水分胁迫对露花叶片中PEP羧化酶活性及其调节特性的影响

水分胁迫期间露花叶片中PEP羧化酶的活力随胁迫时间的延长明显增加,复水后PEPC同工酶的活力下降。从露花叶片中分离到3个具有不同动力学和物理学特性的PEPC同工酶(PCⅠ,PCⅡ,PCⅢ),其中同工酶PCⅠ只存在于水分胁迫下露花叶片中,复水后消失。 这3个同工酶的K_m(PEP)值不相同;PCⅠ的K_m(PEP)值介于PCⅡ与PCⅢ之间,它在PAGE上的相对迁移率(Rm)比PCⅡ和PCⅢ大,对效应剂G—6—P及Mal的反应不敏感,分子量为PCⅡ之半;PCⅡ被G—6—P激活和被Mal抑制的程度介于PCⅠ与PCⅢ之间,它在PAGE上的相对迁移率和分子量与PCⅢ极相近。     

On the cellular localization of phosphoenolpyruvate carboxylase in Sorghum leaves

The localization of phosphoenol pyruvate carboxylase (EC 4.1.1.3.1.) in the leaf cells of Sorghum vulgare was investigated by using three techniques: the conventional aqueous and non aqueous methods gave conflicting results; the immunocytochemical techniques clearly showed that the enzyme is predominantly located in the cytoplasm of mesophyll cells.Abbreviations PEP phosphoenol pyruvate - PAG polyacrylamide gel - NADP MDH NADP malate dehydrogenase - FITC fluorescein isothiocyanate - SAB serum albumine bovine - DTT dithiothreitol - MDH malate dehydrogenase - ME malic enzyme - PBS phosphate buffer saline - PAP peroxidase anti-peroxidase     

植物磷酸烯醇式丙酮酸羧化酶的可逆胍变性

纯化的高梁叶片磷酸烯醇式丙酮酸羧化酶(PEP羧化酶)经不同浓度的盐酸胍处理变性失活后,在试验的蛋白浓度范围内,它的失活时间进程的动力学分析表明为一级反应。0.4 M盐酸胍处理25分钟后(O℃),酶的催化活性完全丧失,酶蛋白的远紫外圆二色性光谱、内源荧光光谱及免疫特异性等测定均表明酶的结构发生了深刻变化。甘油及PEP羧化酶的变构效应剂G6P和甘氨酸对酶在盐酸胍溶液中的变性作用有一定的保护效果。变性酶用复性缓冲液稀释20倍后,在最佳条件下,再经30分钟保温,酶的催化活性能恢复70％以上。G6P和甘氨酸能促进变性酶的复性,甘油亦有明显效果。随着酶活性的恢复,它的远紫外圆二色性、内源荧光及免疫特异性也随之恢复,变性酶的复性速率在常温下(25℃)比在低温下(0℃)要快得多。     

By-product formation during exposure of respiring Saccharomyces cerevisiae cultures to excess glucose is not caused by a limited capacity of pyruvate carboxylase

Upon exposure to excess glucose, respiring cultures of Saccharomyces cerevisiae produce substantial amounts of ethanol and acetate. A possible role of a limited anaplerotic capacity in this process was investigated by overexpressing pyruvate carboxylase and by replacing it with a heterologous enzyme (Escherichia coli phosphoenolpyruvate carboxylase). Compared to the wild-type, neither the pyruvate carboxylase (Pyc)-overexpressing nor the transgenic strain exhibited reduced by-product formation after glucose pulses to aerobic glucose-limited chemostat cultures. An increased intracellular malate concentration was observed in the two engineered strains. It is concluded that by-product formation in S. cerevisiae is not caused by a limited anaplerotic capacity.     

高粱叶片磷酸烯醇式丙酮酸羧化酶溶液构象状态的紫外差示吸收光谱研究

PEP诱导产生的差光谱在237nm是一强负峰,在252nm附近呈宽负峰。Mg~(2+)产生的差光谱在275nm附近为正的阔峰,在237nm处为一负峰。PEP、Mg~(2+)共同与酶作用的差光谱在263nm附近呈宽的负峰。正效应剂G6P、Gly及GG分别存在条件下PEP羧化酶的差光谱亦各具明显差异,在270nm以下光区内尤其显著。在284nm和291nm为两个负峰,Gly诱导的峰强度大于G6P的,而GG复合效应剂对此两峰的影响表现很大的协同作用。Mal作用于酶的差光谱在246nm处有一负峰。     

温度、pH及效应剂对高粱PEP羧化酶活性的协同作用

纯化的高粱PEP羧化酶活性随pH升高(pH6.6～8.0)而增大。在G6P和Mal存在下,酶活性仍有随pH升高而增大的趋势,但G6P对酶的激活百分率和Mal的抑制百分率随pH升高而降低。高粱PEP羧化酶活性的最适温度高于40℃、酶的催化效率(V_(max)/K_m)随温度升高而增大。高温下,反应激活能降低,Mal对酶活性抑制百分率亦随温度升高而下降,I_(0.5)值增大,Mal增大K_m(PEP)的效应变小。     

几种CAM植物PEP羧化酶同工酶的比较研究

比较研究几种兼性和专一性ＣＡＭ植物材料的ＰＥＰＣ同工酶表明：经自然干旱诱导,兼性ＣＡＭ植物露花（Ｍｅｓｅｍｂｒｙａｎｔｈｅｍｕｍｃｏｒｄｉｆｏｌｉｕｍ）、长药景天（Ｓｕｄｕｍｓｐｅｃｔａｂｉｌｅ）有新的ＰＥＰＣ同工酶的出现,诱导前后各同工酶的天然分子量变化不大;而土三七（Ｓｅｄｕｍａｉｚｏｏｎ）则没有新的ＰＥＰＣ同工酶出现,但诱导后其同工酶的天然分子量有所增大。以上几种兼性ＣＡＭ植物的ＰＥＰＣ同工酶酶谱无明显昼夜变化。专一性ＣＡＭ植物的ＰＥＰＣ酶谱和天然分子量均较一致,亦无昼夜差异。     

Influence of light intensity during growth on photosynthesis and activity of several key photosynthetic enzymes in a C4 plant (Zea mays)

Maize ( Zea mays L. Hybrid Sweet Corn, Royal Crest), a C₄ plant, was grown under different light regimes, after which the rate of photosynthesis and activities of several photosynthetic enzymes (per unit leaf chlorophyll) were measured at different light intensities. Plants were grown outdoors under direct sunlight or 23% of direct sunlight, and in growth chambers at photosynthetic photon flux densities of about 20% and 8% of direct sunlight. The plants grown under direct sunlight had a higher light compensation point than plants grown under lower light. At a light intensity about 25% of direct sunlight, plants from all growth regimes had a similar rate of photosynthesis. Under saturating levels of light the plants grown under direct sunlight had a substantially higher rate of photosynthesis than plants grown under the lower light regimes. The higher photosynthetic capacity in the plants grown under direct sunlight was accompanied by an increased activity of several photosynthetic enzymes and in the amount of the soluble protein in the leaf. Among five photosynthetic enzymes examined, RuBP carboxylase (EC 4.1.1.39) and pyruvate, Pi dikinase (EC 2.7.9.1) were generally just sufficient to account for rates of photosynthesis under saturating light; thus, these may be rate limiting enzymes in C₄ photosynthesis. Pyruvate, Pi dikinase and NADP-malate dehydrogenase (EC 1.1.1.82) were the only enzymes examined which were light activated and increased in activity with increasing light intensity. In the low light grown plants the activity of pyruvate, Pi dikinase closely paralleled the photosynthetic rate measured under different light levels. With the plants grown under direct sunlight, as light intensity was increased the activation of pyruvate, Pi dikinase and NADP⁺-malate dehydrogenase proceeded more rapidly than photosynthesis.     

菠萝叶片的稳定碳同位素比与PEP羧化酶及PEP羧激酶活性

专性景天酸代谢（ＣＡＭ）植物菠萝（Ａｎａｎａｓｃｏｍ ｏｓｕｓ （Ｌ．） Ｍｅｒｒ．）的下数第５ 到第３５ 位叶为材料,研究稳定性碳素同位素（δ１３Ｃ）值,磷酸稀醇式丙酮酸（ＰＥＰ）羧化酶和ＰＥＰ羧激酶活性的变化。１１ 个不同叶位叶片的δ１３Ｃ值平均为－ １２．９４‰,最大变幅相差－ ２．０６‰。两个酶的活性呈单峰形变化,第８—１１ 位叶的活性最高,低位叶的酶活下降。本试验条件下,ＰＥＰ羧激酶平均活性比ＰＥＰ羧化酶高３．４ 倍。结果表明,ＣＡＭ 植物的暗下羧化与光下脱羧之间具有一定的协调性。老叶虽然酶活较低,但总的看来,ＣＡＭ 水平变化不大     

大豆根瘤的PEP羧化酶活性和体外CO_2的固定

豆科植物根瘤固氮的第一产物是谷氨酸和谷氨酰胺(Kennedy 1966a,1966b),但在大豆茎和根瘤的伤流液中发现根瘤中氮固定的最终产物是天冬酰胺(Streeter 1972,Wong和Evans 1971),并且运至植物的地上部分。而反应中需要的草酰乙酸则来自PEP的羧化作用。     

Stable Carbon Isotope Ratio and Activities of PEP Carboxylase and PEP Carboxykinase in Pineapple Leaves

Slight flutuation in carbon isotope values were found in counted from top dounward to the 35th in pineapple, Ananas comosus (L.) Merr., but more negative δ13C value (less heavier 13C) was observed in lower position leaves. The average δ 13C value was –12.94‰ in 11 leaves with maximum range of variation as –2.06‰. Similar single peak curves were found between PEPCase and PEP carboxykinase activities with leaves at various positions. Both enzymes reached the maximum activity in 8—11th leaves, then declined in others at lower positions. PEP carboxykinase activity was 3.4 folds higher than PEPCase activity under the present experimental condition (25—30 ℃). The results indicated that metabolic coordination evisted between dark carboxylation and light decarboxylation. For the obligate CAM plant, pineapple, though the carboxylation and decarxylation activities did occur in old leaves, the CAM level change did much, however.     

The enzymatic determination of bicarbonate and CO2 in reagents and buffer solutions

A method for the determination of bicarbonate in buffer solutions between pH 7.5 and 8.75 and in stock solutions of NaHCO3 is described. The HCO-3 is reacted with phosphoenolpyruvate (PEP) in the presence of PEP carboxylase (EC 4.1.1.31) and the oxaloacetate formed reduced to malate by NADH in the reaction catalyzed by malate dehydrogenase (EC 1.1.1.37). The extent of oxidation of NADH is measured spectrophotometrically. Experiments using standard solutions show that 1 mol of NADH is oxidized per mol of HCO-3 added. The method was used to establish the precautions needed to prepare buffer solutions containing less than 1% of the bicarbonate which would be present in the same buffers in equilibrium with air.     

Products of photosynthetic 14CO2 fixation and related enzyme activities in fruiting structures of chickpea

Fruiting structures of a number of legumes including chickpea are known to carry out photosynthetic CO₂ assimilation, but the pathway of CO₂ fixation and particularly the role of phosphoenolpyruvate carboxylase (EC 4.1.1.31) in these tissues is not clear. Activities of some key enzymes of the Calvin cycle and C₄ metabolism, rates of ¹⁴CO₂ fixation in light and dark, and initial products of photosynthetic ¹⁴CO₂ fixation were determined in podwall and seedcoat (fruiting structures) and their subtending leaf in chickpea (Cicer arietinum L.). Compared to activities of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) and other Calvin cycle enzyme, viz. NADP⁺-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13), NAD⁺-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) and ribulose-5-phosphate kinase (EC 2.7.1.19), the levels of phosphoenolpyruvate carboxylase and other enzymes of C₄ metabolism viz. NADP⁺-malate dehydrogenase (EC 1.1.1.82), NAD⁺-malate dehydrogenase (EC 1.1.1.37), NADP⁺ malic enzyme (EC 1.1.1.40), NAD⁺-malic enzyme (EC 1.1.1.39), glutamate oxaloacetate transaminase (EC 2.6.1.1) and glutamate pyruvate transaminase (EC 2.6.1.2), were generally much higher in podwall and seedcoat than in the leaf. Podwall and seedcoat fixed ¹⁴CO₂ in light and dark at much higher rates than the leaf. Short-term assimilation of ¹⁴CO₂ by illuminated fruiting structures produced malate as the major labelled product with less labelling in 3-phosphoglycerate, whereas the leaf showed a major incorporation into 3-phosphoglycerate. It seems likely that the fruiting structures of chickpea utilize phosphoenolpyruvate carboxylase for recapturing the respired carbon dioxide.     

Carbon dioxide fixation in orchid aerial roots

Acidity fluctuation, CO₂ gas exchange, δ¹³C value, PEP carboxylase and RuBP carboxylase activities in aerial roots of selected thick-leaved orchid hybrids ( Arachnis and Aranthera ) were studied. Both aerial roots and leaves showed acidity fluctuation over a 24 h period. Dark acidification in aerial roots was enhanced at low temperature (15°C). Aerial roots had δ¹³C values close to those of leaves which have been previously demonstrated to possess crassulacean acid metabolism. Variation in δ¹³C values along the length of the roots was observed; the root tip having a less negative δ¹³C value (—13.34%‰) than the older portions of the roots (—14.55%‰). There was no net CO₂ fixation by aerial root, although ¹⁴³²CO₂ fixation was observed in light and in darkness. The pattern of fluctuation in activities of PEP carboxylase and RuBP carboxylase in aerial roots was similar to that obtained for the leaves. In both aerial roots and leaves, PEP carboxylase activity was several times higher than that of RuBP carboxylase.     

Adaptation to sub- and supraoptimal temperatures of inbred maize lines differing in origin with regard to seedling development and photosynthetic traits

Six inbred lines of maize ( Zea mays L.) from cool temperate regions (C) and from warm regions (W) were grown at 14, 22, 30 and 38°C up to the same physiological age, the full expansion of the third leaf. Generally, plants developed smaller shoot dry weights and leaf areas at extreme temperatures. The shoot:root ratio was lowest at 22°C and highest at 30°C. Most lines had a minimum for specific leaf dry weight at 30°C, but W lines had a second lower minimum at 14°C. Phosphofructokinase activity scarcely reacted to temperature between 22° and 38°C; at 14°C one C line and all W lines had rather low activities. Generally, the chlorophyll content increased steeply from 14 to 22°C and decreased somewhat from 30 to 38°C. In C lines the carotenoid level decreased from 14 to 38°C. No uniform temperature response was found for PEP carboxylase activity, but the highest activity was mostly attained at 38°C. RuBP carboxylase activity increased considerably from 14 to 22°C and remained comparatively constant at higher temperatures. The highest activity of NADP malate dehydrogenase was found at 22°C, with a decrease up to 38°C and with second lowest values at 14°C. C lines possessed larger leaf areas, shoot dry weights and higher shoot:root ratios than W lines at 14 and 22°C, and higher specific leaf dry weights over the whole temperature range. The genotypic pattern of shoot dry weight at 14°C corresponded reasonably well with that of phosphofructokinase activity. A better adaptation of C lines to suboptimal temperatures was mostly clearly indicated for photosynthetic traits which have a well proven relationship with the chloroplast membranes: chlorophyll, carotenoids and RuBP carboxylase. The least distinct effects of origin were observed at 38°C; a tendency prevailed for a better performance of C lines with regard to phosphofructokinase, carotenoids, RuBP carboxylase and NADP malate dehydrogenase.     

Phosphoenolpyruvate carboxylase in soybean root nodules: An immunochemical study

The activity of phosphoenolpyruvate carboxylase (E.C. 4.1.1.31) strongly increased during the maturation of soybean (Glycine max L. Weber) root-nodules. By using a specific immune serum it was shown that this increase was the consequence of an elevated population of enzyme molecules whose appearance preceded the emergence of nitrogen fixing capacity. Whether or not the phenomenon could be ascribed to the formation of a specific isoenzyme is not known. The location of the enzyme was also investigated. Immunocyto-fluorescence experiments established that phosphoenolpyruvate carboxylase was present in the cytoplasmic compartment of both infected and uninfected cells of nodules.Abbreviation PEPCase phosphoenolpyruvate carboxylase     

高粱的光合特性和杂种优势

从杂交高粱及其亲本的PEP羧化酶、丙酮酸磷酸二激酶、NADP-苹果酸酶和NAD-苹果酸脱氢酶活性比较了它们的光合碳代谢特性,也比较了它们在光合强度、CO_2补偿点和产量指标上的差异,分析了不同组合的杂种的叶面积和产量构成因素在各生育期的变化。高产的杂交种比其亲本有较高的关键酶活性、高光合强度和低的CO_2补偿点,其光合特性具有超亲优势,并在这些优势和增加体内物质向穗内分配的基础上增加了穗粒数。     

低温对玉米叶片PEP羧化酶及其调节特性的影响

低温贮存期间,玉米叶片PEP羧化酶活性随贮存时间的延长而明显降低,对效应剂Gly的敏感性也减弱。多羟基醇(甘油和山梨醇)以及PEP羧化酶的正效应剂G-6-P在与Gly和甘油同时作用时,对PEP羧化酶在低温贮存期间的活性和对Gly的敏感性均有保护效应,且对两者的保护程度相一致,表明低温贮存期间PEP羧化酶对效应剂敏感性减弱与其低温失活有直接关系。     

Phosphoenolpyruvate carboxylase from Acetobacter aceti

In Acetobacter aceti growing on pyruvate as the only source of carbon and energy, oxaloacetate (OAA) is produced by a phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31). The enzyme was purified 122-fold and a molecular weight of about 380,000 was estimated by gel filtration.The optimum pH was 7.5 and the K _m values for PEP and NaHCO₃ were 0.49 mM and about 3 mM, respectively. The enzyme needed a divalent cation; the K _m for Mn²⁺, Co²⁺ and Mg²⁺ were 0.12, 0.26 and 0.77 mM, respectively. Maximal activity was only obtained with Mg²⁺. Mn²⁺ and Co²⁺ became inhibitory at high concentrations.The activity was inhibited by succinate and, to a lesser extent, by fumarate, citrate, -ketoglutarate, aspartate and glutamate.As compared with the corresponding enzyme from A. xylinum, the PEP carboxylase of A. aceti showed the following differences: a) It had an absolute requirement for acetyl CoA (K _a 0.18 mM) or propionyl CoA (K _a 0.2 mM). b) It was not affected by ADP. c) It was sensitive to thiol blocking agents.Abbreviations PEP phosphoenolpyruvate - OAA oxaloacetate - MW molecular weight - TEMG buffer 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 5 mM MgCl₂, 1 mM glutathione - HEPES N-2-hydroxyethylpiperazine-N-ethanesulfonic acid