The effect in vivo of high nutrient levels of copper (240 micromolar) on the activity of different metalloenzymes conaining Cu, Mn, Fe, and Zn, distributed in chloroplasts, peroxisomes, and mitochondria, was studied in leaves of two varieties ofPisum satiRvum L. plants with different sensitivity to copper. The metalloenzymes studied were: cytochrome c oxidase, Mn-superoxide dismutase (Mn-SOD) and Cu,Zn-superoxide dismutase I (Cu,Zn-SOD I1), for mitochondria; catalase and Mn-SOD, for peroxisomes; and isozyme C,Zn-SOD II for chloroplasts. The activity of mitochondrial SOD isozymes (Mn-SOD and Cu,Zn-SOD I) was very similar in Cu-tolerant and Cu-sensitive plants, whereas cytochrome c oxidase was lower in Cu-sensitive plants. Chloroplastid Cu,Zn-SOD activity was the same in the two plant varieties. In contrast, the peroxisomal Mn-SOD activity was considerably higher in Cu-tolerant than in Cu-sensitive plants, and the activity of catalase was also increased in peroxisomes of Cu-tolerant plants. The However, there is a paucity of information on the activity response of metalloenzymes present in different cell compartments of plant leaves to an enhanced pool of nutrient copper. Comparative studies of the activity of metalloenzymes in different cell organelles of copper tolerant and nontolerant plants grown in high Cu nutrient levels would allow deeper insights into the molecular mechanisms ofintracellular responses to plant toxicity and metal tolerance. These types of studies may also give information on possible alterations of metalloenzyme characteristics in metal-tolerant plants and on the adaptive nature of enzymes to metal stress in certain plants.The induction of a Mn-SOD3 in leaves of pea plants by high nutrient levels of zinc and manganese has been recently demonstrated (7), as well as the induction of Fe-superoxide dismutases in lemon leaves by iron (25). This suggested a possible involvement of the metalloenzyme family of SODs in the mechanism of plant tolerance to metal toxicity. SODs (EC 1.15.1.1) are a group of metalloenzymes that catalyze the disproportionation of superoxide free radicals (02O), produced in certain cellular loci, and appear to play an important role in protecting cells against the indirect lethal effect of O2 radicals (17). There are essentially three types of SODs containing either Mn, Fe, or Cu plus Zn as metal prosthetic groups (17).Leaves of pea plants contain three electrophoretically distinct SODs, a Mn-containing and two Cu,Zn-containing isozymes, named I and II in order of increasing mobility (7). The isozyme Mn-SOD has been fully characterized (24), as well as the two pea leaf Cu,Zn-SODs (9). By studies of subcellular distribution of SODs in pea leaves the presence of Mn-SOD has been demonstrated both in mitochondria and peroxisomes (6, 19) whereas isozyme Cu,Zn-SOD II was located in chloroplasts (15), and Cu,Zn-SOD I appeared to be distributed between mitochondria and the cytosol (19). This location of SOD isozymes in different cell compartments makes this metalloenzym...
The effect of different Mn levels on the isozyme pattern of superoxide dismutase was investigated. Pisum sativum L. plants were grown in nutrient solutions containing three Mn concentrations: 0.005 μg/ml (deficient), 0.05 μg/ml (low), and 0.5 μg/ml (optimum). Leaf extracts contained three electrophoretically distinct superoxide dismutases (SOD), two of which were inhibited by cyanide and were probably Cu-Zn-SODs, while the third one was CN-insensitive and could be either an Mn- or an Fe-SOD. At 0.005 μg/ml Mn supply the CN-insensitive SOD was significantly depressed at 15, 30, and 45 days of growth, whereas at 0.05 μg/ml Mn this isozyme was significantly decreased only at 45 days growth. The two CN-sensitive SODs were inversely related to the CN-resistant enzyme, the activities of the former enzymes being significantly increased at Mn-deficient levels throughout plant growth. Metal determinations of the plants showed that at low concentrations of Mn in the nutrient media, copper and zinc content of leaves increased: the lower the Mn level, the higher the increase produced. The CN-resistant SOD activity, as judged by its dependency on Mn, appears to be an Mn-SOD rather than an Fe-SOD. In the light of the results obtained, the use of the enzyme system superoxide dismutase for the study of the role and interactions between Mn, Cu, and Zn in the plant cell is proposed.
A cyanide-insensitive superoxide dismutase was purified to apparent homogeneity from lemon leaves (Citrus limonum R). The enzyme was isolated from leaf extracts by ammonium sulfate salting-out, and ion-exchange, gel filtration and hydroxylapatite column chromatography. The purified Fe-SOD had a specific activity of about 1,500 U/mg and represents approximately 1.6% of the total soluble protein in lemon leaf extracts. A molecular weight of 47,500 was determined for the enzyme. Analytical gel electro-focusing of the purified preparation revealed the presence of two isozymes with pI values of 5.13 and 4.98. Metal analysis showed the presence of 1 g-atom of iron and 0.5 g-atom of manganese per mol of enzyme. The visible and UV absorption spectra of the Citrus enzyme were similar to those reported for other iron-containing SODs from different origins. The significance of the presence of Fe-SOD in higher plants is briefly discussed.
The effect of high nutrient levels of copper on the low-molecular-weight copper-proteins of leaves from plants of two cultivars of Pisum sativum L., with different sensitivity to copper, was investigated. Gel-filtration chromatography of leaf extracts from Cu-tolerant and Cu-sensitive plants grown with 1 μM Cu(II), showed the presence of only two copper peaks (I and II), but growth of plants with 240 μM Cu(II) induced two additional copper fractions (III and IV). Fractions II and III were purified by solvent extraction, gel-filtration and ion-exchange chromatography, and their molecular weights, subunit sizes, absorption spectra, metalprotein stoichiometry and amino-acid contents were determined. Fraction II was a polypeptide of Mr 15000 composed of a single chain. The purification of fraction III produced a copper-containing fraction (III-1) of Mr 3700, and a copper-protein (III-2) with an Mr, by sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis, of 66000. The metal contents of fractions III-1 and III-2 were higher in Cu-tolerant than in Cu-sensitive plants. On the basis of amino-acid analyses, fraction III-1 appeared to be complexes of Cu(II)-poly-isoleucine and Cu(II)-poly-leucine. The results rule out the existence, in pea leaves, of any protein similar to either animal metallothioneins or to any of the low-molecularweight metal-binding proteins or peptides described in other plants and reported to be involved in metal tolerance. In the mechanism of copper tolerance at the leaf level, fractions III-1 (Mr 3700), III-2 (Mr 66000), and IV (Mr 2000) appear to have a role, fraction IV being specifically induced in the tolerant cultivar by Cu(II). Fractions III-1 and III-2 could participate in a different mechanism, adaptive in character, involving an enhanced capacity to bind copper in Cu-tolerant plants.
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