Some kinetic properties of a purified glutamine synthetase from bacteroids of Glycine max

Abstract A purified glutamine synthetase was prepared from bacteroids of Rhizobium japonicum from nodules of Glycine max . For the biosynthetic assay the K _m values (mM) were l -glutamate 12.9, NH₄Cl 8.9 and ATP 14.3. When the enzyme was assayed by the γ-glutamyltransferase reaction the K _m values (mM) were l -glutamine 11.1 and hydroxylamine 3.3 compared with 7.7 and 1.2, respectively, for the purified enzyme from Rhizobium japonicum grown in culture. The enzyme prepared from bacteroids of Glycine max was 80% adenylylated.     

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.     

Inhibition of acid phosphatase isoforms purified from mature soybean (Glycine max) seeds

The four soybean seed acid phosphatase isoforms AP1, AP2, AP3A and AP3B were competitively inhibited by phosphate, vanadate, fluoride and molybdate, using p-nitrophenylphosphate as substrate. The four isoforms were not significantly affected by compounds that can interact with SH residues or by pyridoxal phosphate. These results indicated that cysteine and lysine residues are not present in the active site of the four soybean seed acid phosphatase isoforms. The inhibition constant values for phosphate, vanadate, fluoride and molybdate at pH 5.0 were respectively: API (250, 12.8, 1.7, 0.05 microM). AP2 (800, 10, 500, 0.025 microM), AP3A (250, 24.2,250, 0.032 microM ), AP3B (2400 36.9, 750, 0.05 microM).     

A novel alkaline alpha-galactosidase from melon fruit with a substrate preference for raffinose

The cucurbits translocate the galactosyl-sucrose oligosaccharides raffinose and stachyose, therefore, alpha-galactosidase (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) is expected to function as the initial enzyme of photoassimilate catabolism. However, the previously described alkaline alpha-galactosidase is specific for the tetrasaccharide stachyose, leaving raffinose catabolism in these tissues as an enigma. In this paper we report the partial purification and characterization of three alpha-galactosidases, including a novel alkaline alpha-galactosidase (form I) from melon (Cucumis melo) fruit tissue. The form I enzyme showed preferred activity with raffinose and significant activity with stachyose. Other unique characteristics of this enzyme, such as weak product inhibition by galactose (in contrast to the other alpha-galactosidases, which show stronger product inhibition), also impart physiological significance. Using raffinose and stachyose as substrates in the assays, the activities of the three alpha-galactosidases (alkaline form I, alkaline form II, and the acid form) were measured at different stages of fruit development. The form I enzyme activity increased during the early stages of ovary development and fruit set, in contrast to the other alpha-galactosidase enzymes, both of which declined in activity during this period. In the mature, sucrose-accumulating mesocarp, the alkaline form I enzyme was the major alpha-galactosidase present. We also observed hydrolysis of raffinose at alkaline conditions in enzyme extracts from other cucurbit sink tissues, as well as from young Coleus blumei leaves. Our results suggest different physiological roles for the alpha-galactosidase forms in the developing cucurbit fruit, and show that the newly discovered enzyme plays a physiologically significant role in photoassimilate partitioning in cucurbit sink tissue.     

Fermentation of stachyose and raffinose by hind-gut bacteria of the weanling pig

Two pigs were weaned at 28 d of age, and one pig each was placed on a corn-soy (CS) or a corn-soy diet containing 40% lactose (CSL). After 28 d a fecal sample was taken from each pig. The fecal bacterial community was fractionated and used as a source of inoculum to determine if high levels of lactose added to CS diets would modify the structure of the hind-gut microbial community and the in vitro breakdown of stachyose (soy molasses served at the source of stachyose) and raffinose. Bacterial growth rate tended to the higher with the CSL diet. Higher growth rates for bacteria from the CSL-fed pig were supported by the higher acetate: propionate production when compared to the CS diet. All the stachyose and raffinose disappeared during the 48 h fermentation. To our knowledge, this is the first report that stachyose or raffinose are completely fermented by the hind-gut bacteria of the weanling pig, and that this process can be affected by the addition of high levels of lactose to the diet.     

Localization of galactinol,raffinose, and stachyose synthesis in Cucurbita pepo leaves

The biochemical pathway of stachyose synthesis was localized by immunocytochemical and ¹⁴C-labeling techniques in mature Cucurbita pepo L. leaves. Galactinol synthase (GaS; EC 2.4.1.123), the first unique enzyme in this pathway, was immunolocalized within the intermediary cells of minor veins in conventionally fixed and cryo-fixed, resin-embedded sections using polyclonal anti-GaS antibodies and protein A-gold. Intermediary cells are specialized companion cells with extensive symplastic connections to the bundle sheath. Gold particles were not seen over the non-specialized companion cells of larger veins or over intermediary cells in young leaves prior to the sink-source transition. In another approach to localization, radiolabel was measured in isolated mesophyll tissue and whole tissue of leaves that were lyophilized following a 90-s exposure to ¹⁴CO₂. Mesophyll, obtained by abrasion of the leaf surface, contained labeled sucrose, galactinol, raffinose and stachyose. However, the latter three labeled compounds constituted a smaller proportion of the neutral fraction than in whole-tissue samples, which also contained minor veins. We conclude that synthesis of galactinol, raffinose, and stachyose occurs in both mesophyll and intermediary cells, predominantly the latter.Abbreviations GaS galactinol synthase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We thank John Pierce, Phillip Kerr, and Brace Schweiger for the gift of anti-GaS antibody and M.K. Kandasamy for helpful discussions. This research was supported by National Science Foundation grant DCB-9104159, U.S. Department of Agriculture Competetive Grant 90000854, and Hatch funds.     

Degradation of stachyose,raffinose, melibiose and sucrose by different tempe-producing Rhizopus fungi

Forty-six strains of tempe-forming Rhizopus species were screened for their ability to grow on raffinose as the sole carbon source. Six of the strains showed good growth and sporulation. These isolates were one Rhizopus oligosporus, one Rhizopus microsporus var. chinensis, three Rhizopus oryzae and one Rhizopus stolonifer. These six moulds and R. oligosporus strain NRRL 2710 were investigated for their metabolism of the raffinose family of -galactoside carbohydrates. Degradation experiments were performed in submerged culture in a medium containing soybean -protein, sodium phytate and either stachyose, raffinose or melibiose. R. oryzae and R. stolonifer completely consumed the tested carbohydrates as carbon source. R. microsporus var. chinensis failed to hydrolyse the -galactosidic bonds of raffinose, stachyose or melibiose, whereas it was able to use sucrose and the fructose moiety of raffinose or stachyose for growth. R. oligosporus NRRL 2710 was unable to hydrolyse any of the tested carbohydrates. The results of the oligosaccharide degradation experiments could be verified during tempe production from soybeans with the selected fungal species.     

Isolation and Characterization of Thiamin Pyrophosphotransferase from Glycine max Seedlings

Thiamin:ATP pyrophosphotransferase (EC2.7.6.2) activity from soybean (Merr.) seedlings grown for 48 hours was determined by measuring the rate of [2-¹⁴C]thiamin incorporation into thiamin pyrophosphate. With partially purified (11-fold) enzyme, optimal activity occurred between pH 7.1 and 7.3, depending on the buffer system that was used. Assays were routinely conducted at a final pH of 8.1 in order to minimize interference from competing reactions. Enzyme activity required the presence of a divalent cation, and a number of nucleoside triphosphates proved to be active as pyrophosphate donors. Apparent K_m values of 18.3 millimolar and 4.64 micromolar were obtained for Mg·ATP and thiamin, respectively. Among the compounds tested, pyrithiamin and thiamin pyrophosphate were most effective in inhibiting thiamin pyrophosphotransferase activity. Based on Sephadex G-100 gel filtration, soybean thiamin pyrophosphotransferase has a molecular weight of 49,000.     

Molecular characterisation of coproporphyrinogen oxidase from Glycine max and Arabidopsis thaliana

Coproporphyrinogen III oxidase (CPO; E.C. 1.3.3.3 ) is an enzyme of haem and chlorophyll synthesis. Biochemical studies have indicated that the majority of CPO activity is present in plastids, with no detectable levels in mitochondria. However, this approach cannot rule out low (less than 5%) activity in the mitochondria, nor the possible presence of CPO in the cytosol, where it is found in yeast (Saccharomyces cerevisiae). We have studied this question further using molecular techniques. A cDNA encoding the mature protein of soybean (Glycine max L.) CPO was used to overexpress the enzyme 200-fold in Escherichia coli. The recombinant enzyme, purified to homogeneity in three steps, is a dimer, with a K_m for coproporphyrinogen III of 0.25 ± 0.03 μM and a V_max of 1.48 pkat. Antibodies raised against the purified soybean CPO were used in western blots to show that the enzyme is present in etioplasts but not in mitochondria. In the completely sequenced genome of Arabidopsis thaliana, we identified two genes encoding CPO, but only one of them (AtCPO-I ) was able to complement a yeast mutant defective in the enzyme; the other is likely to be a pseudogene. A construct encoding the first 92 residues of AtCPO-I fused to green fluorescent protein (GFP) was introduced into Arabidopsis plants by Agrobacterium-mediated transformation. Confocal microscopy demonstrated that the CPO–GFP fusion protein was confined exclusively to plastids in leaves and roots, with no GFP seen in the mitochondria or cytosol.     

Isolation of xyloglucans from etiolated Glycine max and Vigna sesquipedalis hypocotyls

Xyloglucans isolated from cell walls of etiolated Glycine maxand Vigna sesquipedalis hypocotyls were subjected to fragmentationanalysis with cellulase for structural comparison with thosederived from Phaseolus aureus hypocotyls. The xyloglucans fromG. max and V. sesquipedalis had glucose, xylose, galactose andfucose in the approximate molar ratio of 10:6:4:1 and 10:7:3:1,respectively. However, the results of cellulase fragmentationanalysis of xyloglucans from the three species suggested thatthe basic structure of the xyloglucans in the cell walls ofthese bean-hypocotyls is almost the same; the structure is basedon two repeating oligosaccharide units, one of which consistsof glucose and xylose and the other of glucose, xylose, galactoseand fucose. ¹ Present address: Toppan Printing Co., Ltd., Okaji, Sendai980, Japan. (Received February 3, 1977; )     

Isolation and characterization of ferritin from soyabeans (Glycine max)

Ferritin from the soyabean Glycine max was isolated and characterized. The protein has many features in common with ferritin from mammalian systems, including extensive sequence homology, as determined by two-dimensional peptide mapping. No immunocross-reactivity between the plant and animal proteins was detected. The ferritin isolated by MgCl2 precipitation has a single subunit of 28 kDa, whereas the ferritin remaining in the supernatant exhibits marked heterogeneity, with a main subunit of 22 kDa. This form of the protein appears to be the result of specific proteolytic processing that is not affected by serine protease inhibitors, and appears only after the seeds have been soaked long enough to induce germination. The appearance of the 22-kDa form corresponds to the appearance of "crystalline arrays" of ferritin in the amyloplasts of the plant cotyledons and may represent a plant form of hemosiderin. In support of this hypothesis, the 22-kDa protein appears to be incompletely assembled, as determined by sucrose gradient centrifugation and iron uptake studies. Although ferritin is normally quite resistant to proteolysis, the 22-kDa protein is easily generated from the 28-kDa form by treatment with subtilisin, suggesting the presence of a specific, protease-sensitive sequence on the protein's surface, possibly used to mark the phytoferritin for conversion to hemosiderin and construction of ferritin crystalline arrays.     

Nodulins and nodulin genes of Glycine max

Summary Nodulins are organ-specific plant proteins induced during symbiotic nitrogen fixation. Nodulins play both metabolic and structural roles within infected and uninfected nodule cells. In soybean, several nodulin genes, coding for abundant nodulins, have been identified and isolated. Structural analysis of some of these genes has revealed their possible mode of regulation and the subcellar location of the protein product. Studies of ineffective symbiosis based on cultivar-strain genotype differences suggested that both partners influence the expression of nodulin genes. Concomitant with nodule organogenesis, the Rhizobium undergoes substantial differentiation leading to the accumulation of nodule-specific bacterial proteins, bacteroidins. The major structural alteration occuring in the infected cell is the formation of a membrane enclosing the bacteroid (peribacteroid membrane). A number of nodulins are specifically targetted to this membrane during endosymbiosis. The induction of nodulins and bacteroidins leads to the formation of an effective nodule. Nodulin genes can be induced in vitro by factors derived from nodules suggesting that trans-activators may be involved in derepression of the host genes necessary for Rhizobium-legume symbiosis.     

大豆异戊烯焦磷酸异构酶基因的电子克隆及生物信息学分析

利用电子克隆方法获得大豆异戊烯焦磷酸异构酶基因(IPI)的cDNA序列,并采用生物信息学方法对该基因及其编码蛋白的一般理化性质、疏水性、信号肽、二级结构和亚细胞定位等方面进行了预测和分析。结果表明,大豆异戊烯焦磷酸异构酶基因的cDNA序列全长1384bp,包含一个906bp的ORF,编码301个氨基酸。大豆IPI酶为一亲水性的非分泌蛋白,α-螺旋和无规则卷曲是其主要的二级结构,该酶定位于叶绿体。     

高质量提取大豆叶片总RNA的改良方法

豆科植物总RNA的提取相对比较困难,大豆叶片富含多糖、蛋白质,传统方法提取效果均不理想.在总RNA的提取方法Trizol法和热酚-氯化锂沉淀法的基础上,针对大豆叶片的特殊性,做了适当的改良,从而获得了完整性好,高纯度的大豆叶片总RNA,并成功进行了基因片段的RT-PCR实验,结果证实所获得的RNA完全可以适应后续实验要求.     

Purification and Characterization of S-Adenosyl-L-Methionine Nicotinic Acid-N-Methyltransferase from Leaves of Glycine max

Trigonelline (TRG), which act as a cell cycle regulator and a compatible solute in response to salinity and water-stress, is the N-methyl conjugate of nicotinic acid the formation of which is catalyzed by S-adenosyl-L-methionine nicotinic acid-N-methyltransferase. The enzyme was purified 2650-fold from soybean (Glycine max L.) leaves with a recovery of 4 %. The purification procedure included ammonium sulfate (45 – 60 %) precipitation, linear gradient DEAE-Sepharose chromatography, adenosine-agarose affinity chromatography, hydroxyapatite chromatography and gel filtration (Sephacryl-S-200). The purified enzyme preparation showed a major band with a molecular mass of 41.5 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis that is related to the enzyme activity. The native enzyme had a molecular mass of about 85 kDa as estimated by gel filtration. The K_m values for S-adenosyl-L-methionine and nicotinic acid were 31 and 12.5 M, respectively. The purified enzyme showed optimum activity at pH 6.5 and temperature of 40 – 45 °C. High concentration of dithiothreitol (10 mM) and glycerol (20 %) stabilize the enzyme during purification and storage. Hg²⁺ strongly inhibits enzyme activity.     

Purification and Properties of Arginase from Soybean, Glycine max, Axes

Arginase (EC 3.5.3.1) was purified to homogeneity from cytosol of soybean, Glycine max, axes by chromatographic separations on Sephadex G-200, DEAE-sephacel, hydroxyapatite, and arginine-affinity columns. The molecular weight of the enzyme estimated by pore gradient gel electrophoresis was 240,000, while sodium dodecyl sulfate polyacrylamide gel electrophoresis gave a single band at the molecular weight of 60,000. The optimal pH for activity was 9.5 and the K_m value was 83 millimolar. The enzyme was stimulated by polyamines such as putrescine.     

Identification and characterization of SSRs from soybean (Glycine max) ESTs

Microsatellites, or simple sequence repeats (SSRs), are very useful molecular markers for a number of plant species. We used a new publicly available module (TROLL) to extract microsatellites from the public database of soybean expressed sequence tag (EST) sequences. A total of 12,833 sequences containing di- to penta-type SSRs were identified from 200,516 non-redundant soybean ESTs. On average, one SSR was found per 7.25?kb of EST sequences, with the tri-nucleotide motifs being the most abundant. Primer sequences flanking the SSR motifs were successfully designed for 9,638 soybean ESTs using the software primer3.0 and only 59 pairs of them were found in earlier studies. We synthesized 124 pairs of the primers to determine the polymorphism and heterozygosity among eight genotypes of soybean cultivars, which represented a wide range of the cultivated soybean cultivars. PCR amplification products with anticipated SSRs were obtained with 81 pairs of primers; 36 PCR products appeared to be homozygous and the remaining 45 PCR products appeared to be heterozygous and displayed polymorphism among the eight cultivars. We further analysed the EST sequences containing 45 polymorphic EST-SSR markers using the programs BLASTN and BLASTX. Sequence alignment showed that 29 ESTs have homologous sequences and 15 ESTs could be classified into a Uni-gene cluster with comparatively convincing protein products. Among these 15 ESTs belonging to a Uni-gene cluster, 9 SSRs were located in 3'-UTR, 4 SSRs were located in the intron region and 2 SSRs were located in the CDS region. None of these SSRs was located in the 5'-UTR. These novel SSRs identified in the ESTs of soybean provide useful information for gene mapping and cloning in future studies.     

Crystallization and preliminary X-ray crystallographic analysis of plant ferritin from Glycine max

The iron storage protein ferritin from soybean (Glycine max) was expressed in E. coli and crystallized using the hanging drop vapor diffusion method with sodium tartrate as the precipitant. The crystals belong to the tetragonal I4(1)22 space group, with unit cell parameters a=b=324.0, c=182.7 A. The diffraction data were collected up to a resolution of 3.0 A with a multi-wire area detector.