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1.
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
2.
We did this work to discover the pathway of CO 2 fixation into sugars in the dark during gluconeogenesis by the cotyledons of 5-day-old seedlings of Cucurbita pepo L. We paid particular attention to the possibility of a contribution from ribulosebisphosphate carboxylase. The detailed distribution of 14C after exposure of excised cotyledons to 14CO 2 in the dark was determined in a series of pulse and chase experiments. After 4s in 14CO 2, 89% of the 14C fixed was in malate and aspartate. In longer exposures, and in chases in 12CO 2, label appeared in alanine, phosphoenolpyruvate, 3-phosphoglycerate and sugar phosphates, and accumulated in sugars. The transfer of label from C-4 acids to sugars was restricted by inhibition of phosphoenolpyruvate carboxykinase in vivo by 3-mercaptopicolinic acid. We conclude as follows. Initial fixation of CO 2 in the dark is almost entirely into phosphoenolpyruvate, probably via phosphoenolpyruvate carboxylase (EC 4.1.1.31) which we showed to be present in appreciable amounts. Incorporation into sugars occurs chiefly, if not completely, as a result of randomization of the carboxyl groups of the C-4 acids and subsequent conversion of the oxaloacetate to sugars via the accepted sequence for gluconeogenesis. Ribulosebisphosphate carboxylase appears to make very little contribution to sugar synthesis from fat. 相似文献
3.
Barley, Panicum milioides and Panicum maximum were exposed to 14CO 2 near their photosynthetic CO 2 compensation points and their respective 14C-products were determined. In short exposure times Panicum maximum had 100% of its 14C in malate and aspartate whereas Panicum milioides and barley had 16 and 3% of their respective 14C in C 4 organic acids. Near the respective CO 2 compensation points a linear relationship occurs in plotting the ratio of glycine, serine, and glycerate to C 4 organic acids. The ratio of ribulose 1,5-bisphosphate oxygenase to phosphoenolpyruvate carboxylase is linear with their CO 2 compensation points. The photosynthetic CO 2 compensation point apparently is controlled by the activity of enzymes producing photorespiration metabolites and the activity of phospheonolpyruvate carboxylase. 相似文献
4.
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
5.
The photosynthetic assimilation of 14CO 2 has been studied in healthy and mildew-infected barley. The parasite was separated from the host by removing the mycelium with a camel's hair brush. The ethanol soluble metabolites of the parasite, infected host and healthy host were extracted, separated by paper chromatography and individually identified. From this work it appears that there is a rapid movement of label from host to parasite mainly in the form of sucrose which is then quickly metabolized into many compounds. The majority is converted into mannitol, and lesser amounts are converted into trehalose, arabitol, aspartic acid, and glutamic acid. In conidia the major carbon reserve is arabitol instead of mannitol, with lesser amounts of trehalose and mannitol. Photosynthetic uptake of 14CO2 by the complex decreases steadily after inoculation as compared with healthy leaves. However, the ethanol soluble metabolites of the infected host tissue differ only slightly from those of healthy host tissue. The major differences are a slight decrease in the amount of sucrose and increases in malic acid and serine. 相似文献
6.
The pathway of autotrophic CO 2 fixation was studied in the phototrophic bacterium Chloroflexus aurantiacus and in the aerobic thermoacidophilic archaeon Metallosphaera sedula. In both organisms, none of the key enzymes of the reductive pentose phosphate cycle, the reductive citric acid cycle, and the reductive acetyl coenzyme A (acetyl-CoA) pathway were detectable. However, cells contained the biotin-dependent acetyl-CoA carboxylase and propionyl-CoA carboxylase as well as phosphoenolpyruvate carboxylase. The specific enzyme activities of the carboxylases were high enough to explain the autotrophic growth rate via the 3-hydroxypropionate cycle. Extracts catalyzed the CO 2-, MgATP-, and NADPH-dependent conversion of acetyl-CoA to 3-hydroxypropionate via malonyl-CoA and the conversion of this intermediate to succinate via propionyl-CoA. The labelled intermediates were detected in vitro with either 14CO 2 or [ 14C]acetyl-CoA as precursor. These reactions are part of the 3-hydroxypropionate cycle, the autotrophic pathway proposed for C. aurantiacus. The investigation was extended to the autotrophic archaea Sulfolobus metallicus and Acidianus infernus, which showed acetyl-CoA and propionyl-CoA carboxylase activities in extracts of autotrophically grown cells. Acetyl-CoA carboxylase activity is unexpected in archaea since they do not contain fatty acids in their membranes. These aerobic archaea, as well as C. aurantiacus, were screened for biotin-containing proteins by the avidin-peroxidase test. They contained large amounts of a small biotin-carrying protein, which is most likely part of the acetyl-CoA and propionyl-CoA carboxylases. Other archaea reported to use one of the other known autotrophic pathways lacked such small biotin-containing proteins. These findings suggest that the aerobic autotrophic archaea M. sedula, S. metallicus, and A. infernus use a yet-to-be-defined 3-hydroxypropionate cycle for their autotrophic growth. Acetyl-CoA carboxylase and propionyl-CoA carboxylase are proposed to be the main CO 2 fixation enzymes, and phosphoenolpyruvate carboxylase may have an anaplerotic function. The results also provide further support for the occurrence of the 3-hydroxypropionate cycle in C. aurantiacus. 相似文献
7.
The degree of C 4 photosynthesis was assessed in four hybrids among C 4, C 4-like, and C 3-C 4 species in the genus Flaveria using 14C labeling, CO 2 exchange, 13C discrimination, and C 4 enzyme activities. The hybrids incorporated from 57 to 88% of the 14C assimilated in a 10-s exposure into C 4 acids compared with 26% for the C 3-C 4 species Flaveria linearis, 91% for the C 4 species Flaveria trinervia, and 87% for the C 4-like Flaveria brownii. Those plants with high percentages of 14C initially fixed into C 4 acids also metabolized the C 4 acids quickly, and the percentage of 14C in 3-phosphoglyceric acid plus sugar phosphates increased for at least a 30-s exposure to 12CO 2. This indicated a high degree of coordination between the carbon accumulation and reduction phases of the C 4 and C 3 cycles. Synthesis and metabolism of C 4 acids by the species and their hybrids were highly and linearly correlated with discrimination against 13C. The relationship of 13C discrimination or 14C metabolism to O 2 inhibition of photosynthesis was curvilinear, changing more rapidly at C 4-like values of 14C metabolism and 13C discrimination. Incorporation of initial 14C into C 4 acids showed a biphasic increase with increased activities of phosphoe nolpyruvate carboxylase and NADP-malic enzyme (steep at low activities), but turnover of C 4 acids was linearly related to NADP-malic enzyme activity. Several other traits were closely related to the in vitro activity of NADP-malic enzyme but not phosphoe nolpyruvate carboxylase. The data indicate that the hybrids have variable degrees of C 4 photosynthesis but that the carbon accumulation and reduction portions of the C 4 and C 3 cycles are well coordinated. 相似文献
8.
A new method is presented for measurement of the CO 2/O 2 specificity factor of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The [ 14C]3-phosphoglycerate (PGA) from the Rubisco carboxylase reaction and its dilution by the Rubisco oxygenase reaction was monitored by directly measuring the specific radioactivity of PGA. 14CO 2 fixation with Rubisco occurred under two reaction conditions: carboxylase with oxygenase with 40 micromolar CO 2 in O 2-saturated water and carboxylase only with 160 micromolar CO 2 under N 2. Detection of the specific radioactivity used the amount of PGA as obtained from the peak area, which was determined by pulsed amperometry following separation by high-performance anion exchange chromatography and the radioactive counts of the [ 14C]PGA in the same peak. The specificity factor of Rubisco from spinach ( Spinacia oleracea L.) (93 ± 4), from the green alga Chlamydomonas reinhardtii (66 ± 1), and from the photosynthetic bacterium Rhodospirillum rubrum (13) were comparable with the published values measured by different methods. 相似文献
9.
Ribulose-l,5-bisphosphate carboxylase (E.C. 4.1.1.39) isolated from Chromatium strain D contains 64 free cysteinyl -SH groups per mol ( Mr 5.11 × 10 5) as determined using three different titrants: p-[ 14C]chloromercuribenzoate, the Ellman reagent, and [ 14C]iodoacetamide.Distribution of -SH groups in the two constituent subunits (A and B) isolated from spinach and Chromatium ribulose-1,5-bisphosphate carboxylases was determined to be for spinach, 9 in A and 3 in B; and for Chromatium, 7 in A and 1 in B.The relationship between the numbers of -SH groups blocked vs residual activities of both the ribulose-1,5-bisphosphate carboxylase and oxygenase reactions was examined by titration with p-chloromercuribenzoate. In both spinach and Chromatium enzymes, antisigmoidal curves were obtained for the degree of the enzyme activity loss in relation to the numbers of -SH groups masked. However, at alkaline pH the Chromatium enzyme shows a sharp decline in both carboxylase and oxygenase activities, apparently due to the alkali dissociation of the enzyme molecule accompanied by its structural deformation. The functional role of -SH groups in the ribulose-1,5-bisphosphate carboxylase molecule is discussed in relation to two constituent enzyme reactions, and it is concluded that in both enzyme sources the active sites are probably the same for the two reactions. 相似文献
10.
After a 5-second exposure of illuminated bermudagrass ( Cynodon dactylon L. var. `Coastal') leaves to 14CO 2, 84% of the incorporated 14C was recovered as aspartate and malate. After transfer from 14CO 2-air to 12CO 2-air under continuous illumination, total radioactivity decreased in aspartate, increased in 3-phosphoglyceric acid and alanine, and remained relatively constant in malate. Carbon atom 1 of alanine was labeled predominantly, which was interpreted to indicate that alanine was derived from 3-phosphoglyceric acid. The activity of phosphoenolpyruvate carboxylase, alkaline pyrophosphatase, adenylate kinase, pyruvate-phosphate dikinase, and malic enzyme in bermudagrass leaf extracts was distinctly higher than those in fescue ( Festuca arundinacea Schreb.), a reductive pentose phosphate cycle plant. Assays of malic enzyme activity indicated that the decarboxylation of malate was favored. Both malic enzyme and NADP +-specific malic dehydrogenase activity were low in bermudagrass compared to sugarcane ( Saccharum officinarum L.). The activities of NAD +-specific malic dehydrogenase and acidic pyrophosphatase in leaf extracts were similar among the plant species examined, irrespective of the predominant cycle of photosynthesis. Ribulose-1, 5-diphosphate carboxylase in C 4-dicarboxylic acid cycle plant leaf extracts was about 60%, on a chlorophyll basis, of that in reductive pentose phosphate cycle plants. 相似文献
11.
Parthenocissus quinquefolia (L.) Planch., commonly known as Virginia Creeper, is a vitaceous tartrate-accumulating vine that exhibits C-4/C-5 cleavage of l-ascorbic acid (AA) to produce l-tartaric acid (TA) from the C 4 fragment and carbohydrate pool material from the C 2 fragment. Experiments in which detached leaves were supplied d-[5- 3H,1- 14C]glucose or d-[5- 3H,6- 14C]glucose yielded AA devoid of 3H whereas the l-threonic acid (ThA) and TA recovered from the same tissues still retained some 3H. These comparative experiments also indicated that the ThA was derived from carbons 3 through 6 of d-glucose. ThA was shown to be a natural constituent of P. quinquefolia but apparently not an intermediate between AA and TA. Results are consistent with a biosynthetic pathway from d-glucose to AA that involves a hydrogen-exchanging epimerization at C-5 as reported earlier for the geraniaceous plant Pelargonium crispum, but differing from P.crispum in biosynthesis and metabolism of ThA.When l-[6- 14C]idonate or its lactone was supplied to P. quinquefolia leaves, about 80% of the 14C appeared in the carbohydrates, an observation remarkably similar to previous observations with [6- 14C]AA-labeled leaves. l-Idonate and its lactone appear to have an intermediate role in AA metabolism in vitaceous plants. 相似文献
12.
Abstract: CO 2 fixation was measured in cultured astrocytes isolated from neonatal rat brain to test the hypothesis that the activity of pyruvate carboxylase influences the rate of de novo glutamate and glutamine synthesis in astrocytes. Astrocytes were incubated with 14CO 2 and the incorporation of 14C into medium or cell extract products was determined. After chromatographic separation of 14C-labelled products, the fractions of 14C cycled back to pyruvate, incorporated into citric acid cycle intermediates, and converted to the amino acids glutamate and glutamine were determined as a function of increasing pyruvate carboxylase flux. The consequences of increasing pyruvate, bicarbonate, and ammonia were investigated. Increasing extracellular pyruvate from 0 to 5 m M increased pyruvate carboxylase flux as observed by increases in the 14C incorporated into pyruvate and citric acid cycle intermediates, but incorporation into glutamate and glutamine, although relatively high at low pyruvate levels, did not increase as pyruvate carboxylase flux increased. Increasing added bicarbonate from 15 to 25 m M almost doubled CO 2 fixation. When 25 m M bicarbonate plus 0.5 m M pyruvate increased pyruvate carboxylase flux to approximately the same extent as 15 m M bicarbonate plus 5 m M pyruvate, the rate of appearance of [ 14C]glutamate and glutamine was higher with the lower level of pyruvate. The conclusion was drawn that, in addition to stimulating pyruvate carboxylase, added pyruvate (but not added bicarbonate) increases alanine aminotransferase flux in the direction of glutamate utilization, thereby decreasing glutamate as pyruvate + glutamate →α-ketoglutarate + alanine. In contrast to previous in vivo studies, the addition of ammonia (0.1 and 5 m M) had no effect on net 14CO 2 fixation, but did alter the distribution of 14C-labelled products by decreasing glutamate and increasing glutamine. Rather unexpectedly, ammonia did not increase the sum of glutamate plus glutamine (mass amounts or 14C incorporation). Low rates of conversion of α-[ 14C]ketoglutarate to [ 14C]glutamate, even in the presence of excess added ammonia, suggested that reductive amination of α-ketoglutarate is inactive under conditions studied in these cultured astrocytes. We conclude that pyruvate carboxylase is required for de novo synthesis of glutamate plus glutamine, but that conversion of α-ketoglutarate to glutamate may frequently be the rate-limiting step in this process of glutamate synthesis. 相似文献
13.
Flower buds of `Valencia' orange ( Citrus sinensis [L.] Osbeck) were able to fix 14CO 2 into a number of compounds in their own tissues under both light and dark conditions. The total incorporation, however, was about 4-fold higher in the light than in the dark. In the light, 50% of the total 14C label was found in the neutral fraction (sugars), 22% in the basic fraction (amino acids), and 26% in the acid-1 fraction (organic acids). In the dark, about 95% of the 14C label was incorporated into the basic and acid-1 fractions. Activities of ribulose bisphosphate carboxylase and phosphoenolpyruvate carboxylase (expressed in micromoles CO 2 per milligram protein per hour) averaged 1.95 and 8.87 for the flower buds, and 28.5 and 3.6 for the leaves, respectively. The ability of orange flower buds to fix ambient CO 2 into different compounds suggests that this CO 2 assimilation may have some regulatory role during the early reproductive stages in determining citrus fruit initiation and setting. 相似文献
14.
—Previously published equations for analysis of [ 14C]glucose metabolism assumed that products of glycolysis enter the citric acid cycle only through acetyl-CoA (Larrabee , 1978). These equations are now extended to include entrance into the citric acid cycle through the pyruvate carboxy-lase reaction as well as via acetyl-CoA and are applied to previously reported data from dorsal root ganglia of 15-day-old chicken embryos. The rate of output of labelled CO 2 in the presence of [2- 14C] glucose could not be accounted for if the flux rate into the citric acid cycle through the pyruvate carboxylase reaction was assumed to be more than about 10–15% of that through acetyl-CoA. It is concluded (1) that the pyruvate carboxylase reaction is a relatively minor source of material for the citric acid cycle in these ganglia and (2) that the previous conclusions about [ 14C]glucose metabolism, which ignored the pyruvate carboxylase reaction, need not be modified in the light of this reanalysis. 相似文献
15.
Using a tracer technique, the nutritional relationship between Striga senegalensis and its host ( Sorghum vulgare) has been studied. Preliminary trials with aqueous eosin dye indicated that a mechanism exists for the passage of watery solutions from host to parasite but not vice versa. Use of tracers like C 14O 2, urea-C 14 and sulphur-35 confirmed that minerals as well as organic compounds are translocated from host to parasite. However, when these tracers were applied to the parasites, translocation of labelled products into the host was insignificant. When C 14O 2 was used to label the photosynthate in Sorghum, the bulk of activity appeared in sucrose, glucose and fructose, part of which was presumably obtained by the parasite. The photosynthetic ability of the green tissues of Striga was confirmed. Thus the damaging effect of Striga on the host appears to be due to removal of considerable quantities of water, minerals and organic compounds from the latter. The pattern of translocation of photosynthates and minerals from host to parasite suggests a possible application of selective, systemic weed-killers by aerial spray on the host leaves for control of the parasite. 相似文献
16.
13C/ 12C ratios have been found in totally combusted leaves of Crassulacean acid metabolism plants to range from −14 to −33 δ 13C‰ compared with a limestone standard. Crassulacean acid metabolism plants apparently utilize both ribulose-1, 5-diphosphate carboxylase and phosphoenolpyruvate carboxylase to assimilate atmospheric CO 2 and, depending on environmental conditions, have 13C/ 12C ratios indicative of either carboxylase or to any intermediate value. The degree of discrimination against 13C and the resultant 13C/ 12C ratio from the photosynthetically fixed CO 2 is influenced by environmental conditions and is not a specific and fixed characteristic of a Crassulacean acid metabolism plant. Certain Crassulacean acid metabolism plants may shift their ratios as much as 17 δ 13C‰ in specific environments. 相似文献
17.
Two dual label methods were used to investigate kinetic variability of ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (EC 4.1.1.39). In addition to using [1- 14C,5- 3H]RuBP (method 1), we describe here the detailed assay with 14CO 2 and [5- 3H]RuBP (method 2), which generates [ 3H, 14C]3-phosphoglyceric acid and unlabeled (noncontaminating) phosphoglycolate; the carboxylase/oxygenase activity ratio ( vc/vo) is calculated from 3H/ 14C ratios of substrates and products. vc/vo was found to be a linear function of [CO 2]/[O 2], constant over a 4-minute assay interval, and invariant of the degree of enzyme activity. Accurately measurable vc/vo ratios range from approximately 0.3 to 6. The Km and Vmax of both enzymes may be determined as a composite constant, VcKo/VoKc. By method 2, the directly compared, relative values at 40 micromolar CO 2 and 1240 micromolar O 2 were: Spinacia oleracea (74), Chlorella pyrenoidosa (31), Plectonema boryanum (32), and Rhodospirillum rubrum (8). With method 1, the values for S. oleracea and R. rubrum were 75, and 9, respectively. Under tight experimental controls, the absolute value for S. oleracea was 69 ± 3. 相似文献
18.
The role of phosphoenolpyruvate carboxylase in photosynthesis in the C 3 plant Nicotiana tabacum has been probed by measurement of the 13C content of various materials. Whole leaf and purified ribulose bisphosphate carboxylase are within the range expected for C 3 plants. Aspartic acid purified following acid hydrolysis of this ribulose bisphosphate carboxylase is enriched in 13C compared to whole protein. Carbons 1-3 of this aspartic acid are in the normal C 3 range, but carbon-4 (obtained by treatment of the aspartic acid with aspartate β-decarboxylase) has an isotopic composition in the range expected for products of C 4 photosynthesis (−5‰), and it appears that more than half of the aspartic acid is synthesized by phosphoenolpyruvate carboxylase using atmospheric CO 2/HCO 3−. Thus, a primary role of phosphoenolpyruvate carboxylase in C 3 plants appears to be the anapleurotic synthesis of four-carbon acids. 相似文献
19.
Photosynthetic carboxylating enzymes and the effects of light and temperature on 14CO 2 efflux in two species of gymnosperm leaves were studied. The activity of RuBP carboxylase was high and that of PEP carboxylase was very low when compared to C 4 plants. The CO 2 compensation point was high. 14CO 2 efflux was greater in light than in darkness and the ratio (L/D) increased with increase in temperature and light intensity. The inhibitors of glycolate metabolism showed decreased 14CO 2 evolution in light while dark respiration was unaffected. It is concluded that both Cycas circinalis, L. and Cycas beddomei Dyer are C 3 plants. 相似文献
20.
The effect of O 2 and pH on the in vitro synthesis of 14C-labeled ureides from [8- 14C]hypoxanthine in a cell-free system from cowpea nodules was investigated. Under conditions which suppressed uricase (EC 1.7.3.3) activity, namely low O 2 concentrations and low pH, ureide synthesis was inhibited and the 14C label incorporated into uric acid was increased. Conversely, conditions which increased uricase activity, namely high O 2 concentrations and high pH, also stimulated ureide synthesis, and the 14C label was incorporated principally into allantoin. The overall response of the system to O 2 concentration and pH indicated that the per cent distribution of total 14C label incorporated into uric acid was inversely related to that into allantoin. In the present study there was evidence that uricase (EC 1.7.3.3) controlled the in vitro rate of ureide synthesis in the cell-free system. Adenine and guanine inhibited xanthine dehydrogenase (EC 1.2.1.37) and as a consequence ureide synthesis from [8- 14C]hypoxanthine was also inhibited. 相似文献
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