Purification and characteristics of glutamate dehydrogenase (GDH) from triticale roots

In the investigated 14 day old triticale seedlings a much higher GDH activity was observed in roots than in leaves. The enzyme from the roots was purified up to the state of homogeneity (about 400 fold). The purified enzyme showed a higher activity in the presence of reduced coenzyme forms (NAD(P)H) than their oxidated forms. In the presence of NAD(P)H the enzyme showed absolute specificity to 2-oxoglutarate and in cooperation with NAD(P)⁺ to L-glutamate. The Km values determined for particular substrates indicate a high affinity of NADPH-GDH to ammonium ions. Optimum pH, temperature and thermostability of GDH depended on the type and form of the coenzyme. Molecular mass of purified enzyme was 257 kDa. It seems that native GDH is composed of six identical subunits of the molecular mass 42.5 kDa.     

过表达大豆类受体蛋白激酶基因RLPK2促进转基因拟南芥叶片的衰老北大核心CSCD

大豆RLPK2基因(GenBank登录号:AY687391)是一个编码N-末端富含亮氨酸重复序列的类受体蛋白激酶基因。为分析大豆RLPK2基因的功能,该研究以野生型拟南芥和大豆RLPK2基因过表达拟南芥植株为材料,通过农杆菌介导法转化野生型拟南芥,构建了大豆RLPK2基因过表达载体,分析了叶片衰老过程中叶绿素荧光参数、抗氧化酶活性及衰老相关基因表达量的变化。结果表明:(1)无论是野生型还是转基因拟南芥,随着叶片衰老进程的进行,光系统Ⅱ(PSⅡ)的最大光化学效率(F_(v)/F_(m))、PSⅡ实际光化学效率(Φ_(PSⅡ))、光化学淬灭系数(qP)和光合电子传递速率(ETR)均呈下降趋势,但后者下降趋势更明显;(2)激发压(1-qP)在叶片衰老前期的变化较为平稳,后期则急剧增加,且转基因型比野生型拟南芥增加更明显;(3)在叶片衰老的各个时期,转基因拟南芥叶片丙二醛(MDA)含量均显著高于野生型,而超氧化物岐化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性均显著低于野生型;(4)实时荧光定量PCR检测结果表明,RLPK2转基因拟南芥中衰老标志基因ATSAG12,衰老关键转录因子ATNAP、ATWRKY6和叶绿素降解关键酶编码基因ATACD1表达量显著上调。综上认为,大豆类受体蛋白激酶基因RLPK2参与调控植物叶片衰老进程,其表达对叶片衰老具有促进作用。     

野生大豆与栽培大豆种子差异蛋白质组学研究

运用蛋白质组学方法比较研究3个野生大豆(Glycinesoja)和3个栽培大豆(Glycinemax)的种子贮藏蛋白差异情况.结果发现,在考马斯亮蓝染色的双向电泳pH4～7的胶上,经过PDQuest图像分析软件平均可检测到550个左右的蛋白质点.进一步分析发现,表达量变化2.5倍以上的点有10个,其中大部分蛋白质仅在栽培大豆中检测到.对这10个蛋白质点进行了胶内酶解,用基质辅助激光解析电离飞行时间质谱测定均得到了肽质量指纹图谱.搜索大豆UniGene库和NCBI库共鉴定出5个蛋白质,主要是与大豆抗性、抗营养以及种子萌发相关的蛋白质,包括大豆血凝素,种子成熟蛋白PM24,糖结合蛋白,胰蛋白酶抑制剂p20以及成熟多肽.对这些蛋白质可能的作用进行了讨论.     

Immunolocalization of glyoxysomal malate synthase from soybean cotyledons (Glycine max L)

Summary— Malate synthase (MS; EC 4.1.3.2), an enzyme specific to the glyoxylate cycle, was studied in cotyledons of dark-grown soybean (Glycine max L) seedlings with light and electron microscopy techniques. Immunogold localization confirmed biochemical evidence that MS from soybean is a glyoxysomal matrix enzyme.     

NAD(P)H dehydrogenase from rabbit and rat liver: Purification and some properties

• 1.1. NAD(P)H dehydrogenase from rabbit liver was purified to electrophoretic homogeneity using a procedure also found applicable for the rat liver enzyme.
• 2.2. Rabbit and rat liver enzymes showed different behaviour in isoelectric focusing and different K_m values and turnover numbers.
• 3.3. Both enzymes were inhibited to similar extents by warfarin.
• 4.4. The rabbit enzyme is composed of two subunits of mol. wt 27,000 and contained 1 FAD group per subunit.
• 5.5. Some absorption and circular dichroism properties of the rat enzyme are shown.

     

Salinity induced nuclear and DNA degradation in meristematic cells of soybean (Glycine max (L.)) roots

Changes in the nuclei of meristematic root cells of soybean (Glycine max (L.) Merr. cv. Acme) in response to severe salinity were studied. Root growth was inhibited by 200 mM NaCl, when 1 mM CaCl_2 was present in the culture media. Increasing CaCl_2 up to 5 mM partially prevented this inhibition. However, inhibition also occurred with 100~mM NaCl without CaCl_2. We examined the meristematic cells under a series of NaCl treatments. Nuclear deformation of the cells occurred with 24 h of 150 mM or higher NaCl, and was followed by degradation of nuclei in the apical region of the root. TEM observation and agarose gel electrophoretic analysis confirmed that root tip nuclear DNA deformed or degraded with 150 mM or higher NaCl concentrations.     

Sampling strategy for wild soybean ( Glycine soja ) populations based on their genetic diversity and fine-scale spatial genetic structure

A total of 892 individuals sampled from a wild soybean population in a natural reserve near the Yellow River estuary located in Kenli of Shandong Province (China) were investigated. Seventeen SSR (simple sequence repeat) primer pairs from cultivated soybeans were used to estimate the genetic diversity of the population and its variation pattern versus changes of the sample size (sub-samples), in addition to investigating the fine-scale spatial genetic structure within the population. The results showed relatively high genetic diversity of the population with the mean value of allele number (A) being 2.88, expected heterozygosity (He) 0.431, Shannon diversity index (I) 0.699, and percentage of polymorphic loci (P) 100%. Sub-samples of different sizes (ten groups) were randomly drawn from the population and their genetic diversity was calculated by computer simulation. The regression model of the four diversity indexes with the change of sample sizes was computed. As a result, 27–52 individuals can reach 95% of total genetic variability of the population. Spatial autocorrelation analysis revealed that the genetic patch size of this wild soybean population is about 18 m. The study provided a scientific basis for the sampling strategy of wild soybean populations. __________ Translated from Journal of Fudan University (Natural Science), 2006, 45(3): 322–327 [译自: 复旦学报 (自然科学版)]     

Unusual regulatory properties of sucrose-phosphate synthase purified from soybean (Glycine max) leaves

Sucrose-phosphate (SPS) from source leaves of soybean ( Glycine max (L.) Merr. cv. Ransom II) was purified 74-fold to a final specific activity of 1.8 U (mg protein)¹. The partially purified preparation was free from phosphoglucoseisomerase (EC 5.3.1.9), pyrophosphatase (EC 3.6.1.1), phosphoenolpyruvate-phosphatase (EC 3.1.3.-), phosphofructokinase (EC 2.7.1.11), and uridine diphosphatase (EC 3.6.1.6), and was used for characterization of the kinetic and regulatory properties of the enzyme. The enzyme showed hyperbolic saturation kinetics for both fructose-6-phosphate (K_m=0.57 m M ) and UDPGlucose (UDPG) (K_m=4.8 m M ). The activity of SPS was inhibited by the product UDP. In vitro this inhibition could be partially overcome by the presence of Mg²⁺. Inorganic orthophosphate was only slightly inhibitory (35% inhibition at 25 m M phosphate). Glucose-6-phosphate (up to 20 m M ) had no effect on activity, and did not show any significant interaction with phosphate inhibition. A range of potential effectors was tested and had no effect on SPS activity: Glucose-1-phosphate, fructose-1, 6-bisphosphate, α-glycero-phosphate, dihydroxyacetone-phosphate, 3-phosphoglyceric acid, (all at 5 m M ), sucrose at 100 m M and pyrophosphate at 0.1 m M . The apparent lack of allosteric regulation of soybean SPS makes this enzyme markedly different from SPS previously characterized from spinach and maize.     

Regeneration of NAD(H) by alcohol dehydrogenase in gel-entrapped yeast cells

Anaerobically grown cells of Saccharomyces cerevisiae entrapped in polyacrylamide gel have been shown to provide a stable source of alcohol dehydrogenase [(ADH) alcohol:NAD⁺ oxidoreductase, EC 1.1.1.1] for effective regeneration of NAD(H). This system was able to provide the coenzyme required for the operation of other dehydrogenases, such as lactate dehydrogenase [(LDH) l-lactate: NAD⁺ oxidoreductase, EC 1.1.1.27] and malate dehydrogenase [(MDH) l-malate:NAD⁺ oxidoreductase, EC 1.1.1.37]. Yeast cells coimmobilized with a dehydrogenase are capable of the reversible regeneration of the reduced or oxidized coenzyme, depending on the additions made. A two-cell system can also be constituted using the same strain of yeast, adapted differently. Cells grown anaerobically and aerobically as sources of ADH and MDH, respectively, can operate efficiently on coimmobilization. The system can be used repeatedly without measurable loss of efficiency.     

Purification and characterization of an iron-induced ferritin from soybean (Glycine max) cell suspensions.

Ferric citrate induces ferritin synthesis and accumulation in soybean (Glycine max) cell suspension cultures [Proudhon, Briat & Lescure (1989) Plant Physiol. 90, 586-590]. This iron-induced ferritin has been purified from cells grown for 72 h in the presence of either 100 microM- or 500 microM-ferric citrate. It has a molecular mass of about 600 kDa and is built up from a 28 kDa subunit which is recognized by antibodies raised against pea (Pisum sativum) seed ferritin and it has the same N-terminal sequence as this latter, except for residue number 3, which is alanine in pea seed ferritin instead of valine in iron-induced soybean cell ferritin. It contains an average of 1800 atoms of iron per molecule whatever the ferric citrate concentration used to induce its synthesis. It is shown that the presence of 100 microM- or 500 microM-ferric citrate in the culture medium leads respectively to an 11- and 28-fold increase in the total intracellular iron concentration and to a 30- and 60-fold increase in the ferritin concentration. However, the percentage of iron stored in the mineral core of ferritin remains constant whatever the ferric citrate concentration used and represents only 5-6% of cellular iron.     

Polyamine synthesis in plants. Purification and properties of amidinotransferase from soybean (Glycine max) axes

Three-day-old soybean (Glycine max) seedlings were exposed to 0.4 M sorbitol solution for 4 h to induce amidinotransferase activity, with the corresponding enzyme being purified to homogeneity by chromatographic separation on DEAE-Sephacel, Sephacryl S-300 and L-arginine Sepharose 4B. The purified enzyme used L-arginine and L-glycine as the major donor/acceptor of the amidino group, respectively, with formation of guanidinoacetic acid and ornithine products being confirmed by ESI-MS. The enzyme is a tetrameric protein having a molecular mass of 240,000 Da, whose thiol group is needed for enzymatic activity. The K(M)s for arginine and glycine were 3.8 and 0.89 mM, respectively, with optimal temperature and pH being 37 degrees C and 9.5, respectively. The soybean amidinotransferase could be indirectly involved in nitrogen metabolism, as suggested by the observation that arginine:glycine amidinotransferase in soybean axes is indirectly involved in putrescine biosynthesis and displays feedback control at high levels of an endogenous regulator, putrescine.     

烟草赤星病菌拮抗性大豆根瘤内生菌的筛选及抑制作用

以烟草赤星病菌为供试病原菌,采用对峙法、代谢液培养法对分离自大豆根瘤的内生细菌进行抑菌筛选,对筛选菌株作用的病原菌菌丝进行显微观察,研究筛选菌株细胞培养特征及生理生化特性、16S rDNA测序序列、系统发育,以及进行温室接种防病试验.结果表明: 大豆根瘤内生菌经初筛、复筛,对烟草赤星病菌抑菌率42%以上的有7株菌,分别属于芽孢杆菌属、假单胞菌属、中华根瘤菌属和寡养单胞菌属.显微观察表明,在拮抗性内生菌作用下病原菌菌丝末端出现明显畸形,呈珊瑚状分枝、球状膨大等;代谢液试验表明,内生菌对病原菌的抑制作用主要是内生菌代谢产生的胞外物质在发挥作用;对峙试验表明,芽孢杆菌可迅速形成生物薄膜,有效阻碍病原菌丝生长和延伸.盆栽防病试验表明,拮抗性内生菌处理下烟草病情指数显著低于对照,表明筛选菌株可作为烟草赤星病的生防菌株资源.     

转基因高蛋氨酸大豆对根际土壤主要有机元素和酶活性的影响

【目的】为了评估转基因高蛋氨酸大豆ZD91对土壤生态系统的安全性,开展了其对土壤主要有机元素和酶活性影响的实验。【方法】连续2年,在大豆苗期、花期、鼓粒期和成熟期,采用抖落法采集根际土壤样品,通过室内测定,分析了转基因大豆ZD91对根际土壤含水量、pH、主要有机元素和酶活性的影响。【结果】转基因大豆ZD91较之对应的非转基因大豆对根际土壤含水量、pH、主要有机元素和酶活性无显著影响,但同一种酶活在不同年份和不同生育期存在显著差异。【结论】转基因大豆ZD91对土壤生态系统具有安全性。     

Purification and some properties of NAD(P)H dehydrogenase from Saccharomyces cerevisiae: Contribution to glycolytic methylglyoxal pathway

NAD(P)H dehydrogenase was purified approximately 480-fold from Saccharomyces cerevisiae with 6.5% activity yield. The enzyme was homogeneous on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 40,000–44,000 by gel filtration on Sephadex G-150 column chromatography and SDS-polyacrylamide gel electrophoresis. The K_m values for NADPH and NADH were 7.3 μM and 0.1 mM, respectively. The activity of the enzyme increased approximately 4-fold with Cu²⁺. FAD, FMN and cytochrome c were not effective as electron acceptors, although Fe(CN)₆³⁻ was slightly effective. NADH generated by the reaction of lactaldehyde dehydrogenase in the glycolytic methylglyoxal pathway will be reoxidized by NAD(P)H dehydrogenase. NAD(P)H dehydrogenase thus may contribute to the reduction/oxidation system in the glycolytic methylglyoxal pathway to maintain the flux of methylglyoxal to lactic acid via lactaldehyde.