints Services
Date Deposited: 10 Apr 2018 17:20
Last Modified: 15 May 2018 07:16
URI: http://shura.shu.ac.uk/id/eprint/19818

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