Yang B, LéJohn H B
Manitoba Institute of Cell Biology, Winnipeg, Canada.
J Biol Chem. 1994 Feb 11;269(6):4506-12.
An NAD(+)-specific glutamate dehydrogenase (NAD-GDH) that is inducible by L-glutamine was isolated from Achlya klebsiana and purified to electrophoretic homogeneity. The enzyme is only partially active in vitro unless NADP+ (an activator) is present in both its oxidative deamination and reductive amination reactions. This type of enzyme was reported (LéJohn, H.B. (1971) Nature 231, 164-168) to be widespread among the amorphous group of algae-related fungi classified as Oomycota. The enzyme retained its dependence on NADP+ at all stages of its purification. NADP+ decreased the Km of substrates 3-fold and increased the Vmax 4-fold. M(r) of the undernatured enzyme was 480,000, and, denatured, only a single subunit of M(r) 120,000 was seen. A polyclonal antibody raised in rabbit against purified enzyme subunit excised from SDS-polyacrylamide gel electrophoresis gels immunoprecipitated the M(r) 120,000 polypeptide, the undenatured enzyme, and a physically distinct polypeptide of M(r) 74,000. The antibody, purified against the M(r) 120,000 enzyme subunit as anchored antigen on Sepharose, still immunoprecipitated the M(r) 74,000 polypeptide. The M(r) 74,000 polypeptide was found to be a subunit of a M(r) 220,000 native protein.
从克氏壶菌中分离出一种可被L-谷氨酰胺诱导的NAD(+)特异性谷氨酸脱氢酶（NAD-GDH），并将其纯化至电泳纯。该酶在体外仅部分有活性，除非在其氧化脱氨和还原胺化反应中都存在NADP+（一种激活剂）。据报道（LéJohn，H.B.（1971年）《自然》231卷，164 - 168页），这种类型的酶在被归类为卵菌纲的与藻类相关的无定形真菌类群中广泛存在。该酶在纯化的各个阶段都保持对NADP+的依赖性。NADP+使底物的Km降低了3倍，使Vmax增加了4倍。未变性酶的M(r)为480,000，变性后，仅可见一个M(r)为120,000的亚基。用从SDS - 聚丙烯酰胺凝胶电泳凝胶上切下的纯化酶亚基免疫家兔制备的多克隆抗体免疫沉淀了M(r)为120,000的多肽、未变性的酶以及一个物理性质不同的M(r)为74,000的多肽。以固定在琼脂糖上的M(r)为120,000的酶亚基作为抗原纯化的抗体，仍然能免疫沉淀M(r)为74,000的多肽。发现M(r)为74,000的多肽是一种M(r)为220,000天然蛋白质的亚基。
