Maître M, Ciesielski L, Cash C, Mandel P
Eur J Biochem. 1975 Mar 3;52(1):157-69. doi: 10.1111/j.1432-1033.1975.tb03983.x.
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 mumol times min -1, x 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 Km for 2-oxoglutarate at saturating 4-aminobutyrate extrapolated to saturating 2-oxoglutarate concentration is 4 mM. 2-Oxoglutarate competitively inhibits the enzyme with respect to 4-aminobutyrate, with a Ki of 1.8 times 10(-4) M. The same phenomenon is seen for the reverse reaction where the Ki is 6.6 times 10(-4) M for succinic semi-aldehyde.
采用多种色谱方法,相对于最初的脑匀浆,大鼠脑内的4-氨基丁酸:2-氧代戊二酸转氨酶已纯化了2400倍。纯化后的蛋白质比活性为10 μmol·min⁻¹·mg⁻¹,经丙烯酰胺凝胶电泳和等电聚焦显示为单一谱带。其分子量为105000±5000,等电点为6.8。在0.1%十二烷基硫酸钠存在下,聚丙烯酰胺凝胶上可见单一蛋白质条带,对应分子量为57000±5000。N端分析显示两条链具有相同的N端氨基酸,因此该酶可被视为由两个相同亚基组成的二聚体。酶活性的最适pH为8.5。酶促反应研究表明,其一般机制为乒乓双底物模型。在饱和4-氨基丁酸条件下,将2-氧代戊二酸浓度外推至饱和时,其Km值为4 mM。2-氧代戊二酸对4-氨基丁酸而言对该酶有竞争性抑制作用,Ki为1.8×10⁻⁴ M。在逆反应中也观察到相同现象,琥珀酸半醛的Ki为6.6×10⁻⁴ M。