The in Vivo and in Vitro Inhibition of Catalase from Leaves of Nicotiana sylvestris by 3-Amino-1,2,4-Triazole

Abstract

Seedlings of tobacco (Nicotiana sylvestris) were treated in vivo with 0.03 to 20 millimolar 3-amino-1,2,4-triazole (aminotriazole). There was a rapid loss of catalase (EC 1.11.1.6) activity over the first 5 hours followed by a slower decrease for the next 4 hours to a level that was 15 to 20% of the initial activity, with little or no change for periods up to 3 days. Fifty percent loss of catalase activity occurred at 0.10 to 0.15 millimolar inhibitor (18-hour incubation). The isozymes of tobacco catalase differed in sensitivity to the inhibitor. Enhanced-peroxidatic catalase (EP-CAT) (Havir EA, McHale NA, [1989] Plant Physiol 91: 812-815) decreased 35% under conditions in which the major isozyme decreased 85%. The resistance to aminotriazole inhibition demonstrated in vivo by EP-CAT was also observed in vitro. The times for 50% inhibition at 0.67, 3.33, 5.0, 10.0, and 15 millimolar aminotriazole were 15, 5, 2.6, 2.5, and 1.5 minutes, respectively, for the major isozyme of catalase and 60, 18.5, 5.1, 4, and 3.0 minutes, respectively, for EP-CAT. Increasing H₂O₂ concentration did not change the sensitivity of EP-CAT to aminotriazole. The major form of catalase contained 4.0 ± 0.4 moles of heme per mole enzyme and EP-CAT 3.4 ± 0.3. Thus, the resistance of EP-CAT to aminotriazole is probably not due to lowered affinity for H₂O₂ or alteration in heme content but to structural changes that impair inhibitor binding.