Purification and Studies on Some Properties of the 4‐Aminobutyrate: 2‐Oxoglutarate Transaminase from Rat Brain

Abstract

Using various chromatographic procedures, 4-aminobutyrate: 2-oxoglutarate transaminase from rat brain has been purified 2400 times with respect to the initial brain homogenate. The purified protein, which has a specific activity of 10 μmol × min⁻¹× mg⁻¹, gave a single band by acrylamide gel electrophoresis and isoelectric focusing. It has a molecular weight of 105000 ± 5000 and an isoelectric point of 6.8 In the presence of 0.1% sodium dodecylsulphate, a single protein band is seen on polyacrylamide gel, corresponding to a molecular weight of 57000 ± 5000. N-terminal analysis reveals two chains with the same N-terminal amino acid, thus the enzyme may be considered as a dimer consisting of two identical subunits. The pH optimum for enzyme activity is 8.5 Studies of the enzymic reaction show that the general mechanism is of the ping-pong bi-bi model. The K_m for 2-oxoglutarate at saturating 4-aminobutyrate concentration is 5.5 mM and V is 22 μmol × min⁻¹× mg⁻¹ whereas the k_m for 4-aminobutyrate extrapolated to saturating 2-oxoglutarate concentration is 4 mM. 2-Oxoglutarate competitively inhibits the enzyme with respect to 4-aminobutyrate, with a K_i of 1.8 × 10⁻⁴ M. The same phenomenon is seen for the reverse reaction where the K_i is 6.6 × 10⁻⁴ M for succinic semi-aldehyde.