John R A, Fowler L J
Biochem J. 1976 Jun 1;155(3):645-51. doi: 10.1042/bj1550645.
Purification and 4-aminobutyrate-2-oxoglutarate aminotransferase (EC 2.6.1.19) from rabbit brain is described. The method was used as a routine to give between 5 and 10mg of pure enzyme from 750 g of rabbit brain. The enzyme is a dimer made up of subunits each with a mol. wt. of 58000. An absorption spectrum of the freshly prepared enzyme shows peaks at 415 and 330 nm. Treatment of the enzyme with the substrate 4-amino-butyrate or glutamate produces a decrease in the 415 nm and an increase in the 330 nm peak. This conversion, which is attributed to an aldimine into ketimine step in the reaction, is sufficiently slow when 4-aminobutyrate is the substrate to allow it to be followed by stopped-flow spectrophotometry. A first-order rate constant was determined for this step (12s-1) and compared with the turnover number for the enzyme derived by steady-state methods (9.5S-1). The first-order rate constant when glutamate was used as substrate was estimated to be approx. 30s-1.
本文描述了从兔脑中纯化4-氨基丁酸-2-氧代戊二酸氨基转移酶（EC 2.6.1.19）的方法。该方法作为常规方法，可从750克兔脑中获得5至10毫克纯酶。该酶是一种二聚体，由分子量为58000的亚基组成。新制备的酶的吸收光谱在415和330纳米处有峰值。用底物4-氨基丁酸或谷氨酸处理该酶会导致415纳米处的峰值降低，330纳米处的峰值升高。这种转变归因于反应中醛亚胺到酮亚胺的步骤，当4-氨基丁酸作为底物时，该转变足够缓慢，可用停流分光光度法跟踪。测定了该步骤的一级速率常数（12秒⁻¹），并与通过稳态方法得出的酶的周转数（9.5秒⁻¹）进行了比较。当谷氨酸用作底物时，一级速率常数估计约为30秒⁻¹。
