Patel R N, Hou C T, Felix A, Lillard M O
J Bacteriol. 1976 Jul;127(1):536-44. doi: 10.1128/jb.127.1.536-544.1976.
Procedures for the purification of catechol 1,2-dioxygenase from extracts of Acinetobacter calcoaceticus strain ADP-96 are described. The purified enzyme was homogeneous as judged by ultracentrifugation and acrylamide gel electrophoresis. The enzyme contained 2 g-atoms of iron per mol of protein. The enzyme had a broad substrate specificity and catalyzed the oxidation of catechol, 4-methylcatechol, 3-methylcatechol, and 3-isopropyl catechol. The activity of the enzyme was inhibited by heavy metals, sulfhydryl inhibitors, and substrate analogues. The molecular weight of the enzyme was 85,000 as estimated by filtration on Bio-Gel agarose and 81,000 as estimated by sedimentation equilibrium analysis. The subunit size determined by sodium dodecyl sulfate-gel electrophoresis was 40,000. The amino terminal amino acid was methionine. The amino acid composition and spectral properties of 1,2-dioxygenase are also presented. Antisera prepared against the purified enzyme cross-reacted and inhibited enzyme activity in crude extracts from the other strain of A. calcoaceticus, but failed to cross-react and inhibit isofunctional enzyme from organisms of the genera Pseudomonas, Alcaligenes, and Nocardia.
本文描述了从醋酸钙不动杆菌ADP - 96菌株提取物中纯化儿茶酚1,2 -双加氧酶的方法。通过超速离心和聚丙烯酰胺凝胶电泳判断，纯化后的酶是均一的。该酶每摩尔蛋白质含有2克原子铁。该酶具有广泛的底物特异性，能催化儿茶酚、4 -甲基儿茶酚、3 -甲基儿茶酚和3 -异丙基儿茶酚的氧化反应。重金属、巯基抑制剂和底物类似物可抑制该酶的活性。通过在生物凝胶琼脂糖上过滤估算，该酶的分子量为85,000，通过沉降平衡分析估算为81,000。经十二烷基硫酸钠 - 凝胶电泳测定，亚基大小为40,000。氨基末端氨基酸为甲硫氨酸。文中还给出了1,2 -双加氧酶的氨基酸组成和光谱特性。针对纯化酶制备的抗血清能与醋酸钙不动杆菌另一菌株的粗提物发生交叉反应并抑制其酶活性，但不能与假单胞菌属、产碱菌属和诺卡氏菌属生物体中的同功能酶发生交叉反应及抑制其活性。
