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11.
Purification and characterization of glutathione reductase from corn mesophyll chloroplasts 总被引:3,自引:0,他引:3
Differences in the apparent molecular weights of the subunits of glutathione reductase (EC 1.6.4.2) from pea chloroplasts and corn mesophyll chloroplasts have been recently reported. In order to more fully describe the differences between the enzymes from these two sources, glutathione reductase from the mesophyll chloroplasts of corn seedlings ( Zea mays L. cv. G-4507) has been purified 200-fold by affinity chromatography using adenosine 2',5'-disphosphate agarose. The purified enzyme had a specific activity of 26 μmol NADPH oxidized (mg protein)-1 min-1 . The native enzyme had a relative molecular weight of 190 ± 30 kDa and exhibited polypeptides of 65, 63, 34, and 32 kDa when separated on sodium dodecylsulfate-polyacrylamide gels. Comparisons of the results from electroblotting, native molecular weight and subunit molecular weight analyses suggest that the enzyme exists as a heterotetramer. Optimal enzyme activity was obtained at pH 8 in N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (HEPES-NaOH) buffer. The sulfhydryl reagent, n -ethylmaleimide, inhibited enzymatic activity when incubated in the presence of NADPH while no inhibition was detected with oxidized glutathione in the incubation mixture. Reduced glutathione (5 m M ) inactivated the enzyme by 50%. This inactivation followed first order kinetics with a rate constant of 0.0028 s-1 . The enzyme was also inactivated by NADPH. The inactivation reached ca 90% within 30 min and followed first order kinetics with a rate constant of 0.0015 s-1 . 相似文献
12.
The hydrogen reactions of nitrogenase 总被引:2,自引:0,他引:2
Frank B. Simpson 《Physiologia plantarum》1987,69(1):187-190
13.
P. lanceolata andP. major were grown in culture solutions with nitrate or ammonium as the nitrogen source. Dry matter accumulation in the shoot was
faster with nitrate than with ammonium, whilst that of the roots was not affected by the nitrogen source. As a consequence,
the shoot-to-root ratio was lower with ammonium than with nitrate. InP. lanceolata, dry matter percentage of shoot and root tissue was lower with nitrate nutrition, suggesting better elongation growth than
with ammonium. However, in shoot tissue ofP. major the opposite was found. The rate of root respiration declined with time, and this was almost completely due to a declining
activity of the alternative path, which amounted to about 30–60% of total root respiration. Respiration via the cytochrome
path was for a part of time slightly increased by ammonium, whereas the activity of the alternative path was strongly enhanced.
The concentration of ethanol-soluble carbohydrates (SC) in the roots of both species was higher when nitrate was used, but
no difference in the concentration of starch was found. When the plants were transferred from one nitrogen source to the other,
many parameters, including the concentration of nitrate and chloride, and the shoot to root ratio, adjusted to the new situation
in both species.
Grassland Species Research Group, Publication no. 116. 相似文献
14.
Growth of 2-month-old nonnodulatedHippophaë rhamnoides seedlings supplied with combined N was compared with that of nodulated seedlings grown on zero N. Plant growth was significantly better with combined N than with N2 fixation and, although not statistically significant for individual harvests, tended to be highest in the presence of NH 4 + , a mixture of NH 4 + and NO 3 ? producing the highest yields. Growth was severely reduced when solely dependent on N2 fixation and, unlike the combined-N plants, shoot to root ratios had only slightly increased after an initial decrease. An apparently insufficient nodule mass (nodule weight ratio <5 per cent) during the greater part of the experimental period is suggested as the main cause of the growth reduction in N2-fixing plants. Thein vivo nitrate reductase activity (NRA) of NO 3 ? dependent plants was almost entirely located in the roots. However, when grown with a combination of NO 3 ? and NH 4 + , root NRA was decreased by approximately 85 per cent.H. rhamnoides demonstrated in the mixed supply a strong preference for uptake of N as NH 4 + , NO 3 ? contributing only for approximately 20 per cent to the total N assimilation. Specific rates of N acquisition and ion uptake were generally highest in NO 3 ? +NH 4 + plants. The generation of organic anions per unit total plant dry weight was approximately 40 per cent less in the NH 4 + plants than in the NO 3 ? plants. Measured extrusions of H+ or OH? (HCO 3 ? ) were generally in good agreement with calculated values on the basis of plant composition, and the acidity generated with N2 fixation amounted to 0.45–0.55 meq H+. (mmol Norg)?1. Without acidity control and in the presence of NH 4 + , specific rates of ion uptake and carboxylate generation were strongly depressed and growth was reduced by 30–35 per cent. Growth of nonnodulatedH. rhamnoides plants ceased at the lower pH limit of 3.1–3.2 and deterioration set in; in the case of N2-fixing plants the nutrient solution pH stabilized at a value of 3.8–3.9 without any apparent adverse effects upon plant performance. The chemical composition of experimental and field-growing plants is being compared and some comments are made on the nitrogen supply characteristics of their natural sites. 相似文献
15.
The mechanism of hypercholesterolemia effect of Cu2+ deficiency was studied in rats. There was increased activity of hepatic hydroxymethylglutaryl-coenzyme A reductase and increased
incorporation of labelled acetate into free cholesterol of liver in the Cu2+ deficient rats. Incorporation of label into ester cholesterol was however decreased in the liver. Concentration of bile acids
in the liver was not significantly altered. Increase in the incorporation of labelled acetate into serum cholesterol and increase
in the concentration of cholesterol and apo B in the low density lipoproteins + very low density lipoproteins fractions were
observed. Activity of lipoprotein lipase of the extrahepatic tissues decreased in the Cu2+ deficient rats. 相似文献
16.
Cibacron Blue F3G-A, a probe used to monitor nucleotide binding domains in enzymes, inhibited sheep liver 5,10-methylenetetrahydrofolate
reductase competitively with respect to 5-methyltetrahydrofolate and NADPH. TheK
i values obtained by kinetic methods and theK
d value for the binding of the dye to the enzyme estimated by protein fluorescence quenching were in the range 0.9–1.2 μM.
Another triazine dye, Procion Red HE-3B interacted with the enzyme in an essentially similar manner to that observed with
Cibacron Blue F3G-A. These results as well as the interaction of the dye with the enzyme monitored by difference spectroscopy
and intrinsic protein fluorescence quenching methods indicated that the dye was probably interacting at the active site of
the enzyme by binding at a hydrophobic region. 相似文献
17.
Activity of key nitrogen assimilating enzymes was studied in developing grains of high-lysine opaque sorghum P-721 and normal
sorghum CSV-5. The higher percentage of protein in opaque sorghum was mainly due to lower starch content since protein per
grain was less than in CSV-5. During grain development, albufn and globulin decreased while prolafne and glutelin increased.
Prolafne content in CSV-5 was higher than in opaque sorghum. Average nitrate reductase activity in flag and long leaf were
similar in both the varieties. The nitrate reductase activity decreased during grain development. Glutamate dehydrogenase
activity was higher during early development and lower at later stages in opaque sorghum than in CSV-5. Glutamate oxaloacetate
transaminase activity was higher and glutamine synthetase lower in opaque sorghum than in CSV-5 grains during development.
Glutamate synthase activity was higher in opaque sorghum up to day 20 and lower thereafter than in CSV-5. It is suggested
that reduced activities of glutamine synthetase as well as glutamate synthase in opaque sorghum as compared to CSV-5 during
later stages of development may restrict protein accumulation in the former. 相似文献
18.
The relationship between nitrification potential and nitrogen accumulation was studied in an early successional sere on Mt.
Fuji. Soil organic nitrogen accumulated with the invasion ofPolygonum cuspidatum and successively withMiscanthus oligostachyus and other species. Laboratory incubation experiments showed a higher nitrification potential at theM. oligostachyus state. The numbers of nitrifying bacteria increased with the progress of succession. No significant difference in nitrate
reductase activity was found between pioneer and succeeding species. The soil solution at theM. oligostachyus stage contained a lower level of nitrate than rainwater, while that of the bare ground and theP. cuspidatum stage contained a higher nitrate level than rainwater. It was concluded that the high nitrate levels in the soil solution
of the bare ground and theP. cuspidatum stage were due to lower nitrate-absorbing activity, leading to loss of nitrogen with precipitation, while the lower nitrate
levels at theM. oligostachyus stage when higher nitrification activity occurred were due to higher nitrate-absorbing activity, preventing net loss of nitrogen
from the ecosystem. 相似文献
19.
A flavoenzyme which showed NADPH-cytochrome c reductase (NADPH-cytochrome c oxidoreductase EC 1.6.2.4) and transhydrogenase (NADPH-NAD+ oxidoreductase, EC 1.6.1.1) activities was purified to an electrophoretically homogeneous state from Nitrobacter winogradskyi. The reductase was a flavoprotein which contained one FAD per molecule but no FMN. The oxidized form of the enzyme showed absorption maxima at 272, 375 and 459 nm with a shoulder at 490 nm, its molecular weight was estimated to be 36,000 by SDS polyacrylamide gel electrophoresis, and the enzyme seemed to exist as a dimer in aqueous solution. The enzyme catalyzed reduction of cytochrome c, DCIP and benzylviologen by NADPH, oxidation of NADPH with menadione and duroquinone, and showed transhydrogenase activity. NADH was less effective than NADPH as the electron donor in the reactions catalyzed by the enzyme. The NADPH-reduction catalyzed by the enzyme of N. winogradskyi cytochrome c-550 and horse cytochrome c was stimulated by spinach ferredoxin. The enzyme reduced NADP+ with reduced spinach ferredoxin and benzylviologen radical.Abbreviations DCIP
dichlorophenolindophenol
- Tris
trishydroxy-methylaminomethane
- Mops
3-(N-morpholino) propanesulfonic acid
- SDS
sodium dodecylsufate 相似文献
20.
Anin situ method, derived from anin vivo method, was used to determine nitrate reductase activity (NRA) in:i) excised barley and corn shoots and excised soybean leaves
during a N-depletion experiment and; ii) roots and shoots of N-depleted barley and corn seedlings during induction of nitrate,
reductase (NR). Nitrate reduction, calculated from thesein situ RNA measurements, was compared with estimates of each organ's nitrate reduction in light aerobic conditions from NO
3
−
consumption and a15N model (Gojonet al., 1986b).
Thein situ RNA of roots strongly underestimated their15NO
3
−
reduction. In contrast, in barley and corn shoots and in the first trifoliolate leaves from 26-day-old, soybean, thein situ NRA assay gave a fair approximation of the true NO
3
−
reduction rate (relative differences ranging from −14 to +32%). In young soybean leaves (from 20-day-old plants), however,
thein situ NRA strongly underestimated the actual NO
3
−
reduction. The physiological significance of thein situ NRA assay in shoots and roots, and its value for field studies are discussed from these results. 相似文献