Havir E A, McHale N A
Department of Biochemistry and Genetics, Connecticut Agricultural Experiment Station, New Haven 06504.
Arch Biochem Biophys. 1990 Dec;283(2):491-5. doi: 10.1016/0003-9861(90)90672-l.
Two isozymes of catalase (EC 1.11.1.6), one with typically low peroxidatic activity (CAT-1) and the other with enhanced-peroxidatic activity (EP-CAT or CAT-3) have been purified to electrophoretic homogeneity from tobacco (Nicotiana sylvestris) seedlings and antibodies prepared against each. The isozyme proteins showed no immunological cross-reactivity. The subunit Mr was 55,300 +/- 750 for CAT-1 and 53,300 +/- 850 for CAT-3 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the catalatic reaction, the apparent Km values for CAT-1 and CAT-3 were 0.057 and 0.054 M, respectively, and the kcat values were 4.8 x 10(7) and 3.0 x 10(6) min-1, respectively. In the peroxidatic reaction, both have similar apparent Km's for H2O2. The apparent Km values for CAT-3 for the series methyl, ethyl, propyl, butyl, and allyl alcohols were 2.48, 5.6, 38.6, 429, and 16.3 mM, respectively. For CAT-1, the values were 697, 55.8, no detectable reaction with propyl and butyl, and 163 mM, respectively. Neither isozyme utilized dianisidine or guaiacol in the peroxidatic reaction. Catalase activity (CAT-2) which eluted in an intermediate position between CAT-1 and CAT-3 from a chromatofocusing column was composed of only one subunit whose Mr coincided with CAT-1, and only the antibody to CAT-1 reacted with CAT-2 protein. Thus, CAT-2 and CAT-1 appear closely related while CAT-3 is distinctly different.
已从烟草（野生烟草）幼苗中纯化出过氧化氢酶的两种同工酶（EC 1.11.1.6），一种具有典型的低过氧化物酶活性（CAT-1），另一种具有增强的过氧化物酶活性（EP-CAT或CAT-3），并分别制备了针对它们的抗体。这些同工酶蛋白没有免疫交叉反应。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定，CAT-1的亚基Mr为55,300±750，CAT-3的亚基Mr为53,300±850。在催化反应中，CAT-1和CAT-3的表观Km值分别为0.057和0.054 M，kcat值分别为4.8×10⁷和3.0×10⁶ min⁻¹。在过氧化物酶反应中，两者对H₂O₂的表观Km值相似。CAT-3对甲基、乙基、丙基、丁基和烯丙醇系列的表观Km值分别为2.48、5.6、38.6、429和16.3 mM。对于CAT-1，相应的值分别为697、55.8、与丙醇和丁醇无可检测反应以及163 mM。在过氧化物酶反应中，两种同工酶均不利用联茴香胺或愈创木酚。在色谱聚焦柱上从CAT-1和CAT-3之间的中间位置洗脱的过氧化氢酶活性（CAT-2）仅由一个亚基组成，其Mr与CAT-1一致，并且只有针对CAT-1的抗体与CAT-2蛋白发生反应。因此，CAT-2和CAT-1似乎密切相关，而CAT-3则明显不同。
