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1.
Partially purified ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) was isolated from diploid and tetraploid cultivars of ryegrass ( Lolium perenne L.) using two separate methods. The apparent Km (CO 2) values for the enzymes prepared by either method did not differ significantly between diploid and tetraploid when assayed by two separate techniques. The unpurified enzymes from freshly lysed (and fully functional) protoplasts of both diploid and tetraploid cultivars gave virtually identical apparent Km (CO 2) values. There was no indication of large differences in affinity for CO 2 of illuminated intact protoplasts from the two cultivars. 相似文献
2.
The exchange properties of the activator CO 2 of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase were characterized both in vitro with the purified enzyme, and in situ within isolated chloroplasts. Carboxyarabinitol-1,5-bisphosphate, a proposed reaction intermediate analog for the carboxylase activity of the enzyme, was used to trap the activator CO 2 on the enzyme both in vitro and in situ. Modulation of ribulose-1,5-bisphosphate carboxylase/oxygenase activity in intact chloroplasts during a light/dark cycle was associated with a similar modulation in carboxyarabinitol-1,5-bisphosphate-trapped CO 2. The exchange kinetics of the activator CO 2 were monitored by activation of the enzyme to steady state in the presence of 12CO 2, followed by addition of 14CO 2 and determination of the amount of labeled CO 2 trapped on the enzyme by carboxyarabinitol-1,5-bisphosphate. Rate constants ( Kobs) for exchange with both the purified enzyme (0.45 min −1) and in illuminated chloroplasts (0.18 min −1) were comparable to the observed rate constants for enzyme activation under the two conditions. A similar exchange of the activator CO 2 was not observed in chloroplasts in the dark. Kinetic analysis of the exchange properties of the purified enzyme were consistent with an equilibrium between active and inactive forms of the enzyme during steady state activation. 相似文献
3.
CO 2 fixation during photosynthesis is regulated by the activity of ribulose bisphosphate carboxylase (Rubisco). This conclusion
became more apparent to me after CO 2-fixation experiments using isolated spinach chloroplasts and protoplasts, purified Rubisco enzyme, and intact leaves. Ribulose
bisphosphate (RuBP) pools and activation of Rubisco were measured and compared to 14CO 2 fixation in light. The rates of 14CO 2 assimilation best followed the changes in Rubisco activation under moderate to high light intensities. RuBP pool sizes regulated
14
2 assimilation only in very high CO 2 levels, low light and in darkness. Activation of Rubisco involves two separate processes: carbamylation of the protein and
removal of inhibitors blocking carbamylation or blocking RuBP binding to carbamylated sites before reaction with CO 2 or O 2.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
4.
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
5.
A mass spectrometric method combining 16O/ 18O and 12C/ 13C isotopes was used to quantify the unidirectional fluxes of O 2 and CO 2 during a dark to light transition for guard cell protoplasts and mesophyll cell protoplasts of Commelina communis L. In darkness, O 2 uptake and CO 2 evolution were similar on a protein basis. Under light, guard cell protoplasts evolved O 2 (61 micromoles of O 2 per milligram of chlorophyll per hour) almost at the same rate as mesophyll cell protoplasts (73 micromoles of O 2 per milligram of chlorophyll per hour). However, carbon assimilation was totally different. In contrast with mesophyll cell protoplasts, guard cell protoplasts were able to fix CO 2 in darkness at a rate of 27 micromoles of CO 2 per milligram of chlorophyll per hour, which was increased by 50% in light. At the onset of light, a delay observed for guard cell protoplasts between O 2 evolution and CO 2 fixation and a time lag before the rate of saturation suggested a carbon metabolism based on phospho enolpyruvate carboxylase activity. Under light, CO 2 evolution by guard cell protoplasts was sharply decreased (37%), while O 2 uptake was slowly inhibited (14%). A control of mitochondrial activity by guard cell chloroplasts under light via redox equivalents and ATP transfer in the cytosol is discussed. From this study on protoplasts, we conclude that the energy produced at the chloroplast level under light is not totally used for CO 2 assimilation and may be dissipated for other purposes such as ion uptake. 相似文献
6.
Activities of ribulose-1,5-bisphosphate carboxylase and rates of photosynthetic O 2 evolution were measured in guard-cell and mesophyll protoplasts from Vicia faba. The ribulose-1,5-bisphosphate carboxylase activity of guard-cell protoplasts was 30% of that of mesophyll protoplasts; however, the O 2 evolution rate was 3 times higher in guard-cell protoplasts than in mesophyll protoplasts on a chlorophyll basis. When the dark-adapted, guard-cell protoplasts were illuminated by red light, O 2 was evolved with an induction period, which became shorter when the protoplasts were reilluminated. High activity of irreversible NADP-glyceraldehyde-3-phosphate dehyrogenase was found in guard-cell protoplasts. Several lines of evidence revealed that there was virtually no contamination by mesophyll cells in guard-cell preparations. These results indicate that guard cells fix CO 2 photosynthetically and imply that the cells utilize a considerable proportion of reducing equivalents from water for reactions other than CO 2 fixation. 相似文献
7.
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
8.
Photosynthesis was examined in leaves of Flaveria brownii A. M. Powell, grown under either 14% or 100% full sunlight. In leaves of high light grown plants, the CO 2 compensation point and the inhibition of photosynthesis by 21% O 2 were significantly lower, while activities of ribulose 1,5-bisphosphate carboxylase/oxygenase and various C 4 cycle enzymes were considerably higher than those in leaves grown in low light. Both the CO 2 compensation point and the degree of O 2 inhibition of apparent photosynthesis were relatively insensitive to the light intensity used during measurements with plants from either growth conditions. Partitioning of atmospheric CO 2 between Rubisco of the C 3 pathway and phosphoenolpyruvate carboxylase of the C 4 cycle was determined by exposing leaves to 14CO 2 for 3 to 16 seconds, and extrapolating the labeling curves of initial products to zero time. Results indicated that ~94% of the CO 2 was fixed by the C 4 cycle in high light grown plants, versus ~78% in low light grown plants. Thus, growth of F. brownii in high light increased the expressed level of C 4 photosynthesis. Consistent with the carbon partitioning patterns, photosynthetic enzyme activities (on a chlorophyll basis) in protoplasts from leaves of high light grown plants showed a more C 4-like pattern of compartmentation. Pyruvate, Pi dikinase and phosphoenolpyruvate carboxylase were more enriched in the mesophyll cells, while NADP-malic enzyme and ribulose 1,5-bisphosphate carboxylase/oxygenase were relatively more abundant in the bundle sheath cells of high light than of low light grown plants. Thus, these results indicate that F. brownii has plasticity in its utilization of different pathways of carbon assimilation, depending on the light conditions during growth. 相似文献
9.
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
10.
Photosynthetically active bundle sheath strands capable of assimilating up to 8 micromoles CO 2 per milligram chlorophyll per hour have been isolated from fully expanded leaves of Zea mays L. Mesophyll cell contamination of the preparations was negligible, as evidenced by light and electron microscopy and by a high ratio of chlorophyll a to chlorophyll b in the strands. Ribose 5-phosphate markedly stimulated the rate of photosynthetic 14CO 2 fixation by the isolated strands. In contrast, both pyruvate and phosphoenolpyruvate had a comparatively small stimulatory effect on bundle sheath 14CO 2 fixation. After 5 minutes of photosynthesis in 14C-bicarbonate, 95% of the incorporated 14C was found in compounds other than C 4-dicarboxylic acids, most notably in 3-phosphoglycerate and sugar phosphates. A similar distribution of 14C was observed in the presence of exogenous ribose 5-phosphate. Extracts of bundle sheath strands contained high specific activities of “malic” enzyme, phosphoglycolate phosphatase, hydroxypyruvate reductase, and ribulose 1,5-diphosphate carboxylase, whereas the specific activities of NADP +-malate dehydrogenase and phosphopyruvate carboxylase were extremely low. These results indicate that the Calvin cycle occurs in the bundle sheath cells of maize. 相似文献
11.
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
12.
The regulation of phosphoenolpyruvate carboxylase (PEPCase, EC. 4.1.1.31) and PEPCase kinase was investigated using barley ( Hordeum vulgare L.) mesophyll protoplasts. Incubation of protoplasts in the light resulted in a reduction in the sensitivity of PEPCase to the inhibitor L-malate; PEPCase from protoplasts incubated in the light for 1 h was inhibited 48±2% by 2mM malate, whereas the enzyme from protoplasts incubated for 1 h in the dark was inhibited by 67±2%. Light-induced reduction of sensitivity of PEPCase to malate was decreased by cycloheximide (CHM), indicating the involvement of protein synthesis. The PEPCase kinase in protoplasts increased with time after isolation in darkness, and increased still further following light treatment. The increase in kinase activity in the light was sensitive to CHM. When protoplasts were illuminated in the presence of EGTA and the calcium ionophore A23187 to reduce intracellular Ca 2+, the reduction in the senstivity of PEPCase to malate was enhanced, though no more PEPCase kinase activity was detected than in protoplasts illuminated in the absence of EGTA and A23187. Incubation with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) had no effect on the light-induced reduction of sensitivity of PEPCase to malate inhibition or on light-activation of PEPCase kinase. These results indicate that there is a constitutive PEPCase kinase activity in C 3 leaf tissue, that there is another kinase which is light-activated in a CHMsensitive way, that the sensitivity of PEPCase to its inhibitor may not always be correlated with apparent PEPCase kinase actvity, and that PEPCase and PEPCase kinase are regulated in a different manner in C 3 protoplasts than in C 4 protoplasts or leaf tissue.Abbreviations CAM
Crassulacean acid metabolism
- Chl
chlorophyll
- CHM
cycloheximide
- DCMU
3-(3,4-dichloro-phenyl)-1,1-dimethylurea
- PEP
phosphoenolpyruvate
- PEPCase
PEP carboxylase 相似文献
13.
Photosynthetic CO 2-fixation in isolated pea ( Pisum sativum L., cv Little Marvel) chloroplasts during induction is markedly inhibited by 0.4 millimolar sulfite. Sulfate at the same concentration has almost no effect. The 14CO 2-fixation pattern indicates that the primary effect of sulfite is inhibition of the reaction catalyzed by ribulose bisphosphate carboxylase and a stimulation of export of intermediates out of the chloroplasts. Inhibition of light modulation of stromal enzyme activity does not appear to account for the toxicity of SO 2 in this Pisum variety. Arsenite at 0.2 millimolar concentrations inhibits light activation and inhibits photosynthetic CO 2 fixation. The 14CO 2-fixation pattern indicates that the primary effect of arsenite is inhibition of light activation of reductive pentose phosphate pathway enzyme activity. 相似文献
14.
Rates of carbon fluxes and pool sizes of photosynthetic metabolites in different cellular compartments of barley protoplasts were calculated from the time curves of their labeling in the medium of 14CO 2. Using membrane filtration procedure, kinetics of 14C incorporation into the products of steady-state photosynthesis was determined separately in chloroplasts, mitochondria and cytosol of barley protoplasts illuminated for different periods in the air containing 14CO 2. To extract the quantitative information, analytical labeling functions P( t) describing the dependence of 14C content in the primary, intermediate and end products of a linear reaction chain upon the duration of tracer feeding have been derived. The parameters of these functions represent pool sizes of metabolites and rates of carbon fluxes. The values of these parameters were determined by fitting the experimental labeling curves to the functions P( t) by means of non-linear regression procedure. To elucidate the possible effects of fractionation on the photosynthetic carbon metabolism, the parameters of protoplasts were compared with corresponding values in intact leaves of barley. 相似文献
15.
A procedure is described for isolating and purifying mesophyll protoplasts and bundle sheath protoplasts of the C 4 plant Panicum miliaceum. Following enzymic digestion of leaf tissue, mesophyll protoplasts and bundle sheath protoplasts are released and purified by density centrifugation. The lower density of mesophyll protoplasts allowed rapid separation of the two protoplast types. Evidence for separation of mesophyll protoplasts and bundle sheath protoplasts (up to 95% purity) is provided from light microscopy (based on size difference in both chloroplasts and protoplasts), levels of marker enzymes in the preparations ( i.e. pyruvate, Pi dikinase and phosphoenolpyruvate carboxylase for mesophyll and ribulose-1,5-bisphosphate carboxylase for bundle sheath), and differences in substrate-dependent O 2 evolution by chloroplasts isolated from protoplasts. 相似文献
16.
The nature and sequence of metabolic events during phase II (early morning) Crassulacean acid metabolism in Opuntia erinacea var columbiana (Griffiths) L. Benson were characterized. Gas exchange measurements under 2 and 21% O 2 revealed increased O 2 inhibition of CO 2 fixation with progression of phase II. Malate and titratable acidity patterns indicated continued synthesis of C 4 acids for at least 30 minutes into the light period. Potential activities of phosphoenolpyruvate carboxylase (PEPC) and NADP-malic enzyme exhibited little change during phase II, while light activation of NADP-malate dehydrogenase, pyruvate, orthophosphate dikinase, and ribulose-1,5-bisphosphate carboxylase was apparent. Short-term 14CO 2 fixation experiments showed that the per cent of 14C incorporated into C 4 acids decreased while incorporation into other metabolites increased with time. PEPC exhibited increased sensitivity to 2 millimolar malate, and the Ki(malate) for PEPC decreased markedly with time. Sensitivity of PEPC to malate inhibition was considerably greater at pH 7.5 than at 8.0. The results indicate that decarboxylation and synthesis of malate occur simultaneously during the early morning period, and that phase II acid metabolism is not limited by CO 2 diffusion through stomata. With progression of phase II, CO 2 fixation by PEPC decreases while fixation by ribulose-1,5-bisphosphate carboxylase increases. 相似文献
17.
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
18.
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
19.
Increased photosynthetic CO 2 assimilation by Chlamydomonas reinhardtii cells treated with triacontanol (TRIA) was not due to changes in glycolate excretion, CO 2 compensation point, or the sensitivity of photosynthetic CO 2 assimilation to O 2. Kinetic analysis of TRIA-treated cells showed that the increase in photosynthetic CO 2 assimilation was a result of an increase in the apparent Vmax for intact cells. The total activity of ribulose-P 2 carboxylase/oxygenase was higher in cell lysates from TRIA-treated cells. However quantification of this enzyme concentration by binding of [ 14C]carboxyarabinitol-P 2 did not show an increase in TRIA-treated cells. Thus, there was an increase in the specific activity of ribulose-P 2 carboxylase/oxygenase extracted from Chlamydomonas cells treated with TRIA. TRIA alone had no effect on the activity of the enzyme in cell lysates from Chlamydomonas or purified from spinach ( Spinacia oleracea L.) leaves. The ribulose-P2 pool was 50 to 60% higher in cells treated with TRIA that were assayed for photosynthetic CO2 assimilation at high- and low-CO2. TRIA also increased ribulose-P2 levels in the absence of CO2 in the light with atmospheres of N2 or N2 with 21% O2. 相似文献
20.
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
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