Purification of rat-liver [gamma]-hydroxyglutamate transminase and its probable identity with glutamate-aspartate transminase

Abstract

1. 1. An enzyme which catalyzes the transamination of [gamma]-hydroxyglutamate has been purified over 300-fold from rat-liver homogenates.2. 2. The following observations strongly suggest that [gamma]-hydroxyglutamate transminase is identical with glutamate-aspartate transaminase (-aspartate: 2-oxo-glutarate aminotransferase, EC 2.6.1.1): a, at all stages of purification from either rat-liver or pig-heart extracts, the glutamate to [gamma]-hydroxyglutamate transminase ratios are not significantly different; b, transaminase activity for both substrates declines at the same rate during controlled heat denaturation of the purified rat-liver enzyme; c, transamination of [gamma]-hydroxyglutamate is strongly inhibited by either -glutamate or -aspartate; d, glutarate and maleate function as competitive inhibitors for either glutamate or [gamma]-hydroxyglutamate and determined K1 values for a given inhibitor are the same for either amino acid; and e, the purified rat-liver enzyme has the same pH-activity curve for both substrates.3. 3. The erythro- and threo-isomers of [gamma]-hydroxy--glutamate serve as substrates for both isozymes of the rat-liver enzyme as well as for the pig-heart enzyme, although the former isomer is a somewhat better substrate. The corresponding -diastereo-isomers are enzymically inactive.4. 4. With [gamma]-hydroxyglutamate, only [alpha]-ketoglutarate and oxaloacetate serve as amino group acceptors; pyruvate, [alpha]-ketobutyrate, and [beta]-phenylpyruvate are inactive.5. 5. Other [gamma]-substituted forms of glutamic acid, including [gamma]-methyleneglutamic acid and [gamma]-hydroxy-[gamma]-methylglutamic acid, are also active with the purified enzymes.