| Mutiple forms of superoxide dismutase in bread wheat
Takaji IKUSHIMA Research Reactor Institute, Kyoto University, Kumatori-cho, Sennan-gun, Osaka 590-04, Japan Superoxide dismutase (SOD; E.C. 1.15.1.1) provides a defence mechanism against oxygen toxity such as inhibition of photosynthesis, cell killing and oxygen effects of ionizing radiations, to oxygen-metabolizing cells through catalizing the dismutation of O2- radical to yield hydrogen peroxide and oxygen. Three kinds of metalloproteins with SOD activity have been isolated from a variety of aerobic and anaerobic organisms; manganese, iron and cupper-zinc enzymes.1) A sensitive staining procedure for localizing SOD on polyacrylamide gel electrophoretograms is now applicable to crude extracts from various materials.2) A comparative study of the SOD distribution among tissues of bread wheat has been conducted in the present study from phylogenical and ontogenical interests. The first leaves and roots emerged under sterile conditions of Triticum aestivum cv. Chinese Spring (2n=42) were excised from seeds, and the tissues were homogenized in 0.1 M K-phosphate (pH 7.8) and 0.1 mM EDTA (0.1 ml per leaf or per two roots). The supernatant after centrifugation at 13,000 x g for 10 min at 2C was used as crude SOD extract. SOD activity was assayed photochemically by inhibition of nitro blue tetrazolium (NBT) reduction due to O2- in photoreduction system.2) Reduction of NBT, during 6 min of illumination, was measured in terms of increased absorbance at 560 nm, and inhibition (%) caused by SOD was plotted as function of concentration of this enzyme as shown in Fig. 1. Percent of inhibition is not linear with SOD concentration of leaf extracts. This non-linear relationship was also confirmed with crude extracts from roots or other plant materials.4) Polyacrylamide isoelectric focusing was performed by the procedure of Leaback & Rutter3) with carrier ampholyte (40 % solution, LKB Ampholine, pH 3-10 range). Electrophoresis was run for 3 hrs at 200 V using 0.2 M HCl and 1 % TEMED for the upper cathodal and the lower anodal solution, respectively. Negative staining of SOD bands was achieved according to BEAUCHAM & FRIDOVICH.2) Isoelectric focusing in combination with disc electrophoresis indicates that the SOD activity of the crude extracts is composed of a number of distinct bands. The zymograms of SOD from leaf and root extracts are drawn in Fig.2. In leaves, SOD activities were localized at eight sites of the gel with pI values of 3.9, 4.8, 5.0, 5.1, 5.6, 5.9, 6.9 and 7.8, whereas the same five active bands with apparent quantitative differences excluding bands with pI of 3.9, 5.9 and 6.9 were obtained from root extracts. Any new root-specific band was not observed. The similar change of the band pattern was also visible in two other cereals, barley and diploid oats.4) The above multiple bands may correspond to different proteins with SOD activity, or different isozymes of a single protein, or a combination of both. Cupper-zinc enzymes are sensitive to cyanide, whereas manganese enzymes are resistant to cyanide and sensitive to treatment with a chloroform-ethanol mixture.5) The only band with pI 3.9 was resistant to cyanide (not shown), and this result suggests that this enzyme seems to be a manganese enzyme, all the remaining being isozymes of cupperzinc SOD. More detail analyses of metal ions bounded to each SOD band will be necessary to elucidate the organelle specificity. References 1) FRIDOVICH,I., Ann. Rep. Biochemistry 44 (1975) 147 2) BEAUCHAM, C. and I. FRIDOVICH, Analytical Biochemistry 44 (1971) 276 3) LEABACK, D.H. and A.C. RUTTER, Biochem. Biophys. Res. Comm. 32 (1968) 447 4) IKUSHIMA, T., Japan. J. Breed. 27 Suppl. 2 (1977) 198 5) WEISIGER, R.A. and I. FRIDOVICH, J. Biol. Chem. 248 (1973) 3582 |