Alterations in carbohydrate metabolism of γ-irradiated cavendish banana

γ-Irradiation of preclimacteric banana resulted in a gradual increase in fructose content, which reached a maximum in 6 days. Although the catabolism of glucose-U-¹⁴C was less in irradiated banana, incorporation of label into fructose was high. Initial fructose accumulation in irradiated banana may be due to a shift in glucose utilization from the glycolytic to the pentose phosphate pathway. The ratio of resporatory CO₂ from glucose-6-¹⁴C and glucose-1-¹⁴C was halved in irradiated bananas indicating predominance of the pentose phosphate pathway. The radioactivity of fructose derived from glucose-6-¹⁴C was almost twice that from glucose-1-¹⁴C in irradiated bananas, whilst in control both fruit the labelled precursors yielded equal amounts. Studies on individual enzymes in these two pathways showed an increase in phosphorylase, phosphoglucomutase, glucose-6-phosphate dehydrogenase and fructose-6-phosphatase and a decrease in hexokinase in irradiated banana.     

反应分离耦合技术生产L-苹果酸工艺过程的优化研究

运用生物反应分离耦合原理,以富马酸钙为底物,采用游离延胡索酸酶,直接转化生产苹果酸钙。该法相对目前广泛采用的包埋式固定化方法具有工艺流程短、操作简便、转化率、收率高等特点,研究结果表明,转化温度为40℃,PH为7．0－7．5时,每升延胡索酸酶液能在20－28h间将3．2kg的富马酸钙转化生产成苹果酸钙,转化率高达99．9％,富马酸在产品中的残留在0．1％左右,产品符合美国药典标准,成本与化学合成法生产的DL－苹果酸相当。     

NADP-linked malic enzyme and malate metabolism in ageing tomato fruit

Malate dehydrogenase (oxaloacetate-decarboxylating) (NADP⁺) EC 1.1.1.40, malic enzyme, has been purified 40-fold to a homogeneous state using affinity chromatography and gel permeation chromatography. The M_r is 260–265 K with four subunits each of 64–65 K. The enzyme has some competitive or non-competitive inhibitors, particularly some of the Krebs cycle acids and exhibits a rapid rise in activity at the same time as activity of the enzymes of the Krebs cycle are decreasing in the tomato mitochrondrion. The malic enzyme is restricted to the cytosol. The relevance of this information to malate metabolism in plants is discussed.     

Comparative studies of coupled assays for phosphoenolpyruvate carboxylase

Phosphoenolpyruvate carboxylase (PEPC) from higher plants is usually assayed by using malate dehydrogenase (MDH) as a coupling enzyme. To avoid erroneous readings caused by metal ions, which convert oxaloacetate (OAA) to pyruvate, lactic dehydrogenase can be included. Reporting the total NADH used by both coupling enzymes gives the total OAA production. Microbial PEPC has been assayed by employing citrate synthase (CS) as a coupling enzyme which detects the reaction of CoA with Ellman's reagent. Comparable K_m values for MgPEP are found with the two assays. When MDH alone is used as the coupling system, the V_max value is about 60% larger than the one found with the CS assay. However, when MDH is added to the CS assay without the NADH cofactor, V_max is brought back to the same level as that with the NADH-coupled enzyme. Malate inhibition of PEPC assayed with the CS coupling system is blocked by low concentrations of citrate in the range produced in the assay. High concentrations of citrate inhibit PEPC. Glucose-6-phosphate in concentrations higher than 1 m M blocks the response of PEPC to added MDH in the CS assay.     

丛枝菌根真菌N代谢与C代谢研究进展

N元素是植物需求量最大的元素,丛枝菌根（arbuscular mycorrhizal,AM）真菌可以与大部分植物形成共生关系,通过根外菌丝促进植物对N元素的吸收。C元素也是AM真菌完成其生命过程的必备元素,AM真菌依赖宿主植物提供所必需的C源。近年来对AM真菌的N代谢途径的研究有了新的进展,AM真菌中与代谢途径相关酶的发现,相关基因的克隆,更进一步验证了N元素在AM真菌中的代谢途径。对C代谢及与N代谢的关系也有所涉及。综述近几年文献,总结了AM真菌的最新研究进展。     

多效唑对不同品质类型花生产量、品质及相关酶活性的影响

选用高蛋白品种KB008（KB008）、高脂肪品种花17(H17)和高油酸/亚油酸（O/L）品种农大818（818）,在大田栽培条件下,研究了盛花后期叶面喷施多效唑(PBZ)对不同品质类型花生产量、品质及相关碳、氮代谢酶活性的影响.结果表明：喷施PBZ显著增加了3种品质类型花生荚果产量,原因是增加了单株结果数,降低了千克果数而提高了双仁果率.喷施PBZ不同程度地提高了3种类型花生籽仁脂肪和可溶性糖含量,降低了蛋白质含量,显著增加了高脂肪品种H17的O/L值.PBZ使高O/L值品种818的脂肪含量增加显著,同时其蛋白质含量显著降低,而对其他两品种的蛋白质和脂肪含量影响较小.喷施PBZ均降低了3种类型花生结荚期叶片硝酸还原酶（NR）活性及结荚期和饱果期叶片谷氨酰胺合成酶和谷氨酸脱氢酶活性,818的3种酶活性降低幅度最大,KB008和H17的酶活性降幅较小;喷施PBZ均降低了3种类型花生结荚期和饱果期叶片谷草转氨酶和谷丙转氨酶活性.说明氮代谢酶活性的降低是喷施PBZ降低3种类型花生籽仁蛋白质含量的主要原因.喷施PBZ均提高了3品种结荚期和饱果期叶片蔗糖合成酶和磷酸蔗糖合成酶活性,其中显著提高了818的2种酶活性,而对KB008和H17的活性提高不显著;喷施PBZ提高了3品种结荚期和饱果期的磷酸烯醇式丙酮酸羧化酶和1,5-二磷酸核酮糖羧化酶活性,其中对818在结荚期的活性提高最显著,对H17活性提高较小.碳代谢酶活性的增强是喷施PBZ提高花生籽仁脂肪含量的生理基础.     

Carbohydrate and Metal Analyses of Violet-colored Acid Phosphatase of Sweet Potato

Two malate dehydrogenases (MDH-M1 and MDH-M2) were found in a methanol-using yeast, Candida sp. N-16. MDH-M2 was induced with methanol. These enzymes were purified as electrophoretically and isoelectrophoretically homogeneous proteins. The molecular weights of MDH-M1 and MDH-M2 were estimated to be about 78,000 (homodimer) and 160,000 (homotetramer). Several kinetic properties were significantly different between the two enzymes. The value (2.07) of V_{max(oxaloacetate)}/V_max(malate) and K_cats (555 s^-1 for oxaloacetate, 481 s^-1 for NADH) of MDH-M2 were higher than the ratio (1.37) of V_max and K_cats(241 s^-1 for oxaloacetate, 271 s^-1 for NADH) of MDH-M1, respectively. The activity of MDH-M2 was inhibited by a high concentration of NAD⁺ and the activity of MDH-M1 by oxaloacetate.     

Measurement of key metabolic enzyme activities in mammalian cells using rapid and sensitive microplate‐based assays

Sensitive microplate‐based assays to determine low levels of key enzyme activities in mammalian cells are presented. The enzyme platform consists of four cycling assays to measure the activity of 28 enzymes involved in central carbon and glutamine metabolism. The sensitivity limit of all cycling assays was between 0.025 and 0.4 nmol product. For the detection of glutaminase activity, a new glutamate cycle system involving the enzymes glutamate dehydrogenase and aspartate transaminase was established. The relative standard deviation of the method was found to be 1.7% with a limit of detection of 8.2 pmol and a limit of quantitation of 24.8 pmol. Hence, cell extracts could be highly diluted to reduce interferences caused by other components in the extract, which in addition minimized underestimates or overestimates of actual enzyme activities. Since substrate concentrations could be maintained at a nearly constant level throughout the assay product accumulation during the reaction was low, which minimized product inhibition. As an example, the enzyme platform was used to investigate maximum enzyme activities of stationary‐phase MDCK cells grown in serum‐containing GMEM medium as typically used in influenza vaccine production. Biotechnol. Bioeng. 2010;107: 566–581. © 2010 Wiley Periodicals, Inc.     

不同保护性耕作措施对武威绿洲灌区冬小麦水分利用的影响

2006—2008年通过田间定位试验,研究了武威绿洲灌区不同耕作措施（传统耕作、秸杆翻压、免耕不覆盖、免耕秸杆立茬、免耕秸杆覆盖）对冬小麦土壤水分空间分布、动态变化、作物耗水量、水分利用效率和产量的影响.结果表明：返青至拔节期,免耕秸杆覆盖（NTS）和免耕秸杆立茬（NTSS）处理显著提高了0～30 cm土壤贮水量,处理间差异较大,在小麦拔节后差异变小;返青至成熟期,NTS和NTSS处理30～150 cm土壤贮水量都大于传统耕作处理（T）;播种期,NTS、NTSS和NT（免耕不覆盖）处理0～150 cm土层总贮水量分别比T处理增加29.55～34.69、17.32～21.79和0.89～15.68 mm,收获期分别增加37.59～38.35、5.70～22.14和4.61～13.93 mm,且随着土层深度的增加处理间土壤贮水量差异增大.NTS、NTSS、NT和TIS（秸杆翻压）处理小麦产量分别比T处理提高15.65%～16.84%、6.98%～12.75%、5.88%～11.74%和3.92%～8.16%,水分利用效率分别提高17.15%～17.52%、7.75%～9.65%、8.24%～10.00%和4.17%～9.91%.免耕秸杆覆盖（NTS）和免耕秸杆立茬（NTSS）处理提高了冬小麦产量和水分利用效率,是改善该区域水资源匮乏的有效耕作措施.     

Discovery of PPi-type Phosphoenolpyruvate Carboxykinase Genes in Eukaryotes and Bacteria

Phosphoenolpyruvate carboxykinase (PEPCK) is one of the pivotal enzymes that regulates the carbon flow of the central metabolism by fixing CO₂ to phosphoenolpyruvate (PEP) to produce oxaloacetate or vice versa. Whereas ATP- and GTP-type PEPCKs have been well studied, and their protein identities are established, inorganic pyrophosphate (PP_i)-type PEPCK (PP_i-PEPCK) is poorly characterized. Despite extensive enzymological studies, its protein identity and encoding gene remain unknown. In this study, PP_i-PEPCK has been identified for the first time from a eukaryotic human parasite, Entamoeba histolytica, by conventional purification and mass spectrometric identification of the native enzyme, followed by demonstration of its enzymatic activity. A homolog of the amebic PP_i-PEPCK from an anaerobic bacterium Propionibacterium freudenreichii subsp. shermanii also exhibited PP_i-PEPCK activity. The primary structure of PP_i-PEPCK has no similarity to the functional homologs ATP/GTP-PEPCKs and PEP carboxylase, strongly suggesting that PP_i-PEPCK arose independently from the other functional homologues and very likely has unique catalytic sites. PP_i-PEPCK homologs were found in a variety of bacteria and some eukaryotes but not in archaea. The molecular identification of this long forgotten enzyme shows us the diversity and functional redundancy of enzymes involved in the central metabolism and can help us to understand the central metabolism more deeply.     

Cerebral dicarboxylate transport and metabolism studied with isotopically labelled fumarate,malate and malonate

Transport and metabolism of dicarboxylates may be important in the glial-neuronal metabolic interplay. Further, exogenous dicarboxylates have been suggested as cerebral energy substrates. After intrastriatal injection of [(14) C]fumarate or [(14) C]malate, glutamine attained a specific activity 4.1 and 2.6 times higher than that of glutamate, respectively, indicating predominantly glial uptake of these four-carbon dicarboxylates. In contrast, the three-carbon dicarboxylate [(14) C]malonate gave a specific activity in glutamate which was approximately five times higher than that of glutamine, indicating neuronal uptake of malonate. Therefore, neurones and glia take up different types of dicarboxylates, probably by different transport mechanisms. Labelling of alanine from [(14) C]fumarate and [(14) C]malate demonstrated extensive malate decarboxylation, presumably in glia. Intravenous injection of 75 micromol [U-(13) C]fumarate rapidly led to high concentrations of [U-(13) C]fumarate and [U-(13) C]malate in serum, but neither substrate labelled cerebral metabolites as determined by (13) C NMR spectroscopy. Only after conversion of [U-(13) C]fumarate into serum glucose was there (13) C-labelling of cerebral metabolites, and only at <10% of that obtained with 75 micromol [3-(13) C]lactate or [2-(13) C]acetate. These findings suggest a very low transport capacity for four-carbon dicarboxylates across the blood-brain barrier and rule out a role for exogenous fumarate as a cerebral energy substrate.     

Fluoride-induced alteration of carbon and nitrogen metabolism in developing wheat grains

Effect of fluoride (10 and 50 mM) on the activities of sucrose metabolizing enzymes, alkaline inorganic pyrophosphatase, and transaminases in relation to the accumulation of free sugars, starch, and soluble protein was studied in detached ears of wheat cultured in a liquid medium. Culturing for 5 d in the presence of fluoride reduced the amount of grain starch whereas contents of total free sugars, particularly sucrose, and soluble protein increased. Fluoride inhibited the activities of soluble acid and neutral invertases, as well as sucrose synthase acting in the cleavage direction. Uptake of uniformly labelled ¹⁴C-sucrose or fructose was also drastically reduced by fluoride. Glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT) activities also increased with fluoride addition in correspondence with an increase in soluble protein. Apparently, the wheat grain responds to fluoride-mediated disruption of carbon metabolism by a compensatory effect on nitrogen metabolism. This revised version was published online in July 2006 with corrections to the Cover Date.     

Differential response of carbon and nitrogen metabolism to fluoride application in fruiting structures of chickpea (Cicer arietinum)

The effect of sodium fluoride (10 and 50 mol·m⁻³) on the activities of sucrose metabolizing enzymes, transaminases and glutamine synthetase in relation to the transformation of free sugars to starch and protein in the fruiting structures (pod wall, seed coat, cotyledons) of chickpea was studied by culturing detached reproductive shoots in a liquid medium. Addition of fluoride to the culture medium drastically reduced starch content of the cotyledons and caused a marked build-up of total free sugars comprised mainly of reducing sugars in the pod wall and seed coat, and sucrose in the cotyledons. Concomitantly, the activity of soluble invertase was stimulated in the pod wall but reduced in the cotyledons. However, soluble protein content of both the pod wall and the cotyledons increased in conjunction with an increase in the activities of glutamate-oxaloacetate transaminase, glutamate-pyruvate transaminase and glutamine synthetase. Disruption of starch biosynthesis under the influence of fluoride and the resulting accumulation of free sugars possibly resulted in their favoured utilization in nitrogen metabolism. Labelling studies with [U-¹⁴C]-sucrose showed that the ¹⁴C incorporation into total free sugars was enhanced by fluoride in the pod wall but reduced in the seed coat and cotyledons, possibly due to an inhibitory effect on their translocation to the developing seeds.