Centre de Génétique Moléculaire, C.N.R.S., F-91190 Gif-sur-Yvette, France.
EMBO J. 1983;2(11):1909-14. doi: 10.1002/j.1460-2075.1983.tb01678.x.
Assimilatory nitrate reductase has been purified with 55% recovery from a Neurospora crassa nmr-1 nit-6 mutant, using a modification of a published procedure. It possesses one heme per 240 000 g, and subunits of mol. wt. 68 000. Upon digestion with chymotrypsin, a heme-binding domain was isolated by gel filtration; its visible spectrum was highly similar to that of cytochrome b(5). On SDS gels, the fraction showed two heme-containing bands of 10 000 and 12 5000 daltons; their amino acid composition was not very different, suggesting that they originated from the same region of the polypeptide chain. After S-carboxymethylation, the mixture of bands was submitted to cyanogen bromide cleavage, and the fragments were separated by h.p.l.c. The two largest fragments yielded an identical sequence upon automated degradation. This sequence (39 residues with some gaps) could be easily aligned with that of cytochrome b(5) starting close to the N terminus. These results are discussed in terms of the possible quaternary structure of N. crassa nitrate reductase, whose heme-binding domain proves to be another member of the family of b(5)-like cytochromes.
从一个 Neurospora crassa nmr-1 nit-6 突变体中,用一种改进的已发表的方法,以 55%的回收率纯化了同化硝酸盐还原酶。它每 240000 克含有一个血红素,亚基分子量为 68000。用胰凝乳蛋白酶消化后,通过凝胶过滤分离出一个血红素结合结构域;其可见光谱与细胞色素 b(5)非常相似。在 SDS 凝胶上,该部分显示出两个含有 10000 和 12500 道尔顿的血红素的带;它们的氨基酸组成差异不大,表明它们来自多肽链的同一区域。S-羧甲基化后,混合带经氰化溴裂解,用 h.p.l.c.分离片段。两个最大的片段在自动降解后产生相同的序列。这个序列(39 个残基,有些空缺)可以很容易地与细胞色素 b(5)的序列对齐,从接近 N 端开始。这些结果是根据 Neurospora crassa 硝酸盐还原酶的可能的四级结构讨论的,其血红素结合结构域证明是 b(5)-样细胞色素家族的另一个成员。