Hilliard N P, Hirasawa M, Knaff D B, Shaw R W
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock 79409-1061.
Arch Biochem Biophys. 1991 Nov 15;291(1):195-9. doi: 10.1016/0003-9861(91)90123-z.
Recent preparations of nitrite reductase do not display the heterodimeric quaternary structure obtained previously (total molecular weight 85,000; subunit molecular weights 24,000 and 61,000), but rather yield only the 61,000 molecular weight subunit, even when buffers containing the protease inhibitor phenylmethylsulfonyl fluoride are used. Nevertheless, such preparations retain the high ratio of ferredoxin-linked to methyl viologen-linked enzyme activity which has been previously taken as a characteristic of only the heterodimeric form. These preparations display a siroheme prosthetic group to protein ratio of 1.1. When nitrite reductase samples are frozen during the purification scheme, even though the ferredoxin-linked specific activity does not significantly decrease, enzyme activity-stained native gel electrophoresis of the subsequently purified protein reveals that gels with several bands of activity can be obtained. Further evidence of protein heterogeneity in these preparations comes from N-terminal amino acid analysis which reveals that even nonfrozen preparations contain two major peptides with valine and cysteine as the N-termini. Formation of complexes of purified nitrite reductase with ferredoxin resulted in siroheme difference electronic spectra which resembled those observed previously for monomeric preparations. However, the siroheme midpoint potential of recent preparations of nitrite reductase (-287 mV) is close to that of the heterodimeric preparations. Ultrafiltration studies of crude extracts of the enzyme indicate that, at least at certain stages of the preparation, higher molecular weight forms of the enzyme may exist. We conclude that the 24,000 molecular weight polypeptide is a contaminant and that the heterodimeric quaternary structure model for spinach nitrite reductase is incorrect. Furthermore, the monomeric preparations we do obtain display both significant protein heterogeneity and facile loss of siroheme upon gel filtration.
最近制备的亚硝酸还原酶并不呈现先前得到的异二聚体四级结构（总分子量85,000；亚基分子量分别为24,000和61,000），而是仅产生分子量为61,000的亚基，即便使用了含有蛋白酶抑制剂苯甲基磺酰氟的缓冲液也是如此。然而，这些制剂保留了铁氧还蛋白连接的酶活性与甲基紫精连接的酶活性的高比例，这一比例先前被视为仅异二聚体形式的特征。这些制剂显示出siroheme辅基与蛋白质的比例为1.1。当在纯化方案中将亚硝酸还原酶样品冷冻时，尽管铁氧还蛋白连接的比活性没有显著降低，但随后纯化的蛋白质经酶活性染色的天然凝胶电泳显示，可以得到具有多条活性带的凝胶。这些制剂中蛋白质异质性的进一步证据来自N端氨基酸分析，该分析表明，即使是未冷冻的制剂也含有两种主要肽段，其N端分别为缬氨酸和半胱氨酸。纯化的亚硝酸还原酶与铁氧还蛋白形成的复合物产生了siroheme差分电子光谱，类似于先前在单体制剂中观察到的光谱。然而，最近制备的亚硝酸还原酶的siroheme中点电位（-287 mV）接近异二聚体制剂的中点电位。对该酶粗提物的超滤研究表明，至少在制备的某些阶段，可能存在分子量更高的酶形式。我们得出结论，分子量为24,000的多肽是一种污染物，菠菜亚硝酸还原酶的异二聚体四级结构模型是不正确的。此外，我们得到的单体制剂在凝胶过滤时既显示出显著的蛋白质异质性，又容易失去siroheme。
