Purification and characterization of an isozyme of catalase with enhanced-peroxidatic activity from leaves of Nicotiana sylvestris

Abstract

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 M r 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 K m values for CAT-1 and CAT-3 were 0.057 and 0.054 m, respectively, and the k cat values were 4.8 × 10 7 and 3.0 × 10 6 min −1, respectively. In the peroxidatic reaction, both have similar apparent K m 's for H 2O 2. The apparent K m 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 m m, respectively. For CAT-1, the values were 697, 55.8, no detectable reaction with propyl and butyl and 163 m m, 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 M r 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.