Purification and properties of four species of lysyl oxidase from bovine aorta.
Purification and properties of four species of lysyl oxidase from bovine aorta.
复制标题
牛主动脉四种赖氨酰氧化酶的纯化和性质。
作者：
H. Kagan;K. Sullivan;T. Olsson;A. Cronlund
牛主动脉的赖氨酰氧化酶通过用6 m-尿素中的盐梯度从DEAE-纤维素洗脱而分解成四种酶活性物质，这与用其它组织的酶获得的纯化结果一致[Stassen（1976）Biochim. Biophys. Acta 438，49-60]。在本研究中，四个活性峰中的每一个都通过随后在6 m-尿素中的凝胶过滤介质上的色谱法纯化至表观均一性。每种酶作为具有mol. wt.约30000在这些条件下，虽然赖氨酰氧化酶聚合成一系列的多聚体的分子量范围高达1000000在没有尿素。通过在十二烷基硫酸钠和8 m-尿素中电泳测定的每种酶的表观亚基分子量约为1.5g/L。32000-33000.赖氨酰氧化酶的纯化形式的氨基酸组成彼此相似，尽管存在足够的差异以得出每个是独特的分子种类的结论。将α-甲苯磺酰氟纳入纯化方案中不会改变酶分解成四种物质，这表明分离过程中的蛋白水解不是异质性的基础。每种酶对螯合剂、氨基脲和异烟肼的敏感性相似，表明每种酶都需要金属离子（可能是Cu（2+））和羰基化合物的参与才能发挥酶的功能。本研究描述了一种用于纯化多种赖氨酰氧化酶的方法，并揭示了不同酶形式之间存在显着的化学差异。
Lysyl oxidase of bovine aorta was resolved into four enzymically active species by elution from DEAE-cellulose with a salt gradient in 6m-urea, consistent with purification results obtained with enzyme of other tissues [Stassen (1976) Biochim. Biophys. Acta438, 49-60]. In the present study, each of the four peaks of activity was purified to apparent homogeneity by subsequent chromatography on gel-filtration media in 6m-urea. Each enzyme is eluted as a species with mol.wt. approx. 30000 under these conditions, although lysyl oxidase polymerizes to a series of multimers with molecular weights ranging up to 1000000 in the absence of urea. The apparent subunit molecular weight of each enzyme species determined by electrophoresis in sodium dodecyl sulphate and 8m-urea is approx. 32000-33000. The amino acid compositions of the purified forms of lysyl oxidase are similar to each other, although sufficient differences exist to conclude that each is a unique molecular species. Incorporation of alpha-toluenesulphonyl fluoride into the purification scheme does not alter the resolution of enzyme into four species, suggesting that proteolysis during isolation is not the basis of the heterogeneity. The similar sensitivities of each form of enzyme to chelating agents and to semicarbazide and isoniazid indicate that each requires the participation of a metal ion, presumably Cu(2+), and of a carbonyl compound for enzyme function. The present study describes a method for the purification of multiple species of lysyl oxidase and reveals that significant chemical differences exist between the different enzyme forms.