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1.
The pathway of carbon assimilation in greening roots was compared to the pathway in leaves of Lens culinaris seedlings by means of labelling distribution analysis among the products of 14CO 2 fixation in vivo, and in vitro with ribulose 1,5-diphosphate as the substrate. In green leaves, CO 2 fixation via ribulose 1,5-diphosphate carboxylase predominated largely while, in green roots, this carboxylase activity and the phosphoenolpyruvate carboxylase contributed almost equally to the whole in vivo CO 2 fixation. A participation of the activities of both carboxylases according to the double carboxylation pathway in the synthesis of dicarboxylic acids (malate and aspartate) was demonstrated in vitro after 48 h of greening in roots but seemed to be absent in in vivo experiments. 相似文献
2.
Kinetic properties of soybean net photosynthetic CO 2 fixation and of the carboxylase and oxygenase activities of purified soybean ( Glycine max [L.] Merr.) ribulose 1, 5-diphosphate carboxylase (EC 4.1.1.39) were examined as functions of temperature, CO 2 concentration, and O 2 concentration. With leaves, O 2 inhibition of net photosynthetic CO 2 fixation increased when the ambient leaf temperature was increased. The increased inhibition of CO 2 fixation at higher temperatures was caused by a reduced affinity of the leaf for CO 2 and an increased affinity of the leaf for O 2. With purified ribulose 1,5-diphosphate carboxylase, O 2 inhibition of CO 2 incorporation and the ratio of oxygenase activity to carboxylase activity increased with increased temperature. The increased O 2 sensitivity of the enzyme at higher temperature was caused by a reduced affinity of the enzyme for CO 2 and a slightly increased affinity of the enzyme for O 2. The similarity of the effect of temperature on the affinity of intact leaves and of ribulose 1,5-diphosphate carboxylase for CO 2 and O 2 provides further evidence that the carboxylase regulates the O 2 response of photosynthetic CO 2 fixation in soybean leaves. Based on results reported here and in the literature, a scheme outlining the stoichiometry between CO 2 and O 2 fixation in vivo is proposed. 相似文献
3.
Rhizobium japonicum CJ1 was capable of growing using formate as the sole source of carbon and energy. During aerobic growth on formate a cytoplasmic NAD +-dependent formate dehydrogenase and ribulose bisphosphate carboxylase activity was demonstrated in cell-free extracts, but hydrogenase enzyme activity could not be detected. Under microaerobic growth conditions either formate or hydrogen metabolism could separately or together support ribulose bisphosphate carboxylase-dependent CO 2 fixation. A number of R. japonicum strains defective in hydrogen uptake activity were shown to metabolise formate and induce ribulose bisphosphate carboxylase activity. The induction and regulation of ribulose bisphosphate carboxylase is discussed.Abbreviations hup
hydrogen uptake
- MOPS
3-(N-morpholino)-propanesulphonate
- TSA
tryptone soya agar
- RuBP
ribulose 1,5-bisphosphate
- FDH
formate dehydrogenase 相似文献
4.
Seven day old wheat and maize seedlings were exposed to 1300 or 2000 microeinsteins per square meter per second photosynthetically active radiation in CO 2-free air for 3 hours with either 1% O 2 in N 2 or N 2-only and then returned to normal air of 340 microliters per liter CO 2, 21% O 2 in N 2. Activity of the ribulose bisphosphate carboxylase and amount of the substrate, ribulose 1,5-bisphosphate, were measured during and following the CO 2-free treatments as was photosynthetic CO 2 fixation. Photoinhibition of photosynthesis was observed only with wheat seedlings following the N 2 only treatment. During the CO 2-free treatments, the levels of RuBP rose during all experiments except when wheat was photoinhibited. The activity of the ribulose bisphophate carboxylase, measured directly upon grinding the leaves, declined during the CO 2-free conditions. The carboxylase total activity increased in minutes in the leaf during and following the CO 2-free treatments. The specific activities of the wheat carboxylase went from 0.16 to 1.06 micromoles CO 2 fixed per milligram protein per minute while the maize carboxylase varied from 0.05 to 0.36 micromole CO 2 fixed per millogram protein per minute. This suggests that in these seedlings considerable inactive carboxylase must be stored in a form not activatable in extracts by CO 2 and Mg 2+. Possible mechanisms of regulation of photosynthesis by the ribulose bisphosphate carboxylase must consider not only the amount of active enzyme, but the amount of enzyme which the plant can make activatable upon demand. 相似文献
5.
Photosynthetic carbon assimilation in plants is regulated by activity of the ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase. Although the carboxylase requires CO 2 to activate the enzyme, changes in CO 2 between 100 and 1,400 microliters per liter did not cause changes in activation of the leaf carboxylase in light. With these CO 2 levels and 21% O 2 or 1% or less O 2, the levels of ribulose bisphosphate were high and not limiting for CO 2 fixation. With high leaf ribulose bisphosphate, the Kact(CO 2) of the carboxylase must be lower than in dark, where RuBP is quite low in leaves. When leaves were illuminated in the absence of CO 2 and O 2, activation of the carboxylase dropped to zero while RuBP levels approached the binding site concentration of the carboxylase, probably by forming the inactive enzyme-RuBP complex. The mechanism for changing activation of the RuBP carboxylase in the light involves not only Mg2+ and pH changes in the chloroplast stroma, but also the effects of binding RuBP to the enzyme. In light when RuBP is greater than the binding site concentration of the carboxylase, Mg2+ and pH most likely determine the ratio of inactive enzyme-RuBP to active enzyme-CO2-Mg2+-RuBP forms. Higher irradiances favor more optimal Mg2+ and pH, with greater activation of the carboxylase and increased photosynthesis. 相似文献
6.
A simple approach to determine CO 2/O 2 specificity factor () of ribulose 1,5-bisphosphate carboxylase/oxygenase is described. The assay measures the amount of CO 2 fixation at varying [CO 2]/[O 2] ratios after complete consumption of ribulose 1,5-bisphosphate (RuBP). Carbon dioxide fixation catalyzed by the carboxylase was monitored by directly measuring the moles of 14CO 2 incorporated into 3-phosphoglycerate (PGA). This measurement at different [CO 2]/[O 2] ratios is used to determine graphically by several different linear plots the total RuBP consumed by the two activities and the CO 2/O 2 specificity factor. The assay can be used to measure the amounts of products of the carboxylase and oxygenase reactions and to determine the concentration of the substrate RuBP converted to an endpoint amount of PGA and phosphoglycolate. The assay was found to be suitable for all [CO 2]/[O 2] ratios examined, ranging from 14 to 215 micromolar CO 2 (provided as 1–16 mM NaHCO 3) and 614 micromolar O 2 provided as 50% O 2. The procedure described is extremely rapid and sensitive. Specificity factors for enzymes of highly divergent values are in good agreement with previously published data.Abbreviations HEPPS
N-(2-hydroxyethyl)piperazine-N-(3-propanesulfonic acid)
- L
large subunit of rubisco
- PGA
3-phosphoglyceric acid
- rubisco
ribulose 1,5-bisphosphate carboxylase/oxygenase
- RuBP
d-ribulose 1,5-bisphosphate
- S
small subunit of rubisco
- XuBP
d-xylulose 1,5-bisphosphate 相似文献
7.
Ribulose-1,5-bisphosphate carboxylase activity was found in endosperm of germinating castor bean seed Ricinus communis and was localized in proplastids. The endosperm carboxylase has been extensively purified and is composed of two different subunits. The molecular weights of the native carboxylase and its subunits were 560,000, 55,000, and 15,000 daltons, respectively. The Michaelis-Menten constants, Km, for the endosperm carboxylase with respect to ribulose 1,5-bisphosphate, bicarbonate, CO 2, and magnesium in millimolar are 0.54, 13.60, 0.92, and 0.57, respectively. The endosperm carboxylase was activated by Mg 2+ and HCO 3−. The preincubation of the carboxylase with 1 millimolar HCO 3− and 5 millimolar MgCl 2 resulted in activation by low and inhibition by high concentrations of 6-phosphogluconate. In studies of dark 14CO2 fixation by endosperm slices, [14C]malate and [14C]citrate were the predominantly labeled products after 30 seconds of exposure of the tissue to H14CO3−. In pulse-chase experiments, 87% of the label is malate, and citrate was transferred to sugars after a 60-minute chase with a small amount of the label appearing in the incubation medium as 14CO2. The minimal incorporation of the label from 14CO2 into phosphoglyceric acid indicated a lack of the endosperm ribulose-1,5-bisphosphate carboxylase participation in the endosperm's CO2 fixation system. The activities of key Calvin cycle enzymes were examined in the endosperms and cotyledons of dark-grown castor bean seedlings. Many of these autotrophic enzymes develop in the dark in these tissues. The synthesis of ribulose-1,5-bisphosphate carboxylase in the nonphotosynthetic endosperms is not repressed in the dark, and high levels of enzymic activity appear with germination. All of the Calvin cycle enzymes are present in the castor bean endosperm except NADP-linked glyceraldehyde 3-P dehydrogenase, and the absence of this dehydrogenase probably prevents the functioning of these series of reactions in dark CO2 fixation. 相似文献
8.
Ribulose 1,5-bisphosphate in the chloroplast has been suggested to regulate the activity of the ribulose bisphosphate carboxylase/oxygenase. To generate high levels of ribulose bisphosphate, isolated and intact spinach chloroplasts were illuminated in the absence of CO 2. Under these conditions, chloroplasts generate internally up to 300 nanomoles ribulose 1,5-bisphosphate per milligram chlorophyll if O 2 is also absent. This is equivalent to 12 millimolar ribulose bisphosphate, while the enzyme, ribulose bisphosphate carboxylase, offers up to 3.0 millimolar binding sites for the bisphosphate in the chloroplast stroma. During illumination, the ribulose bisphosphate carboxylase is deactivated, due mostly to the absence of CO 2 required for activation. The rate of deactivation of the ribulose bisphosphate carboxylase was not affected by the chloroplast ribulose bisphosphate levels. Upon addition of CO 2, the carboxylase in the chloroplast was completely reactivated. Of interest, addition of 3-phosphoglycerate stopped deactivation of the carboxylase in the chloroplast while ribulose bisphosphate accumulated. With intact chloroplasts in light, no correlation between deactivation of the carboxylase and ribulose bisphosphate levels could be shown. 相似文献
9.
A rapid method to determine the CO 2/O 2 specificity factor of ribulose 1,5-bisphosphate carboxylase/oxygenase is presented. The assay measures the amount of CO 2 and O 2 fixation at varying CO 2/O 2 ratios to determine the relative rates of each reaction. CO 2 fixation is measured by the incorporation of the moles of 14CO 2 into 3-phosphoglycerate, while O 2 fixation is determined by subtraction of the moles of CO 2 fixed from the moles of RuBP consumed in each reaction. By analyzing the inorganic phosphate specifically hydrolyzed from RuBP under alkaline conditions, the amount of RuBP present before and after catalysis by rubisco can be determined. 相似文献
10.
When the amount of activation of ribulose 1,5-bisphosphate carboxylase has been measured, two forms of the enzyme, not one, are actually determined experimentally. Only the enzyme-activator CO 2-Mg 2+ form can bind ribulose bisphosphate for reaction with substrate CO 2 or O 2. A method is presented which measures only this catalytically active form by stabilizing it with ribulose bisphosphate just before dilution and assay in Mg 2+-free reaction medium. 相似文献
11.
The changes in the levels of intact spinach ( Spinacia oleracea L.) chloroplast adenine nucleotides during the time course of light-dependent CO 2 fixation were determined with respect to the effect of antimycin A. This study demonstrated that antimycin A lowered the rate of ATP formation during the induction period of carboxylation. While the steady state levels of ATP and the energy-charge value also decreased in the presence of antimycin, the concomitant increase of the CO 2 fixation activities insured higher ATP turnover rates. Changes in the labeling of CO 2 fixation products during the lag phase suggested a stepwise activation of the Calvin cycle, with fructose 1,6-diphosphate, and ribulose 5-phosphate kinase being activated before ribulose 1,5-diphosphate carboxylase. The possible mechanisms of the enhancement of CO 2 fixation activity by antimycin A in relation to its action on photophosphorylation during the lag phase are discussed. 相似文献
12.
Equations are developed to describe the reactions of ribulose 1,5-biphosphate carboxylase—oxygenase with ribulose biphosphate (RuP 2), carbon dioxide, and oxygen. It is predicted that at the high concentrations of enzyme sites found in vivo there will be a large proportion of the total RuP 2 bound to the enzyme. The kinetic characteristics of the in vivo reactions with RuP 2 are predicted to be analogous to those which would occur in the presence of a tight-binding substrate. Equations are developed which are applicable when the enzyme is only partially activated by CO 2 and Mg 2+. The response of carboxylase velocity to CO 2 concentration is sigmoidal when Mg 2+ concentration is low. 相似文献
13.
Wheat ( Triticum aestivum L. cv Albis) was grown in open-top chambers in the field and fumigated daily with charcoal-filtered air (0.015 microliters per liter O 3), nonfiltered air (0.03 microliters per liter O 3), and air enriched with either 0.07 or 0.10 microliters per liter ozone (seasonal 8 hour/day [9 am-5 pm] mean ozone concentration from June 1 until July 10, 1987). Photosynthetic 14CO 2 uptake was measured in situ. Net photosynthesis, dark respiration, and CO 2 compensation concentration at 2 and 21% O 2 were measured in the laboratory. Leaf segments were freeze-clamped in situ for the determination of the steady state levels of ribulose 1,5-bisphosphate, 3-phosphoglycerate, triose-phosphate, ATP, ADP, AMP, and activity of ribulose, 1,5-bisphosphate carboxylase/oxygenase. Photosynthesis of flag leaves was highest in filtered air and decreased in response to increasing mean ozone concentration. CO 2 compensation concentration and the ratio of dark respiration to net photosynthesis increased with ozone concentration. The decrease in photosynthesis was associated with a decrease in chlorophyll, soluble protein, ribulose bisphosphate carboxylase/oxygenase activity, ribulose bisphosphate, and adenylates. No decrease was found for triose-phosphate and 3-phosphoglycerate. The ratio of ATP to ADP and of triosephosphate to 3-phosphoglycerate were increased suggesting that photosynthesis was limited by pentose phosphate reductive cycle activity. No limitation occurred due to decreased access of CO 2 to photosynthetic cells since the decrease in stomatal conductance with increasing ozone concentration did not account for the decrease in photosynthesis. Ozonestressed leaves showed an increased degree of activation of ribulose bisphosphate carboxylase/oxygenase and a decreased ratio of ribulose bisphosphate to initial activity of ribulose bisphosphate carboxylase/oxygenase. Nevertheless, it is suggested that photosynthesis in ozone stressed leaves is limited by ribulose bisphosphate carboxylation possibly due to an effect of ozone on the catalysis by ribulose bisphosphate carboxylase/oxygenase. 相似文献
14.
The mechanisms regulating transient photosynthesis by soybean ( Glycine max) leaves were examined by comparing photosynthetic rates and carbon reduction cycle enzyme activities under flashing (saturating 1 s lightflecks separated by low photon flux density (PFD) periods of different durations) and continuous PFD. At the same mean PFD, the mean photosynthetic rates were reduced under flashing as compared to continuous light. However, as the duration of the low PFD period lengthened, the CO 2 assimilation attributable to a lightfleck increased. This enhanced lightfleck CO 2 assimilation was accounted for by a greater postillumination CO 2 fixation occurring after the lightfleck. The induction state of photosynthesis, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco), fructose 1,6-bisphosphatase (FBPase) and ribulose 5-phosphate kinase (Ru5P kinase) activities all responded similarly and were all lower under flashing as compared to constant PFD of the same integrated mean value. However, the fast phase of induction and FBPase and Ru5P kinase activities were reduced more than were the slow phase of induction and rubisco activity. This was consistent with the role of the former enzymes in the fast induction component that limited RuBP regeneration. Competition for reducing power between carbon metabolism and thioredoxin-mediated enzyme activation may have resulted in lower enzyme activation states and hence lower induction states under flashing than continuous PFD, especially at low lightfleck frequencies (low mean PFD).Abbreviations FBPase
fructose 1,6-bisphosphatase (EC 3.1.3.11)
- LUE
lightfleck use efficiency
- P-glycerate
3-phosphoglycerate
- PICF
post-illumination CO 2 fixation
- Ru5P kinase
ribulose 5-phosphate kinase (EC 2.7.1.19)
- RuBP
ribulose 1,5-bisphosphate
- rubisco
ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39)
- SBpase
sedoheptulose 1,7-bisphosphatase (EC 3.1.3.37) 相似文献
15.
Values of δ 13C and levels of phospho enolpyruvate carboxylase and ribulose 1,5-bisphosphate carboxylase/oxygenase were analyzed in segments from the fourth leaf of young maize ( Zea mays L.) plants. The δ 13C values became significantly more negative from the base to the tip of the leaves. Phospho enolpyruvate carboxylase levels and ribulose bisphosphate carboxylase levels both increased from the base to the tip. The principal effect of phospho enolpyruvate carboxylase levels or δ 13C should arise through its effect on the carboxylation/diffusion balance in the mesophyll. In this case, δ 13C values should become more negative as phospho enolpyruvate carboxylase levels increase, unless there are offsetting changes in stomatal aperture. The principal effect of ribulose bisphosphate carboxylase/oxygenase on δ 13C should occur through its effect on the extent of leakage of CO 2 from the bundle sheath cells. In this case, δ 13C values should become more positive as ribulose bisphosphate carboxylase levels increase. Accordingly, the variation in δ 13C values seen in maize leaves appears to be the result of variations in the level of phospho enolpyruvate carboxylase. 相似文献
16.
Nitrogen effects on the regulation of photosynthesis in wheat ( Triticum aestivum L., cv Remia) seedlings were examined. Ribulose 1,5-bisphosphate carboxylase/oxygenase was rapidly extracted and tested for initial activity and for activity after incubation in presence of CO 2 and Mg 2+. Freeze clamped leaf segments were extracted for determinations of foliar steady state levels of ribulose 1,5-bisphosphate, triose phosphate, 3-phosphoglycerate, ATP, and ADP. Nitrogen deficient leaves showed increased ATP/ADP and triose phosphate/3-phosphoglycerate ratios suggesting increased assimilatory power. Ribulose 1,5-bisphosphate levels were decreased due to reduced pentose phosphate reductive cycle activity. Nevertheless, photosynthesis appeared to be limited by ribulose 1,5-bisphosphate carboxylase/oxygenase, independent of nitrogen nutrition. Its degree of activation was increased in nitrogen deficient plants and provided for maximum photosynthesis at decreased enzyme protein levels. It is suggested that ribulose 1,5-bisphosphate carboxylase/oxygenase activity is regulated according to the amount of assimilatory power. 相似文献
17.
d-Ribulose 1,5-diphosphate carboxylase from extracts of the unicellular blue-green alga Aphanocapsa 6308 has been purified by ammonium sulphate precipitation and linear sucrose density gradient centrifugation. The molecular weight was estimated to be 525 000 and the enzyme consisted of two types of sub-unit of molecular weights 51 000 and 15 000. The small sub-units were not detected after purification involving acid precipitation but were observed if the acid precipitation step was omitted. The Michaelis constants for Mg 2+ and CO 2, when tested under air, were 0.35 mM and 0.071 mM respectively. Oxygen acted as a competitive inhibitor with respect to CO 2, suggesting that the enzyme also acts as an oxygenase. This was confirmed by measuring ribulose diphosphate-dependent O 2 uptake. A 1:1 stoichiometry between ribulose diphosphate utilization and O 2 consumption was observed. 6-Phosphogluconate inhibited carboxylase activity both at high (20 mM) and low (1 mM) bicarbonate concentrations. The data are compared with the properties of ribulose diphosphate carboxylase from other autotrophic prokaryotes and from chloroplasts.Abbreviations RuDP
d-Ribulose 1,5-diphosphate
- EDTA
ethylene diamine tetraacetic acid
- GSH
reduced glutathione
- SDS
sodium dodecyl sulphate
- 6PGluc
6-phosphogluconate
- STB
supplemented Tris buffer 相似文献
18.
Temperature, activating metal ions, and amino-acid substitutions are known to influence the CO 2/O 2 specificity of the chloroplast enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase. However, an understanding of the physical basis for enzyme specificity has been elusive. We have shown that the temperature dependence of CO 2/O 2 specificity can be attributed to a difference between the free energies of activation for the carboxylation and oxygenation partial reactions. The reaction between the 2,3-enediolate of ribulose 1,5-bisphosphate and O 2 has a higher free energy of activation than the corresponding reaction of this substrate with CO 2. Thus, oxygenation is more responsive to temperature than carboxylation. We have proposed possible transition-state structures for the carboxylation and oxygenation partial reactions based upon the chemical natures of these two reactions within the active site. Electrostatic forces that stabilize the transition state of the carboxylation reaction will also inevitably stabilize the transition state of the oxygenation reaction, indicating that oxygenase activity may be unavoidable. Furthermore, the reduction in CO 2/O 2 specificity that is observed when activator Mg 2+ is replaced by Mn 2+ may be due to Mg 2+ being more effective in neutralizing the negative charge of the carboxylation transition state, whereas Mn 2+ is a transition-metal ion that can overcome the triplet character of O 2 to promote the oxygenation reaction.Abbreviations CABP
2-carboxyarabinitol 1,5-bisphosphate
- enol-RuBP
2,3-enediolate of ribulose 1,5-bisphosphate
- K c
K mfor CO 2
- K o
K mfor O 2
- Rubisco
ribulose-1,5-bisphosphate carboxylase/oxygenase
- RuBP
ribulose 1,5-bisphosphate
- V c
V
max for carboxylation
- V o
V
max for oxygenation 相似文献
19.
Ribulose 1,5-bisphosphate carboxylase (EC.4.1.1.39) has been obtained from Nicotiana tabacum leaf homogenates with specific activites from 0.5 to 0.8 µmol CO 2 fixed (mg protein min) -1. These activities are reconciled with much lower, previously reported activities. The results suggest that if the tobacco enzyme is assayed under optimum conditions there is little difference in the intrinsic specific activities of tobacco and spinach ribulose 1,5-bisphosphate carboxylase. Several factors affecting activity measurements were examined. 相似文献
20.
Plants with the C 3, C 4, and crassulacean acid metabolism (CAM) photosynthetic pathways show characteristically different discriminations against 13C during photosynthesis. For each photosynthetic type, no more than slight variations are observed within or among species. CAM plants show large variations in isotope fractionation with temperature, but other plants do not. Different plant organs, subcellular fractions and metabolises can show widely varying isotopic compositions. The isotopic composition of respired carbon is often different from that of plant carbon, but it is not currently possible to describe this effect in detail. The principal components which will affect the overall isotope discrimination during photosynthesis are diffusion of CO 2, interconversion of CO 2 and HCO ?3, incorporation of CO 2 by phosphoenolpyruvate carboxylase or ribulose bisphosphate carboxylase, and respiration. Theisotope fractionations associated with these processes are summarized. Mathematical models are presented which permit prediction of the overall isotope discrimination in terms of these components. These models also permit a correlation of isotope fractionations with internal CO 2 concentrations. Analysis of existing data in terms of these models reveals that CO 2 incorporation in C 3 plants is limited principally by ribulose bisphosphate carboxylase, but CO 2 diffusion also contributes. In C 4 plants, carbon fixation is principally limited by the rate of CO 2 diffusion into the leaf. There is probably a small fractionation in C 4 plants due to ribulose bisphosphate carboxylase. 相似文献
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