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Demonstration and Preliminary Characterization of an Enzyme Capable of the Further Metabolism of the Thymidine Catabolite, β‐Aminoisobutyric Acid, in Tetrahymena pyriformis Strain GL 1
Author(s) -
NIEMANW MARILYN A.,
BERECH JOHN
Publication year - 1981
Publication title -
the journal of protozoology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.067
H-Index - 77
eISSN - 1550-7408
pISSN - 0022-3921
DOI - 10.1111/j.1550-7408.1981.tb05318.x
Subject(s) - chemistry , biochemistry , tetrahymena pyriformis , enzyme , d amino acid oxidase , metabolism , aminoisobutyric acid , oxidase test , catabolism , amino acid , tetrahymena
. The enzyme that catalyzes the critical conversion of the proposed reductive thymidine catabolic end product, P‐aminoisobutyric acid, to the initial anabolic reutilization substrate, probably methylmalonic semialdehyde, was investigated to un‐ ambiguously substantiate the operation of a reductive pyrimidine catabolic and reutilization pathway in Tetrahymena pyriformis strain GL. Although most of the studies on the further metabolism of P‐aminoisobutyric acid in other organisms suggested transamination of p‐aminoisobutyric acid to methylmalonic semialdehyde followed by its further oxidation to methylmalonic acid, such a transaminating system could not be demonstrated. By means of a sensitive fluorometric assay system, however, a low, but significant amount of P‐aminoisobutyric acid oxidase activity was detected. This enzymatic activity exhibited the following characteristics in homogenates: good activity in alkaline 0.2 M Tris‐HCI buffer with a rather broad pH optimum ranging from 7.8 to 9.0; optimum activity at a temperature of 37°C; stimulation on the addition of exogenous FAD; inhibition on the addition of divalent cations, EDTA, or PCMB; little stimulation on the addition of detergents; and no increase in activity on repeated freezing and thawing. In addition, crude preparations of this oxidase were found to be relatively stable when stored up to one week either refrigerated or frozen, to have a specific activity of 2.8 nmoles/min/mg of protein, and to have a K, of 3.6 × 10 − M for d,l ‐β‐aminoisobutyric acid. Whether this high K m , is physiologically significant or not, however, will have to await further investigation. Preliminary (NH 4 ) 2 SO 4 fractionation and affinity chromatography studies also indicated that this enzyme appears to be a unique and specific oxidase whose activity is separable from other marker enzymes, including other oxidases.