Soluble γ-Aminobutyric-Glutamic Transaminase from Pseudomonas fluorescens

Abstract

The presence of relatively high concentrations of y-aminobutyric acid in mammalian brain (1, 2) led to the discovery of a reaction utilizing this amino acid by what appeared to be transamination with a-ketoglutaric acid (3, 4). Studies with brain preparations (3, 4) were, however, handicapped by the particulate nature of the system catalyzing the reaction. Although active, soluble extracts were obtained from microorganisms (5-7), these were not further purified. Recent studies have implicated y-aminobutyric-glutamic transaminase (8) and succinic semialdehyde dehydrogenase (8, 9) as the successive, terminal steps in the conversion of pyrrolidine to succinic acid by a strain of Pseudomonas JIuorescens. Preliminary evidence suggested (8) that pyrrolidine was metabolized by way of the following successive intermediates: Al-pyrroline,l y-aminobutyric acid, succinic semialdehyde, and succinic acid. The present report summarizes the data on the purification and properties of y-aminobutyric-glutamic transaminase (Reaction 1) and presents a kinetic analysis of the reaction which it catalyzes. dehydrogenase described by Black (10) .2 Alternatively, the substrate specific, succinic semialdehyde dehydrogenase from P. Jluorescens (9), utilizing either TPN or DPN, was employed. Although the pH of the standard incubation system was not optimal, considerable endogenous activity, i.e. reduction of pyridine nucleotide, found with the cruder fractions at higher pH values was minimized. Activity was followed at l-minute intervals for 5 minutes. Transaminase activity was a linear function of both time and protein concentration at rates of less than 0.05 pmole of DPNH (or TPNH) formed per minute. A unit of activity was defined as that amount of transaminase allowing the formation of 1 pmole of reduced pyridine nucleotide per minute in the standard assay system. Specific activity was expressed in terms of units ner mg. of protein.