Partial Purification and Characterization of Muscle L-Glutamate Dehydrogenase

Relicardo  M. Coloso,; Lourdes  Cruz,

Partial Purification and Characterization of Muscle L-Glutamate Dehydrogenase

Abstract:

A method of partially purifying L-glutamate dehydrogenase, a key enzyme in the biosynthesis of non-essential amino acids, from the muscle of P. monodon juveniles is presented. Enzyme extraction with Tris-acetate buffer, ammonium sulfate fractionation, DEAE-cellulose chromatography, and Sepharose 48 chromatography were used. A 260-fold purification was obtained, but the recovery of enzyme activity was low. Possible modification in the method and the need for further purification are discussed. P. monodon possesses an enzyme which catalyzes the de novo synthesis of glutamate from alpha-ketoglutarate and ammonia. The enzyme has a pH optimum for the reductive amination of alpha-ketoglutarate between pH 8.0 and 8.2. The low Michaelis-Menten constant ( 1.03 x 104M) of the enzyme for alpha-ketoglutarate strongly suggests the physiological importance of this pathway in P. monodon. Substrate inhibition by alphaketoglutarate at high non-physiological levels was observed and discussed .. The molecular weight of the native enzyme estimated by molecular sieve chromatography is 320,000 daltons. Subunit studies using SOS polyacrylamide gel electrophoresis suggest that the enzyme possesses a unique molecular organization compared to those derived from other sources.

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