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1.
FRANZ-ARNOLD AUSTENFELD 《Physiologia plantarum》1976,36(1):82-87
The response of glycolate oxidase from shoots of Salicornia europaea L. and from leaves of Pisum sativum L. to salt treatment during assay was studied by DCPIP reduction and O2 uptake. In Pisum there was found up to five times more glycolate oxidase activity per gram fresh weight than in Salicornia. However, the calculation of the specific activity pointed out that this result was caused only by the high level of enzyme protein in Pisum, and that specific activity from both species was of equal size. By the DCPIP method it was shown that in test media containing up to 1.0 M NaCl or KCl glycolate oxidase of Salicornia was of equal size compared with the control (medium without additional salts). With 2.0 M NaCl or KCl the activity decreased by about 80 and 30% respectively. Glycolate oxidase of Pisum was somewhat more salt sensitive. 1.0 M NaCl or KCl reduced the activity by about 35%. In the presence of 2.0 M NaCl or KCl the enzyme activity from Pisum was inhibited to about 80 and 60% respectively. By substituting sulfates for chlorides the activity of glycolate oxidase from both Salicornia and Pisum was stimulated strongly. 1.5 M Na2SO4 and 0.5 M K2SO4 (both are saturated solutions) caused an increase of glycolate activity from Salicornia of about 225 and 185% respectively, and from Pisum of about 50 and 30% respectively. Studying the response of glycolate oxidase to salt treatment by O2 uptake one must establish that with this method the degree of inhibition of enzyme activity at higher salt concentrations was always more severe than with dye reduction. Addition of 1.0 M NaCl or KCl to the assay medium caused an inhibition of glycolate oxidase activity from Salicornia of about 50% and from Pisum of about 60%. 2.0 M NaCl or KCl reduced the enzyme activity of both Salicornia and Pisum to nearly 10% of control activity. Furthermore, in contrast to DCPIP reduction no stimulating effect of sulfates on glycolate oxidase activity was detectable. Indeed, the inhibitory effect of sulfates was very slight. 1.0 M Na2SO4 caused a mean inhibition of glycolate oxidase activity of only 15% with both species, and in the presence of 1.5 M Na2SO4 50% of control activity was measured. At maximal K2SO4 concentrations (0.5 M) glycolate oxidase from both Salicornia and Pisum was also unaffected. It is supposed that the described salt tolerance of glycolate oxidase in vitro, possibly is due to an adaptation of the enzyme to high salt levels within peroxisomes in vivo. 相似文献
2.
Twenty-seven species of coccoid, zoospore-producing green algae representing 16 genera in the Chlorococcales and Chlorosarcinales were assayed for glycolate oxidase or glycolate dehydrogenase. Only Planophila terrestris Groover & Bold and Fasciculochloris boldii Trainor, contained glycolate oxidase whereas the others contained glycolate dehydrogenase. Representative algae were grown under varying conditions and assayed to determine any effects on these glycolate enzymes. Although specific rates of enzyme activity often varied widely, the form of glycolate enzyme present was not affected. 相似文献
3.
Transgenic Pssu-ipt tobacco with elevated content of endogenous cytokinins grown under in vitro conditions exhibited elevated activities of antioxidant enzymes (i.e. catalase, ascorbate peroxidase, guaiacol and syringaldazine peroxidase, glutathione reductase) and some of enzymes involved in anaplerotic pathways such as glucose-6-phosphate dehydrogenase, glycolate oxidase, NADP-malic enzyme, NADP-isocitrate dehydrogenase, and glutamate dehydrogenase compared to control non-transgenic SR1 tobacco. Higher activities of peroxidases, NADP-malic enzyme, and glutamate dehydrogenase were maintained in transgenic grafts after several weeks of the growth under ex vitro conditions, while transgenic rooted plants showed only the increase in activity of glycolate oxidase compared to control non-transformed tobacco. Total activities of superoxide dismutase were lower in both types of Pssu-ipt tobacco contrary to controls under both growth conditions. The presence of PR-1 protein and proteins with elevated activities of chitinase was proved in the extracellular fluid in both transgenic types under both in vitro and ex vitro conditions. 相似文献
4.
Glycolate oxidase was purified to apparent homogeneity from the brown alga Spatoglossum pacificum Yendo. The 1326-fold purified glycolate oxidase enzyme exhibited a specific activity of 22. 4 micromoles glyoxylate formed ·min?1·mg protein?1. The molecular weight of the native enzyme was estimated to be 230,000 by gel filtration. The subunit molecular weight of the enzyme was determined to be 49,000 by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, suggesting that the native enzyme is a tetramer. There were two absorption peaks at 345 and 445 nm, indicating that glycolate oxidase is a flavoprotein. This enzyme had a high isoelectric point (pI 9.6) and a high pH optimum (pH 8.3). The Km values for glycolate and l -lactate were 0.49 and 5.5 mM, respectively. This enzyme also had a broad specificity for other straight-chain α-hydroxy acids but not for β-hydroxyacids. Cyanide, azide, N-ethylmaleimide, and p-chloromercuribenzoic acid did not affect the enzyme, whereas 2-pyridylhydroxymethanesulfonic acid strongly inhibited it. These properties of glycolate oxidase from the brown alga S. pacificum are similar to the properties of the glycolate oxidasesfrom higher plants. Polyclonal antibodies raised against the polypeptide fragment of Spatoglossum glycolate oxidase could recognize glycolate oxidase from Spinacia oleracea L., although the cross-reactivity was weak. The N-terminal sequence of two internal polypeptide fragments of the enzyme from S. pacificum showed a high degree of similarity to that of glycolate oxidase from higher plants. These results suggest that glycolate oxidase from higher plants and brown algae share the same ancestral protein. 相似文献
5.
Summary This paper describes the first purification method for crystalline inclusions (cores) from plant peroxisomes and an ultrastructural characterization of these isolated cores. 5-day-old sunflower (Helianthus annuus L.) cotyledon fractions which were highly enriched in cores showed negligible activity of the matrix enzyme glycolate oxidase but high catalase activity. As proven by electron microscopy, crystalline particles were surrounded neither by matrix material nor by membranes. Their geometrical outlines and ultrastructure were identical to those of cores in tissue sections, as was their reactivity with three different polyclonal catalase antibodies in the immunogold technique. Three-dimensional reconstruction, based on the geometrical outlines and ultrastructure of sectioned isolated cores from sunflower, suggested that they were quadrangular blocks. Ultrastructural analysis revealed an even periodic arrangement of repeating units which are probably cubes with 20 nm long edges. Isolated peroxisomal cores from potato (Solanum tuberosum L.) tubers had outlines which suggested that they were even rhomboidal prisms. They showed a granular ultrastructure without any repeating units and contained catalase, demonstrated by immunogold labelling and enzyme activity measurement. The results presented here suggested the hypothesis that the structural elements in plant peroxisomal cores are made of enzymatically active catalase, although the substructure may vary from species to species.Abbreviations ACOx
acyl-CoA oxidase
- BSA
bovine serum albumin
- EDTA
ethylenediamine-tetraacetate
- GDH
glutamate dehydrogenase
- GOx
glycolate oxidase
- KPB
potassium phosphate buffer 相似文献
6.
Leaves of Quercus robur L. were infected with conidia of Microsphaera alphitoides Griffon and Maublanc. Crude enzyme extracts of healthy and infected tissue, taken during a 7-day experimental period, were assayed, using spectrophotometric and manometric techniques, for glycolate oxidase, NAD- and NADP-glyoxylate reductase activities. After 2 days, extracts from infected tissue contained significantly higher activities of glycolate oxidase and NAD-glyoxylate reductase than extracts from controls, but the activity of NADP-glyoxylate reductase, after an initial rise, was significantly reduced below that in control extracts. The reduction in glycolate oxidase activity may be partly due to a deficiency in flavin mononucleotide in infected tissue. A possible connection between the reduction in flavoprotein enzyme activity, photorespiration and susceptibility to infection is described. 相似文献
7.
Glycolate oxidase isoforms are distributed between the bundle sheath and mesophyll tissues of maize leaves 总被引:5,自引:0,他引:5
Glycolate oxidase (EC 1.1.3.15) activity was detected both in the bundle sheath (79%) and mesophyll (21%) tissues of maize leaves. Three peaks of glycolate oxidase activity were separated from maize leaves by the linear KCl gradient elution from the DEAE-Toyopearl column. The first peak corresponded to the glycolate oxidase isoenzyme located in the bundle sheath cells, the second peak had a dual location and the third peak was related to the mesophyll fraction. The mesophyll isoenzyme showed higher affinity for glycolate (Km 23 micromol x L(-1)) and a higher pH optimum (7.5-7.6) as compared to the bundle sheath isoenzyme (Km 65 micromol x L(-1), pH optimum 7.3). The bundle sheath isoenzyme was strongly activated by isocitrate and by succinate while the mesophyll isoenzyme was activated by isocitrate only slightly and was inhibited by succinate. It is concluded that although the glycolate oxidase activity is mainly attributed to the bundle sheath, conversion of glycolate to glyoxylate occurs also in the mesophyll tissue of C4 plant leaves. 相似文献
8.
Three peroxisomal enzymes, glycolate oxidase, urate oxidase and catalase were localized cytochemically in Psychotria punctata (Rubiaceae) leaves and Yucca torreyi (Agavaceae) seedling root tips, both of which contain developing and mature calcium-oxalate raphide crystal idioblasts. Glycolate-oxidase (EC 1.1.3.1) and catalase (EC 1.11.1.6) activities were present within leaftype peroxisomes in nonidioblastic mesophyll cells in Psychotria leaves, while urate-oxidase (EC 1.7.3.3) activity could not be conclusively demonstrated in these organelles. Unspecialized peroxisomes in cortical parenchyma of Yucca roots exhibited activities of all three enzymes. Reactionproduct deposits attributable to glycolate-oxidase activity were never observed in peroxisomes of any developing or mature crystal idioblasts of Psychotria or Yucca. Catalase localization indicates that idioblast microbodies are functional peroxisomes. The apparent absence of glycolate oxidase in crystal idioblasts of Psychotria and Yucca casts serious doubt that pathways involving this enzyme are operational in the synthesis of the oxalic acid precipitated as calcium-oxalate crystals in these cells.Abbreviations AMPD
2-amino-2-methyl-1,3-propandiol
- CTEM
conventional transmission electron microscopy
- DAB
3,3-diaminobenzidine tetrahydrochloride
- HVEM
high-voltage electron microscopy 相似文献
9.
N. Roth-Bejerano 《Planta》1980,149(3):252-256
The attachment of glycolate oxidase to the peroxisomal fraction derived from etiolated barley leaves (Hordeum vulgare L. cr. Dvir) is affected by light. The effect of red irradiation is reversed by subsequent far-red irradiation, indicating the involvement of phytochrome. This phytochrome effect is assumed to be related to phytochrome binding. Indeed, prevention by filipin (1.2·10-6 mol g-1 f wt) or cholesterol of phytochrome binding to membranes abolishes the effect of light on the interaction between glycolate oxidase and the peroxisomal fraction. Glycolate oxidase binding is affected by addition of quasi-ionophores such as gramicidin and filipin at a concentration of 0.6·10-3 mol g-1 f wt. This fact indicates that peroxisome-glycolate oxidase interaction may be affected by membrane potential. Since both ion transport and membrane potential are known to be affected by phytochrome, it is proposed that phytochrome acts in the light-induced modulation of glycolate oxidase attachment as a quasi-ionophore.Abbreviations GO
glycolate oxidase
- Pr and Pfr
phytochrome forms absorbing in red and far-red, respectively
- R and F
red and far-red irradiation
- Cumulative 20 Kp
20,000 g pellet obtained by centrifugation of the crude extract
- 1 Kp
1,000 g pellet
- 20 Kp
20,000 g pellet, obtained by centrifugation of 1 Kp supernatant
- 1 Kp, 20 Kp and cumulative 20 Kp
pellets obtained after density centrifugation through a sucrose cushion 相似文献
10.
Microbodies of the algaMougeotia were isolated in a linear sucrose gradient. The organelles, which moved to the density 1.24 g cm–3, contained about 70% of the glycolate oxidase (EC 1.1.3.1) found in this alga. The enzyme oxidized glycolate, utilizing either oxygen or 2,6-dichlorophenolindophenol (DCPIP) as the electron acceptor. L-Lactate was an alternate substrate; almost no D-lactate was utilized. In the presence of O2, a Km of 415 M was determined for glycolate, whereas the Km for L-lactate was about 5,000 M. In the presence of DCPIP, lower concentrations of glycolate and L-lactate were sufficient to obtain the highest rates of enzyme activity.Abbreviations DCPIP
2,6-dichlorophenolindophenol
Supported by the Deutsche Forschungsgemeinschaft 相似文献
11.
Glycolate oxidase extracted from tobacco leaves (Nicotiana tabacum L. cv. NC-95) and assayed by the 2,6-dichlorophe-nolindophenol reduction method was stimulated by chlorogenic acid and other o-diphenols but not by p-diphenols such as hydroquinone. Chlorogenic acid also protected the enzyme against certain enzyme antagonists. A novel assay utilizing horseradish peroxidase with the chromogen o-dianisidine was developed for detecting glycolate oxidase in conjunction with disc electrophoresis. Dissociation of glycolate oxidase into an active monomer during ammonium sulfate fractiona-tion was confirmed electrophoretically. After electrophoresis, flavin mononucleotide was required for monomer activity whereas chlorogenic acid was inhibitory to enzyme band development. 相似文献
12.
The occurrence of glycolate oxidase in addition to glycolate dehydrogenase in Dunaliella salina and D. primolecta, as reported in the literature, could not be confirmed. Both species were demonstrated to possess only glycolate dehydrogenase. After separation of organelles by gradient centrifugation, glycolate dehydrogenase along with hydroxypyruvate reductase was found exclusively in the mitochondria. Thus the peroxisomes from Dunaliella are not of the leaf-type: because of their content of catalase, uricase and hydroxyacyl-CoA dehydrogenase they appear to be of the same type as in Eremosphaera and other chlorophycean algae. No activity of glycolate dehydrogenase was found in the chloroplast fraction when the 2,6-dichlorophenol-indophenol test was used.This work was supported by the Deutsche Forschungsgemeinschaft. 相似文献
13.
A simple method for estimating intactness of spinach leaf protoplasts by glycolate oxidase assay
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A method was developed for the quantitative analysis of intactness of spinach leaf protoplasts using glycolate oxidase activity as an index. Since glycolate does not penetrate into protoplasts at neutral pH, the increase of O2 consumption by the addition of glycolate to protoplast suspension was due to the glycolate oxidase activity released from damaged protoplasts. The proportion of damaged protoplasts in the whole preparation was calculated from the ratio of released and total glycolate oxidase activity. Freshly prepared spinach leaf protoplasts were found to be 80 to 90% intact as estimated by the method. The effect of osmolarity on the respiratory activities of spinach leaf protoplasts was also examined by applying the same principle. 相似文献
14.
The metabolism of glycolate by Eurasian watermilfoil (Myriophyllum spicatum L.), a submersed angiosperm, was studied by feeding radioactive glycolate and glyoxylate and by analysis of glycolate and glycolic acid oxidase. Evidence for operation of the glycolate pathway is given. Glycolic acid oxidase occurs at levels comparable to amounts in species showing photorespiration. This species has a high affinity for CO2 and a possible mechanism for it is described. 相似文献
15.
Serological Characterization of the Glycolate-oxidizing Enzymes from Tobacco, Euglena gracilis, and a Yellow Mutant of Chlorella vulgaris
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An antiserum to tobacco glycolate oxidase has been prepared by injection of the purified enzyme into rabbits. Double gel diffusion tests between the antiserum and purified antigen and also with a crude tobacco preparation gave a single immunoprecipitation band. Crude extracts of Euglena gracilis Z Klebs, containing glycolate dehydrogenase, and of Chlorella vulgaris 211-11h/20, containing glycolate oxidase, also formed single bands with the tobacco antiserum. The algal bands were identical and showed partial identity with the tobacco band. The antiserum inhibited the glycolate oxidase activities of the tobacco and Chlorella extracts but did not affect Euglena glycolate dehydrogenase activity. 相似文献
16.
By ammonium sulfate fractionation and gel filtration an enzyme preparation which catalyzed NAD+-dependent L-lactate oxidation (10-4 kat kg-1 protein), as well as NADH-dependent pyruvate reduction (10-3 kat kg-1 protein), was obtained from leaves of Capsella bursa-pastoris. This lactate dehydrogenase activity was not due to an unspecific activity of either glycolate oxidase, glycolate dehydrogenase, hydroxypyruvate reductase, alcohol dehydrogenase, or a malate oxidizing enzyme. These enzymes could be separated from the protein displaying lactate dehydrogenase activity by gel filtration and electrophoresis and distinguished from it by their known properties. The enzyme under consideration does not oxidize D-lactate, and reduces pyruvate to L-lactate (the configuration of which was determined using highly specific animal L-lactate dehydrogenase). Based on these results the studied Capsella leaf enzyme is classified as L-lactate dehydrogenase (EC 1.1.1.27). It has a Km value of 0.25 mmol l-1 (pH 7.0, 0.3 mmol l-1 NADH) for pyruvate and of 13 mmol l-1 (pH 7.8, 3 mmol l-1 NAD+) for L-lactate. Lactate dehydrogenase activity was also detected in the leaves of several other plants.Abbreviation FMN
flavin adenine mononucleotide 相似文献
17.
植物中草酸积累与光呼吸乙醇酸代谢的关系 总被引:6,自引:1,他引:6
对几种C3 和C4 植物中草酸含量及相应的乙醇酸氧化酶活性测定结果表明 :叶片光呼吸强度及其关键酶活性大小与草酸积累量没有相关性 ;植物根中均能积累草酸 ,但未测出乙醇酸氧化酶活性。烟草根、叶中的草酸含量在不同生长时期差异明显 ,且二者呈极显著正相关 (y =2 .5 6 5lnx 2 .137,r =0 .749,P <0 .0 0 1) ,说明根中草酸可能来自叶片。氧化乙醇酸的酶的活性与氧化乙醛酸的酶的活性呈极显著线性正相关 (y =0 .2 41x 0 .0 0 6 ,r=0 .96 7,P <0 .0 0 0 1) ,进一步证实是乙醇酸氧化酶催化了两种底物的反应。烟草在不同生长期叶片中草酸总含量变化与相应的乙醇酸氧化酶活性变化亦没有相关性 ;低磷胁迫可显著诱导烟草根叶中的草酸形成和分泌 ,但并未影响乙醇酸氧化酶活性 ,进一步证明草酸积累与该酶活性大小无关 相似文献
18.
Seven enzymes participating in glycolate metabolism were demonstrated to be present in crude extract of the brown alga Spatoglossum pacificum Yendo. These were phosphoglycolate phosphatase, glycolate oxidase, glutamate-glyoxylate aminotransferase, serine hydroxymethyltransferase, amino acid-hydroxy-pyruvate aminotransferase, hydroxypyruvate reductase and catalase. Malate synthase, which is involved in glycolate metabolism in the xanthophycean alga, could not be detected. On demonstration of subcellular distribution of glycolate oxidizing enzymes by linear sucrose density gradient centrifugation, glycolate oxidase was detected in the same fraction at a density of 1.23 g cm?3 with catalase: that is, the marker enzyme of peroxisome and serine hydroxymethyltransferase was found in the same fraction at a density of 1.21 g cm?3 with isocitrate dehydrogenase, the marker of mitochondria. From the present data, it is proposed that the brown alga Spatoglossum possesses the ability to metabolize glycolate to glycerate via the pathway which may be similar to that of higher plants. 相似文献
19.
菠菜中的乙醇酸氧化酶是一个同工酶 总被引:8,自引:4,他引:4
徐杰 《中国生物化学与分子生物学报》1998,14(6):800-804
乙醇酸氧化酶(EC.1.1.3.15.GO)是光呼吸途径的关键酶,降低其活性可提高C3植物如水稻的产量,在目前中国乃至世界人口不断增加和可耕种土地日益减少的情况下,对GO的研究具有重要的理论意义和实际应用价值.在光呼吸途径被提出后的几十年间,人们对如... 相似文献