Smith M T, Emerich D W
Department of Biochemistry, University of Missouri, Columbia 65211.
Arch Biochem Biophys. 1993 Aug 1;304(2):379-85. doi: 10.1006/abbi.1993.1365.
Alanine dehydrogenase (ALADH) from soybean nodule bacteriods was purified 184-fold with 14% yield, using ammonium sulfate precipitation, hydroxylapatite, gel filtration, ion exchange, and dye affinity chromatography. The subunit molecular weight was 43,000 and the native molecular weight was approximately 190,000, suggesting that ALADH is a tetramer. ALADH was confined to the bacteroid cytosol fraction only. ALADH is specific for NAD(H) and does not use NADP(H) as a substrate, but it does use glyoxylate and hydroxypyruvate as substrates in lieu of pyruvate. The pH optimum was 8.5 for the amination reaction and 10.0 for the deamination reaction. The apparent Michaelis constants for NADH, NH4+, pyruvate, L-alanine, and NAD were 86 microM, 8.9 mM, 0.49 mM, 1 mM and 200 microM, respectively. High concentrations of pyruvate, L-alanine, or NH4+ caused inhibition of activity with Ki's of 8.6 mM, 6.5-15 mM, and 188 mM, respectively. The amination reaction of ALADH was 95-100% of the control at levels of NADH/NAD corresponding to those measured in isolated bacteroids. The deamination reaction, on the other hand, was only 35-40% of control. Thus, an aminating role for ALADH is possible.
利用硫酸铵沉淀、羟基磷灰石、凝胶过滤、离子交换和染料亲和层析法，从大豆根瘤类菌体中纯化出丙氨酸脱氢酶（ALADH），纯化倍数为184倍，产率为14%。亚基分子量为43,000，天然分子量约为190,000，表明ALADH是一种四聚体。ALADH仅存在于类菌体胞质溶胶组分中。ALADH对NAD(H)具有特异性，不使用NADP(H)作为底物，但它确实使用乙醛酸和羟基丙酮酸作为底物来替代丙酮酸。胺化反应的最适pH为8.5，脱氨反应的最适pH为10.0。NADH、NH4+、丙酮酸、L-丙氨酸和NAD的表观米氏常数分别为86 microM、8.9 mM、0.49 mM、1 mM和200 microM。高浓度的丙酮酸、L-丙氨酸或NH4+会抑制活性，其抑制常数（Ki）分别为8.6 mM、6.5 - 15 mM和188 mM。在与分离的类菌体中测得的NADH/NAD水平相对应时，ALADH的胺化反应为对照的95 - 100%。另一方面，脱氨反应仅为对照的35 - 40%。因此，ALADH可能具有胺化作用。
