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1.
Because glyoxylate inhibits CO 2 fixation by intact chloroplasts and purified ribulose bisphosphate carboxylase/oxygenase, glyoxylate might be expected to exert some regulatory effect on photosynthesis. However, ribulose bisphosphate carboxylase activity and activation in intact chloroplasts from Spinacia oleracea L. leaves were not substantially inhibited by 10 millimolar glyoxylate. In the light, the ribulose bisphosphate pool decreased to half when 10 millimolar glyoxylate was present, whereas this pool doubled in the control. When 10 millimolar glyoxylate or formate was present during photosynthesis, the fructose bisphosphate pool in the chloroplasts doubled. Thus, glyoxylate appeared to inhibit the regeneration of ribulose bisphosphate, but not its utilization. The fixation of CO2 by intact chloroplasts was inhibited by salts of several weak acids, and the inhibition was more severe at pH 6.0 than at pH 8.0. At pH 6.0, glyoxylate inhibited CO2 fixation by 50% at 50 micromolar, and glycolate caused 50% inhibition at 150 micromolar. This inhibition of CO2 fixation seems to be a general effect of salts of weak acids. Radioactive glyoxylate was reduced to glycolate by chloroplasts more rapidly in the light than in the dark. Glyoxylate reductase (NADP+) from intact chloroplast preparations had an apparent Km (glyoxylate) of 140 micromolar and a Vmax of 3 micromoles per minute per milligram chlorophyll. 相似文献
2.
A technique has been developed for the rapid and simple measurement of ribulose 1,5-bisphosphate from isolated spinach chloroplasts. The endogenous ribulose bisphosphate was detected enzymically using (14)CO(2) and ribulose bisphosphate carboxylase/oxygenase released from the chloroplasts. Ribulose 5-phosphate kinase was inhibited with 0.4 to 0.6 millimolar 2,6-dichlorophenol-indophenol and 4 micromolar carbonyl cyanide m-chlorophenylhydrazone. Phosphoenolpyruvate carboxylase activity was low with washed chloroplasts and its labeled product, [(14)C]oxalacetate, was destroyed by heating with 1.0 n HCl at 90 C. The assay method was linear from 0.05 to 0.87 nanomoles ribulose bisphosphate per milliliter. The latter value was determined with chloroplast material having 44 micrograms of chlorophyll per milliliter. This technique was simple and direct, used less chloroplast material, yet provided results comparable to a previously described enzymic technique in which ribulose bisphosphate was determined after the precipitation of chloroplast proteins by perchloric acid. 相似文献
3.
The response of ribulose 1,5-bisphosphate levels and CO(2) fixation rates in isolated, intact spinach chloroplasts to pyrophosphate, triose phosphates, dl-glyceraldehyde, O(2), catalase, and irradiance during photosynthesis has been studied. Within 1 minute in the light, a rapid accumulation of ribulose bisphosphate was measured in most preparations of intact chloroplasts, and this subsequently dropped as CO(2) fixation increased. Pyrophosphate, triose phosphates, and catalase increased CO(2) fixation and also the levels of ribulose bisphosphate. CO(2) fixation was inhibited by dl-glyceraldehyde and O(2) with corresponding decreases in ribulose bisphosphate. When the rate of photosynthesis decreased at limiting irradiances (low light), the level of ribulose bisphosphate in the chloroplast did not always decrease, suggesting that ribulose bisphosphate was not limiting CO(2) fixation under these conditions. When triose phosphates (fructose bisphosphate plus aldolase) were added to suspensions of chloroplasts at low irradiances, ribulose bisphosphate increased while CO(2) fixation decreased. These observations provide considerable evidence that high ribulose bisphosphate levels clearly are not solely sufficient to permit rapid rates of CO(2) fixation, but that factors other than ribulose bisphosphate concentration are overriding the control of photosynthesis.Isolated chloroplasts are capable of using carbon reserves to produce considerable ribulose bisphosphate. Upon illumination in the absence of CO(2) and O(2), intact chloroplasts produced up to 13 millimolar ribulose bisphosphate. 相似文献
4.
At bicarbonate concentrations equivalent to air levels of CO 2, activation of ribulosebisphosphate carboxylase/oxygenase (rubisco) was inhibited by micromolar concentrations of glyoxylate in intact, lysed, and reconstituted chloroplasts and in stromal extracts. The concentration of glyoxylate required for 50% inhibition of light activation in intact chloroplasts was estimated to be 35 micromolar. No direct inhibition by glyoxylate was observed with purified rubisco or rubisco activase at micromolar concentrations. Levels of ribulose 1,5-bisphosphate and ATP increased in intact chloroplasts following glyoxylate treatment. Results from experiments with well-buffered lysed and reconstituted chloroplast systems ruled out lowering of pH as the cause of inhibition. With intact chloroplasts, micromolar glyoxylate did not prevent activation of rubisco at high (10 mM) concentrations of bicarbonate, indicating that rubisco could be spontaneously activated in the presence of glyoxylate. These results suggest the existence of a component of the in vivo rubisco activation system that is not yet identified and which is inhibited by glyoxylate.Abbreviations PEP
phosphoenolpyruvate
- PGA
3-phosphoglycerate
- rubisco
ribulosebisphosphate carboxylase/oxygenase
- RuBP
ribulose 1,5-bisphosphate 相似文献
5.
When isolated spinach chloroplasts are illuminated, photosynthesis and CO 2 fixation die off within 30 to 90 minutes. Even when air levels of CO 2 are used which maintain high and rate-saturating amounts of ribulose 1,5-bisphosphate inside the plastids, CO 2 fixation declines. The decline begins with a drop in activity of the ribulose 1,5-bishosphate carboxylase/oxygenase, specifically loss of the enzyme-activator CO 2-Mg 2+ form. Next, the light reactions cause gradual leakage of the carboxylase and other stromal proteins to the suspending medium. The chloroplast outer envelope appears to reseal and protect the thylakoids since there is little change in the ferricyanide-dependent Hill reaction. In the dark, under otherwise identical conditions, leakage of carboxylase does not occur. 相似文献
6.
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
7.
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
8.
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
9.
The incorporation of 14CO 2 into glycolate by intact spinach leaf ( Spinacia oleracea L. var. Kyoho) chloroplasts exposed to 14CO 2 (NaH 14CO 3, 1 millimolar) in the light was determined as a function of O 2 concentrations in the reaction media. A hyperbolic saturation curve was obtained, apparent Km (O 2) of 0.28 millimolar, indicating that glycolate is produced predominantly by ribulose-1,5-bisphosphate carboxylase/oxygenase. A concentration gradient of glycolate was invariably observed between chloroplast stroma and the outside media surrounding chloroplasts during photosynthetic 14CO 2 fixation under an O 2 atmosphere. 相似文献
10.
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
11.
The light-dependent accumulation of radioactively labeled inorganic carbon in isolated spinach ( Spinacia oleracea L.) chloroplasts was determined by silicone oil filtering centrifugation. Intact chloroplasts, dark-incubated 60 seconds at pH 7.6 and 23°C with 0.5 millimolar sodium bicarbonate, contained 0.5 to 1.0 millimolar internal inorganic carbon. The stromal pool of inorganic carbon increased 5- to 7-fold after 2 to 3 minutes of light. The saturated internal bicarbonate concentration of illuminated spinach chloroplasts was 10- to 20-fold greater than that of the external medium. This ratio decreased at lower temperatures and with increasing external bicarbonate. Over one-half the inorganic carbon found in intact spinach chloroplasts after 2 minutes of light was retained during a subsequent 3-minute dark incubation at 5°C. Calculations of light-induced stromal alkalization based on the uptake of radioactively labeled bicarbonate were 0.4 to 0.5 pH units less than measurements performed with [ 14C]dimethyloxazolidine-dione. About one-third of the binding sites on the enzyme ribulose 1,5-bisphosphate carboxylase were radiolabeled when the enzyme was activated in situ and 14CO 2 bound to the activator site was trapped in the presence of carboxypentitol bisphosphates. Deleting orthophosphate from the incubation medium eliminated inorganic carbon accumulation in the stroma. Thus, bicarbonate ion distribution across the chloroplast envelope was not strictly pH dependent as predicted by the Henderson-Hasselbach formula. This finding is potentially explained by the presence of bound CO 2 in the chloroplast. 相似文献
12.
Ribulose-1,5-bisphosphate (RuBP) carboxylase in lysed spinach ( Spinacia oleracea L. cv virtuosa) chloroplasts that had been partly inactivated at low CO 2 and Mg 2+ by incubating in darkness with 4 millimolar partially purified RuBP was reactivated by light. If purified RuBP was used to inhibit dark activation of the enzyme, reactivation by light was not observed unless fructose-1,6-bisphosphate, ATP, or ADP plus inorganic phosphate were also added. Presumably, ADP plus inorganic phosphate acted as an ATP-generating system with a requirement for the generation of ΔpH across the thylakoid membrane. When the RuBP obtained from Sigma Chemical Co. was used, light did not reactivate the enzyme. There was no direct correlation between ΔpH and activation. Therefore, thylakoids are required in the ribulose-1,5-bisphosphate carboxylase activase system largely to synthesize ATP. Inactivation of RuBP carboxylase in isolated chloroplasts or in the lysed chloroplast system was not promoted simply by a transition from light to dark conditions but was caused by low CO 2 and Mg 2+. 相似文献
13.
A procedure for the facile measurement, in intact cells, of two enzymes unique to the Calvin cycle, ribulose 1,5-bisphosphate carboxylase and phosphoribulokinase, is described. The procedure involved a simple toluene treatment to render phototrophic cells permeable to the necessary substrates, effectors, and cofactors. Whole-cell ribulose 1,5-bisphosphate carboxylase activity quantitatively approximates the activity obtained in cell-free extracts. In addition, the activity measured with toluene-treated whole cells results in a stoichiometric carboxylation of ribulose 1,5-bisphosphate to phosphoglyceric acid. The assay procedures described are most convenient for determining enzyme levels as a function of growth. Moreover, such an assay should open the way to further studies on the regulation of CO 2 assimilation by direct measurement of the enzymes concerned within the cell. 相似文献
14.
Isolated wheat chloroplasts were pre-incubated in the dark inthe presence of various concentrations of inorganic phosphatewith or without carbon dioxide, oxaloacetate, glycerate, and3-phosphoglycerate. The effect of subsequent illumination onphotosynthetic oxygen evolution, ribulose bisphosphate carboxylaseactivity, ATP content, and ribulose bisphosphate content wasinvestigated. Inorganic phosphate had little effect on ribulosebisphosphate carboxylase activity in darkness or during theinitial phase of illumination, but it prevented the declinein activity that occurred during later stages of illumination,when photoreduction of CO 2 was decreasing in rate. Additionof inorganic phosphate to chloroplasts illuminated without phosphaterestored the ribulose bisphosphate carboxylase activity, increasedthe ATP, and decreased the ribulose bisphosphate in the organelles.The responses to CO 2, oxaloacetate, glycerate, and 3-phosphoglyceratesuggest that the decreased activity of ribulose bisphosphatecarboxylase during photosynthesis results from ATP consumption. Purified ribulose bisphosphate carboxylase was activated byinorganic phosphate, but this activation did not occur in thepresence of ATP. ATP inhibited ribulose bisphosphate carboxylasewhen it was present in combination with various photosyntheticmetabolites. Inactivation of ribulose bisphosphate carboxylase in chloroplasts,illuminated in the absence of inorganic phosphate, is not dueto lack of activation by inorganic phosphate or ATP. It mayresult from decreased stromal pH. Key words: Ribulose bisphosphate carboxylase, Chloroplasts, Wheat, Phosphate, ATP 相似文献
15.
A system has been developed for the study of photosynthetic CO 2 fixation by isolated spinach chloroplasts at air levels of CO 2. Rates of CO 2 fixation were typically 20 to 60 micromoles/milligrams chlorophyll per hour. The rate of fixation was linear for 10 minutes but then declined to less than 10% of the initial value by 40 minutes. Ribulose 1,5-bisphosphate (RuBP) levels remained unchanged during this period, indicating that they were not the cause for the decline. The initial activity of the RuBP carboxylase in the chloroplast was high for 8 to 10 minutes and then declined similar to the rate of CO 2 fixation, suggesting that the decline in CO 2 fixation may have been caused by deactivation of the enzyme. 相似文献
16.
Crystalline ribulose 1,5-bisphosphate carboxylase-oxygenase (EC 4.1.1.39) was isolated from tobacco ( Nicotiana tabacum L.) leaf homogenates and the two competing reactions were examined for differential regulation in vitro by temperature pretreatment and chloroplast metabolites. Both the carboxylase and oxygenase activities were inactivated 50% by storing the dissolved protein at 0 °C and fully reactivated by heating the solution at 50 °C in the absence of Mg 2+ and a sulfhydryl reagent. When the heat-activated enzyme was preincubated with physiological levels of various chloroplast metabolites and CO 2 and the two reactions were assayed simultaneously in the same reaction vessel upon initiation with ribulose 1,5-bisphosphate, three classes of effectors were observed: (a) those which stimulated both activities (NADPH, 6-phosphogluco-bisphosphate gluconate, fructose 1,6-bisphosphate, 3-phosphoglycerate glycerate), (b) those which essentially had no effect (fructose 6-phosphate, glucose 6-phosphate), and (c) one, ribose 5-phosphate, which inhibited the two reactions. However, within the limits of experimental error, none of the metabolites examined produced a differential regulation of the ribulose 1,5-bisphosphate carboxylase-oxygenase activities. The similar response of the two competing activities to temperature pretreatment and various chloroplast metabolites is consistent with the notion that both reactions are associated with the same or adjacent catalytic sites on this bifunctional enzyme. 相似文献
17.
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
18.
Partially purified intact chloroplasts were prepared from batch cultures of both wild type ( Wt) and a mutant strain of Chlamydomonas reinhardtii. Protoplasts were generated from log phase cultures of Wt (137c) and the phosphoribulokinase-deficient mutant F60 by incubation of the cells in autolysine. These protoplasts were suspended in an osmoticum, cooled, and then subjected to a 40 pounds per square inch pressure shock using a Yeda pressure bomb. The resulting preparation was fractionated on a Percoll step gradient which separated the intact chloroplasts from both broken chloroplasts and protoplasts. The chloroplast preparation was not significantly contaminated with the cytoplasmic enzyme activity phosphoenolpyruvate carboxylase (>5%), and contained (100%) stromal enzyme activity ribulose-1,5-bisphosphate carboxylase. The chloroplast preparation is significantly contaminated by mitochondria, as determined by succinate dehydrogenase activity. Chloroplasts prepared from Wt cells retained CO2-dependent O2 photoevolution at rates in excess of 60 micromoles per milligram chlorophyll per hour, an activity which is severely inhibited by the addition of 10 millimolar KH2PO4. The chloroplasts are osmotically sensitive as determined by ferricyanide-dependent O2 photoevolution. 相似文献
19.
The activity of ribulose 1,5-bisphosphate (RuBP) car?ylase in intact spinach chloroplasts is shown to depend on light and CO 2. This activity was measured upon lysis of chloroplasts and assay of the initial activity using nonlimiting substrate concentrations. Incubation of chloroplasts at 25 °C in the absence of CO 2 results in a gradual inactivation of the RuBP car?ylase. In the presence of CO 2 the initial activity is preserved or increased. CO 2 is also able to reactivate the chloroplast car?ylase previously inactivated in the absence of CO 2. Upon illumination of the chloroplasts, additional activation was observed. This light activation results from an increased affinity for CO 2 of the chloroplast car?ylase. At pH 7.8, the enzyme in dark-adapted chloroplasts required 112 μ m CO 2 for half activation, while in the light it required 24 μ m CO 2. The light activation was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, carbonylcyanide 3-chlorophenylhydrazone, or dl-glyceraldehyde. Part of the light activation is most likely due to increased Mg 2+ in the stroma. dl-Glyceraldehyde inhibition also suggests that some intermediate of the photosynthetic carbon cycle is involved. These results suggest that photosynthetic CO 2 assimilation in the chloroplast depends upon the amount of activation of the RuBP car?ylase. This activation is regulated by CO 2 and light-induced changes in the chloroplast stroma such as pH, Mg 2+, and intermediates of the photosynthetic carbon cycle. 相似文献
20.
Special culture conditions for Euglena gracilis Z and ZR are described. They induce interactions between the chloroplast and mitochondrial metabolisms leading to paramylon synthesis. When grown in continuous light under pure nitrogen and in the presence of lactate as the sole carbon source, sugar synthesis occurs during the first 24 h of culture with the participation of both mitochondria (using lactate) and of chloroplasts (fixing CO 2 from lactate decarboxylation). The activities of ribulose bisphosphate carboxylase, phosphoenolpyruvate carboxylase, and phosphoenolpyruvate carboxykinase are very high and mitochondria and chloroplasts develop then a common network of vesicles in which paramylon grains can be seen. Electron micrographs demonstrate membrane continuity between the two types of organelles. Occasionally the mitochondrial matrix and the chloroplast stroma are separated by only a unit membrane.Abbreviations Chl
chlorophyll
- OAA
oxaloacetic acid
- PEP
phosphoenolpyruvate
- RuBP
ribulose bisphosphate
- DTT
1,4-dithiothreitol
- PVP
polyvinylpyrrolidone 相似文献
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