The two β-cyanoalanine synthase isozymes of tobacco showed different antioxidative abilities

Abstract

β-Cyanoalanine synthase (CAS, EC 4.4.1.9) catalyzes the conversion of cyanide and cysteine to β-cyanoalanine and plays pivotal roles in cyanide detoxification of plants. Both of tobacco leaves and roots showed some CAS activities, although with a much higher value in leaves. Crude CAS extracted from tobacco leaves or roots using buffers containing such reductants as cysteine (Cys), dithiothreitol (DTT) and ascorbic acid (AsA), produced two CAS bands on gel when detected with lead acetate following non-denaturing polyacrylamide gel electrophoresis (PAGE). They were designated CAS 1 and CAS 2, respectively, CAS 2 with a relatively higher migration rate than CAS 1. CAS 1 had higher activities than CAS 2 in both tobacco leaves and roots. Activity detection on gel showed that both CAS isozymes existed in the isolated cytosol from tobacco leaves, CAS 2 having a higher activity than CAS 1. But only CAS 1 was observed in the isolated mitochondria and no CAS was found in the isolated chloroplast. When measured with N, N-dimethyl- p-phenylenediamine (DMPDA) CAS activities were also found in the isolated cytosol and mitochondria, but not in the isolated chloroplast. Crude CAS extracted from tobacco leaves or roots using buffers without any of the above reductants produced only CAS 1 on gel when detected with lead acetate following non-denaturing PAGE. Reductants also showed significant protective effects on the activity of the CAS extracts when measured with DMPDA. In vitro treatment of the crude CAS samples extracted in the absence of any reductants with cysteine of 10–1000 mM could not produce CAS 2 on gel. These results indicate that CAS 2 isozyme of tobacco is very sensitive to oxidation, but CAS 1 isozyme possesses a distinctly higher antioxidative ability than CAS 2. Reductants such as cysteine, dithiothreitol, and ascorbic acid have significant protective effects on CAS 2 during tissue disruption. The difference in antioxidative ability may be used to distinguish different CAS isozymes, and give new clues for their variant physiological significances in plants.