Holdom M D, Hay R J, Hamilton A J
Dermatology Unit, St. John's Institute of Dermatology, Guy's Hospital, London, United Kingdom.
Infect Immun. 1996 Aug;64(8):3326-32. doi: 10.1128/iai.64.8.3326-3332.1996.
Cu,Zn superoxide dismutases (SODs) have been purified to homogeneity from Aspergillus flavus and A. niger, which are significant causative agents of aspergillosis, and from A. nidulans and A. terreus, which are much rarer causative agents of disease, using a combination of isoelectric focusing and gel filtration fast protein liquid chromatography. The purified enzymes have been compared with the previously described SOD from the most important pathogen in the genus, A. fumigatus (M. D. Holdom, R. J. Hay, and A. J. Hamilton, Free Radical Res. 22:519-531, 1995). The N-terminal amino acid sequences of the four newly purified enzymes were almost identical and demonstrated homology to known Cu,Zn SODs from a range of organisms including that from the previously described SOD from A. fumigatus. SOD activity was detectable in the culture filtrates of all species, and intracellular Cu,Zn SOD activity as a proportion of total protein was highest in early-log-phase cultures. The specific activities of the purified enzymes were similar, and all four of the newly described enzymes were inhibited by potassium cyanide and diethyldithiocarbamate, known Cu,Zn SOD inhibitors. Sodium azide and o-phenanthroline demonstrated inhibition at concentrations from 5 to 30 mM, and EDTA also exhibited a varying degree of inhibition of SOD activity. However, there were differences in the nonreduced molecular masses, the reduced molecular masses, and the isoelectric points of the four newly described SODs and the A. fumigatus enzyme; these varied from 55 to 123 kDa, 17.5 to 19.5 kDa, and 5.0 to 5.9, respectively. Of particular note was the observation that the A. fumigatus enzyme was thermostable compared with the SODs from the other species; in addition, the A.fiumigatus enzyme retained all of its activity at 37 degrees C relative to 20 degrees C, whereas the SODs of A. nidulans and A. terreus lost significant activity at the higher temperature. Aspergillus Cu,Zn SOD plays a hypothetical role in the avoidance of oxidative killing mechanisms, and our data suggest that the thermotolerant A. fumigatus Cu,Zn SOD would be more effective in such a protective system than, for example, the equivalent enzyme from the more rarely pathogenic A. nidulans.
利用等电聚焦和凝胶过滤快速蛋白质液相色谱相结合的方法，从黄曲霉和黑曲霉（曲霉病的重要病原体）以及构巢曲霉和土曲霉（致病性罕见得多的病原体）中纯化出了铜锌超氧化物歧化酶（SOD），使其达到均一状态。已将纯化后的酶与该属最重要的病原体烟曲霉先前描述的SOD进行了比较（M.D.霍尔登、R.J.海和A.J.汉密尔顿，《自由基研究》22:519 - 531，1995年）。四种新纯化酶的N端氨基酸序列几乎相同，并且与一系列生物体中已知的铜锌SOD具有同源性，包括先前描述的烟曲霉SOD。在所有物种的培养滤液中均可检测到SOD活性，且对数生长前期培养物中细胞内铜锌SOD活性占总蛋白的比例最高。纯化酶的比活性相似，所有四种新描述的酶均受到已知的铜锌SOD抑制剂氰化钾和二乙基二硫代氨基甲酸盐的抑制。叠氮化钠和邻菲啰啉在5至30 mM的浓度下表现出抑制作用，EDTA也对SOD活性表现出不同程度的抑制。然而，四种新描述的SOD与烟曲霉的酶在非还原分子量、还原分子量和等电点方面存在差异；这些分别在55至123 kDa、17.5至19.5 kDa和5.0至5.9之间变化。特别值得注意的是，与其他物种的SOD相比，烟曲霉的酶具有热稳定性；此外，烟曲霉的酶在37℃时相对于20℃保留了其所有活性，而构巢曲霉和土曲霉的SOD在较高温度下失去了显著活性。曲霉铜锌SOD在避免氧化杀伤机制中起假设作用，我们的数据表明，耐热的烟曲霉铜锌SOD在这样的保护系统中比例如致病性较低的构巢曲霉的等效酶更有效。
