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.     

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.     

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.     

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.     

An NAD(P) reductase derived from Chlorobium thiosulfa-tophilum: Purification and some properties

A highly purified preparation of an NAD(P) reductase was obtained from Chlorobium thiosulfatophilum and some of its properties were studied. The enzyme possesses FAD as the prosthetic group, and reduces benzyl viologen, 2,6-dichloro-phenolindophenol and cytochromes c, including cytochrome c-555 (C. thiosulfato-philum), with NADPH or NADH as the electron donor. It reduces NADP⁺ or NAD⁺ photosynthetically with spinach chloroplasts in the presence of added spinach ferredoxin. It reduces the pyridine nucleotides with reduced benzyl viologen. The enzyme also shows a pyridine nucleotide transhydrogenase activity. In these reactions, the type of pyridine nucleotide (NADP or NAD) which functions more efficiently with the enzyme varies with the concentration of the nucleotide used; at concentrations lower than approx. 1.0 mM, NADPH (or NADP⁺) is better electron donor (or acceptor), while NADH (or NAD⁺) is a better electron donor (or acceptor) at concentrations higher than approx. 1.0 mM. Reduction of dyes or cytochromes c catalysed by the enzyme is strongly inhibited by NADP⁺, 2′-AMP and and atebrin.     

Growth and onto-morphogenesis of soybean (Glycine max Merril) in an open, naturally CO2-enriched environment

Springs emitting carbon dioxide are frequent in Central Italy and provide a way of testing the response of plants to CO₂ enrichment under natural conditions. Results of a CO₂ enrichment experiment on soybean at a CO₂ spring (Solfatara) are presented. The experimental site is characterized by significant anomalies in atmospheric CO₂ concentration produced by a large number of vents emitting almost pure CO₂ (93%) plus small amounts of hydrogen sulphide, methane, nitrogen and oxygen. Within the gas vent area, plants were grown at three sub-areas whose mean CO₂ concentrations during daytime were 350,652 and 2370 μmol mol^-1, respectively. Weekly harvests were made to measure biomass growth, leaf area and ontogenetic development. Biomass growth rate and seed yield were enhanced by elevated CO₂. In particular, onto-morphogenetic development was affected by elevated CO₂ with high levels of CO₂ increasing the total number of main stem leaf nodes and the area of the main stem trifoliolate leaves. Biochemical analysis of plant tissue suggested that there was no effect of the small amounts of H₂S on the response to CO₂ enrichment. Non-protein sulphydryl compounds did not accumulate in leaf tissues and the overall capacity of leaf extracts to oxidize exogenously added NADH was not decreased. The limitations and advantages of experimenting with crop plants at elevated CO₂ in the open and in the proximity of carbon dioxide springs are discussed.     

Purification and properties of an alcohol dehydrogenase (HUADHII) from Hanseniaspora uvarum K5

An alcohol dehydrogenase HUADHII was purified 43.2-fold from Hanseniaspora uvarum K₅. The enzyme was trimeric with subunits of mol. wt 42 kDa. The N -terminal amino acid sequence of HUADHII has between 45 and 75% identity with part of the sequence of isoenzymes related to group I from Saccharomyces cerevisiae and Kluyveromyces lactis. C2–C4 alcohols and aldehydes were the preferred substrates. The presence of an'α'double bond increased the enzyme activity both for alcohols and aldehydes. It was significantly inhibited by metal-binding agents and thiol reagents. Kinetic studies suggested that HUADHII catalyses the oxidation of ethanol by a random sequential mechanism. It appears that HUADHII, a cytoplasmic fermentative enzyme, is structurally and functionally similar to members of the group I alcohol dehydrogenases.     

Purification and properties of methanol dehydrogenase from Methylophaga marina

Purification of methanol dehydrogenase from Methylophaga marina, in order to avoid the instability observed in crude extracts, was achieved initially by a rapid procedure using mainly an aqueous two-phase partition system composed of polyethylene glycol 1000 (50%, v/v) and potassium phosphate (50%, w/v). The purified enzyme gave a single band of protein after SDS-polyacrylamide gel electrophoresis. Antiserum raised against purified methanol dehydrogenase was used to detect possible protein contaminants in the enzyme preparation. The enzyme is an NAD-independent dehydrogenase containing pyrrolquinoline quinone (PQQ) as a prosthetic group. It is made of two apparently identical subunits giving a total MW of 145,000 for the native enzyme. The isoelectric point is 6.4. In addition to methanol and formaldehyde, multicarbon primary alcohols and aldehydes as well as secondary alcohols can be used as substrates. Except for ammonium chloride, which is a necessary activator in vitro, no effector was found which could modify the rate of enzyme activity under standard conditions of the assay. Although the main properties of this methanol dehydrogenase are similar to those already described in the literature, it does not belong in any of the five categories described by Anthony.     

Purification and identification of an FMN-dependent NAD(P)H azoreductase from Enterococcus faecalis

Azoreductases reduce the azo bond (N=N) in azo dyes to produce colorless amine products. Crude cell extracts from Enterococcus faecalis have been shown to utilize both NADH and NADPH as electron donors for azo dye reduction. An azoreductase was purified from E. faecalis by hydrophobic, anion exchange and affinity chromatography. The azoreductase activity of the purified preparation was tested on a polyacrylamide gel after electrophoresis under native conditions and the protein that decolorized the azo dye (Methyl Red) with both NADH and NADPH was identified by mass spectrometry to be AzoA. Previously, the heterologously expressed and purified AzoA was shown to utilize NADH only for the reduction of Methyl Red. However, AzoA purified from the wild-type organism was shown to utilize both coenzymes but with more than 180-fold preference for NADH over NADPH as an electron donor to reduce Methyl Red. Also, its specific activity was more than 150-fold higher than the previous study on AzoAwhen NADH was used as the electron donor. The catalytic efficiency for Methyl Red reduction by AzoA from E. faecalis was several orders of magnitude higher than other azoreductases that were purified from a heterologous source.