mRNA of Leghemoglobins from Alfalfa Root Nodules

This paper described the methods of isolation, purification, and in vitro translation of alfalfa leghemoglobin mRNA. It was pointed out that the mRNA of alfalfa leghemoglobin accounts for about 1.4% of total RNA, and 24% of poly (A) +-mRNA of alfalfa nodule. This mRNA has 6, 9, 26S value populations with different molecular weights of about 0.15×106, 0.28×106, 0.48×l06 respectively. These S value mRNAs were coded for different components of leghemoglobin.     

Nitrate Metabolism in Alfalfa Root Nodules under Water Stress

Three-month-old plants (vegetative stage) of alfalfa (Medicagosaliva L cv. Aragon) were supplied for one week with 1.0dm³(uniformly distributed) nutrient solutions containing 0 or 20mol m^–3 . One week after initiation of treatment, the plants were subjected to drought by withholding water. Bacteroidsand cytosol of nodules were obtained at different stages ofstress, and used for enzyme assays and for determination of, and . Proteins of bacteroids were more stable than cytosolic proteinswith respect to the detrimental effects of water stress and. Protein contents of bacteroids and cytosol were inversely related to proteolytic activitiesagainst azocasein in both nodule fractions. Specific nitrate reductase activity (NRA) and nitrite reductaseactivity (NiRA) of bacteroids from -treated plants were inhibited by c. 70% and 45%, respectively, as leafwater potential (_w) declined from –0.5 MPa (control) to–1.8 MPa. At still lower _w both activities began to increase:NRA was doubled, whereas NiRA only returned to its control level.Cytosolic NRA was strongly inhibited by drought, but the correspondingNiRA remained constant. Ammonia concentration in bacteroids and nodule cytosol keptbasically constant, whereas accumulated in the cytosol at severe stress, due to the activationof bacteroid nitrate reductase. Results indicate that nitrate and nitrite reductases of thebacteroids and the nodule cytosol act in different form: assimilatory,the cytosolic enzymes; and dissimilatory, the enzymes of bacteroidsat low _w The possibility that assimilation of also occurs in bacteroids at control or mild waterstress conditions is suggested. Key words: Assimilatory and dissimilatory reduction, bacteroids, Medicago saliva, nodule cytosol, water stress     

浑球红假单胞菌（Rhodobacter sphaeroides）谷氨酸合酶的纯化及性质

浑球红假单胞菌菌株601经超声击碎,粗提液通过Triton处理,硫酸铵沉淀,DE—52和DEAE—sephadex A—50柱层析及 Seqhadex G—200凝胶过滤等步骤,将谷氨酸合酶(GOGAT)分离纯化,在聚丙烯酰胺凝胶电泳上呈现一条带。GOGAT表观分子量约为138 kD。该酶最大光吸收在278,375,450 nm和475 nm处,表明GOGAT可能是一种黄素蛋白。纯化的GOGAT对其底物 Gln,α—酮戊二酸和NADPH的表观K_m值分别为830,150和6μmol/L。反应产物Gln和NADP,几种氨基酸对GOGAT活力有不同程度的抑制作用,Gln类似物DON对GOGAT活力有强烈的抑制作用。     

Partial Purification of Digitonin-Solubilized beta-Glucan Synthase from Red Beet Root

An enriched glucan synthase fraction was obtained from red beet root microsomes by sequential extraction with the detergents 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and digitonin. The digitonin suspension was centrifuged on a glycerol gradient, where a glucan synthase peak with a specific activity of 30- to 40-fold over microsomes was recovered. Most protein contaminants were found in the gradient pellet. The glucan synthase-containing fraction was largely free of plasma membrane and tonoplast-derived ATPase activity and was enriched with a protein subunit of 68 kilodaltons.     

Alfalfa Root Nodule Carbon Dioxide Fixation : II. Partial Purification and Characterization of Root Nodule Phosphoenolpyruvate Carboxylase

A nonphotosynthetic phosphoenolpyruvate carboxylase (EC 4.1.1.31) was partially purified from the cytosol of root nodules of alfalfa. The enzyme was purified 86-fold by ammonium sulfate fractionation, DEAE-cellulose, hydroxylapatite chromatography, and reactive agarose with a final yield of 32%. The enzyme exhibited a pH optimum of 7.5 with apparent K_m values for phosphoenolpyruvate and magnesium of 210 and 100 micromolar, respectively. Two isozymes were resolved by nondenaturing polyacrylamide disc gel electrophoresis. Subsequent electrophoresis of these isozymes in a second dimension by sodium dodecyl sulfate slab gel electrophoresis yielded identical protein patterns for the isozymes with one major protein band at molecular weight 97,000. Malate and AMP were slightly inhibitory (about 20%) to the partially purified enzyme. Phosphoenolpyruvate carboxylase comprised approximately 1 to 2% of the total soluble protein in actively N₂-fixing alfalfa nodules.     

Purification of Hemoglobin from the Actinorhizal Root Nodules of Myrica gale L

Hemoglobins are generally absent or present in low concentrations in the nodules of actinorhizal plants. An exception is Casuarina, where a hemoglobin occurs at relatively high concentration. However, this plant is unique in that Frankia, the microsymbiont, lacks the vesicles that are normally the site of nitrogen fixation. The present paper shows that a hemoglobin also occurs at high concentrations in Myrica gale L., an actinorhizal plant in which Frankia does form vesicles. Hemoglobin was extracted from root nodules under anaerobic conditions using a buffer containing CO, detergent, and a reducing agent. Carboxyhemoglobin was purified using gel filtration followed by aerobic ion-exchange chromatography. The optical absorption spectra of the oxy-, deoxy-, and carboxyhemoglobins were similar to those of other hemoglobins. The molecular mass of the native hemoglobin estimated by gel filtration was 38,500 D. The molecular mass of the subunits estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 16,200 D, consistent with the mass of other hemoglobin subunits. Thus, the native hemoglobin is probably a dimer.     

Purification and Characterization of a Trehalose Synthase from the Basidiomycete Grifola frondosa

A trehalose synthase (TSase) that catalyzes the synthesis of trehalose from d-glucose and α-d-glucose 1-phosphate (α-d-glucose 1-P) was detected in a basidiomycete, Grifola frondosa. TSase was purified 106-fold to homogeneity with 36% recovery by ammonium sulfate precipitation and several steps of column chromatography. The native enzyme appears to be a dimer since it has apparent molecular masses of 120 kDa, as determined by gel filtration column chromatography, and 60 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Although TSase catalyzed the phosphorolysis of trehalose to d-glucose and α-d-glucose 1-P, in addition to the synthesis of trehalose from the two substrates, the TSase equilibrium strongly favors trehalose synthesis. The optimum temperatures for phosphorolysis and synthesis of trehalose were 32.5 to 35°C and 35 to 37.5°C, respectively. The optimum pHs for these reactions were 6.5 and 6.5 to 6.8, respectively. The substrate specificity of TSase was very strict: among eight disaccharides examined, only trehalose was phosphorolyzed, and only α-d-glucose 1-P served as a donor substrate with d-glucose as the acceptor in trehalose synthesis. Two efficient enzymatic systems for the synthesis of trehalose from sucrose were identified. In system I, the α-d-glucose 1-P liberated by 1.05 U of sucrose phosphorylase was linked with d-glucose by 1.05 U of TSase, generating trehalose at the initial synthesis rate of 18 mmol/h in a final yield of 90 mol% under optimum conditions (300 mM each sucrose and glucose, 20 mM inorganic phosphate, 37.5°C, and pH 6.5). In system II, we added 1.05 U of glucose isomerase and 20 mM MgSO₄ to the reaction mixture of system I to convert fructose, a by-product of the sucrose phosphorylase reaction, into glucose. This system generated trehalose at the synthesis rate of 4.5 mmol/h in the same final yield.Trehalose (1-α-d-glucopyranosyl-α-d-glucopyranoside) is a nonreducing disaccharide with an α,α-1,1 glycosidic linkage and is widely distributed in plants, insects, fungi, yeast, and bacteria (7). Due to the absence of reducing ends in trehalose, it is highly resistant to heat, pH, and Maillard’s reaction (24). In trehalose-producing organisms, this compound may serve as an energy reserve, a buffer against stresses such as desiccation and freezing, and a protein stabilizer (5, 7, 26, 31, 32). If trehalose can be produced economically, then it has potential commercial applications as a sweetener, a food stabilizer, and an additive in cosmetics and pharmaceuticals (6, 25). Recently, trehalose production through fermentation of yeast (17) and Corynebacterium (30), enzymatic processes from starch (18, 34) and maltose (19, 22, 23, 33), and extraction from transformed plants (10) has been reported.Our approach to trehalose production is to use an enzymatic process to produce trehalose from sucrose, one of the least expensive sugars. Since sucrose is efficiently converted to α-d-glucose 1-phosphate (α-d-glucose 1-P) and fructose by sucrose phosphorylase (SPase), we screened microorganisms for an enzyme that converts α-d-glucose 1-P to trehalose on the assumption that the combination of the putative trehalose synthase (TSase) and SPase would convert sucrose into trehalose. Although similar enzyme activities have been reported in the basidiomycete Flammulina velutipes (11) and in the yeast Pichia fermentans (27), these enzymes have not been well characterized.Our objectives were (i) to screen microorganisms, primarily fungi, for TSase activity; (ii) to purify and characterize the TSase; (iii) to identify the enzymatic process by which trehalose is produced from sucrose; and (iv) to identify an enzymatic process for production of trehalose from sucrose in which the fructose component is also converted to trehalose.     

花生根瘤菌类菌体氢酶的分离提纯及特性

花生根瘤菌类菌体经超声波破碎,ＴｒｉｔｏｎＸ－１００溶解,正已烷－硫酸铵处理后,再经ＤＥＡＥ－纤维素和Ｓｅｐｈａｃｒｙｌ凝胶柱层析等纯化步骤,获得凝胶电泳纯的膜结合态氢酶,比活为７１．４μｍｏｌＨ２ｍｇ－１Ｐｒｏｔｍｉｎ－１,为类菌体吸Ｈ２活性的２１１倍。纯化的氢酶分子量为１１０ｋＤ。经ＳＤＳ－ＰＡＧＥ后,呈现两个蛋白带,分子量分利为６５ｋＤ和３５ｋＤ。纯酶的Ｎｉ含量为０．６２ｍｏｌＮｉ／ｍｏｌ氢酶。在磷酸缓冲液中其活性的最适ｐＨ为６．５。ＤＣＩＰ、亚甲蓝、铁氰化钾、细胞色素Ｃ均可作为氢酶的电子受体,其中以ＤＣＩＰ为最适。     

嗜酸氧化亚铁硫杆菌半肤氨酸合成酶的表达、纯化及其性质鉴定

半胱氨酸合成酶是半胱氨酸合成反应的关键限速酶,催化了半胱氨酸合成的最后一个步骤。以A.ferro-oxidans ATCC 23270基因组为模板,通过PCR扩增得到编码半胱氨酸合成酶(cysM)的基因,与原核表达载体PLM 1构建重组体,转入大肠杆菌DH5α中,测序正确后,加IPTG诱导表达,用一步亲和层析法纯化出了浓度和纯度都较高的半胱氨酸合成酶。由蛋白颜色和紫外分析,确定是以PLP辅基作为活性中心。表达产物进行SDS-PAGE分析,证实分子量为32 kD。     

Characterization of delta-Aminolevulinic Acid Formation in Soybean Root Nodules

Formation of the heme precursor δ-aminolevulinic acid (ALA) was studied in soybean root nodules elicited by Bradyrhizobium japonicum. Glutamate-dependent ALA formation activity by soybean (Glycine max) in nodules was maximal at pH 6.5 to 7.0 and at 55 to 60°C. A low level of the plant activity was detected in uninfected roots and was 50-fold greater in nodules from 17-day-old plants; this apparent stimulation correlated with increases in both plant and bacterial hemes in nodules compared with the respective asymbiotic cells. The glutamate-dependent ALA formation activity was greatest in nodules from 17-day-old plants and decreased by about one-half in those from 38-day-old plants. Unlike the eukaryotic ALA formation activity, B. japonicum ALA synthase activity was not significantly different in nodules than in cultured cells, and the symbiotic activity was independent of nodule age. The lack of symbiotic induction of B. japonicum ALA synthase indicates either that ALA formation is not rate-limiting, or that ALA synthase is not the only source of ALA for bacterial heme synthesis in nodules. Plant cytosol from nodules catalyzed the formation of radiolabeled ALA from U-[¹⁴C]glutamate and 3,4-[³H]glutamate but not from 1-[¹⁴C]glutamate, and thus, operation of the C₅ pathway could not be confirmed.     

Production and Characterization of Monoclonal Antibodies against Aspartate Aminotransferase-P1 from Lupin Root Nodules

Six hybridoma clones were obtained that secreted monoclonal antibodies against the aspartate aminotransferase-P1 (AAT-P1) isoenzyme from root nodules of Lupinus angustifolius [L.] cv Uniharvest. This enzyme is found constitutively in the plant cytosol fraction. The monoclonal antibodies produced were all of the immunoglobulin G1 class, recognized two distinct epitopes on the protein, and represented the major paratopes found in the immunoglobulin fraction of sera taken from mice and rabbits immunized with the pure AAT-P1 protein. One of these epitopes was unique to lupin nodule AAT-P1. The other epitope was shown to be present on enzyme from lupin bean, white clover and tobacco leaves, lupin roots and nodules, and potato tubers. Both epitopes were recognized by the appropriate monoclonal antibodies in both their native and denatured forms. None of the monoclonal antibodies produced reacted with Rhizobium lupini NZP2257, Escherichia coli extracts, or with the inducible aspartate aminotransferase-P2 (AAT-P2) isoform also found in root nodules. A sandwich enzyme-linked immunosorbent assay utilizing two monoclonal antibodies recognizing the two distinct epitopes was developed and was capable of quantitating AAT-P1 in plant extracts. The limit of detection of AAT-P1 was less than 15 pg/mL and AAT-P1 protein could be quantified in the range 80 to 1000 pg/mL. Using this assay, AAT-P1 protein was shown to remain relatively constant during nodule development. Use of an AAT-P2-specific monoclonal antibody that inhibits the enzyme activity of this isoform enabled the direct determination of AAT-P1 enzyme activity in nodule extracts. Using these assays, specific activities of the individual isoforms were calculated; that of the AAT-P1 isoform was shown to be 7.5-fold higher than that of the AAT-P2 isoform.     

Intracellular Localization and Properties of NADH-Glutamate Dehydrogenase from Vitis vinifera L.: Purification and Characterization of the Major Leaf Isoenzyme

Glutamate dehydrogenase was partially purified from grapevine(Vitis vinifera L. cv. Soultanina) tissues and its activityand isoenzymic pattern were studied. Seven anodal migratingisoenzymes were revealed after PAGE. Leaf protoplasts were isolatedfrom in vitro-grown axenic shoot cultures and used to studythe intracellular localization of GDH. Results revealed thatthe enzyme was associated with the mitochondrial fraction. Theisoenzyme with the lowest electrophoretic mobility, which accountedfor 35 to 40% of total activity, was purified 2050-fold to homogeneityfrom leaves. The purification method included ammonium sulphatefractionation, DEAE-cellulose chromatography, Sephadex G-200gel filtration and NAD-sepharose affinity chromatography. Themolecular weight of the native enzyme was estimated to be 252kDa and it consisted of identical 42.5 kDa subunits. pH optimumfor the aminating reaction was 8.0 and for the deaminating reaction9.3. At optimum pH conditions the apparent K_m values for ammonium,as ammonium chloride and ammonium sulphate, -ketoglutarate,NADH, glutamate, and NAD⁺ were 45.0, 13.0, 2.1, 0.069, 18.0,and 0.195 mM, respectively. The amination reaction of GDH wasfully activated with about 100 µM Ca²⁺ while the deaminationreaction was not affected by the addition of Ca²⁺. The isoenzymesof GDH showed different magnitude of their activating responseto calcium ions. Key words: Vitis vinifera L., glutamate dehydrogenase     

Two Isoenzymes of NADH-dependent Glutamate Synthase in Root Nodules of Phaseolus vulgaris L: Purification, Properties and Activity Changes during Nodule Development

The specific activity of plant NADH-dependent glutamate synthase (NADH-GOGAT) in root nodules of Phaseolus vulgaris L. is over threefold higher than the specific activity of ferredoxin-dependent GOGAT. The NADH-GOGAT is composed of two distinct isoenzymes (NADH-GOGAT I and NADH-GOGAT II) which can be separated from crude nodule extracts by ion-exchange chromatography. Both NADH-GOGAT isoenzymes have been purified to apparent homogeneity and shown to be monomeric proteins with similar M_rs of about 200,000. They are both specific for NADH as reductant. An investigation of their kinetic characteristics show slight differences in their K_ms for l-glutamine, 2-oxoglutarate, and NADH, and they have different pH optima, with NADH-GOGAT I exhibiting a broad pH optimum centering at pH 8.0 whereas NADH-GOGAT II has a much narrower pH optimum of 8.5. The specific activity of NADH-GOGAT in roots is about 27-fold lower than in nodules and consists almost entirely of NADH-GOGAT I. During nodulation both isoenzymes increase in activity but the major increase is due to NADH-GOGAT II which increases over a time course similar to the increase in nitrogenase activity. This isoenzyme is twice as active as NADH-GOGAT I in mature nodules. The roles and regulation of these two isoenzymes in the root nodule are discussed.     

Purification and Characterization of Sucrose Synthase from the Cotyledons of Vicia faba L

Partial purification (approximately 270-fold) of sucrose synthase (EC 2.4.1.13) from developing cotyledons of Vicia faba L. cv Maris Bead was achieved by ammonium sulfate fractionation and hydrophobic, affinity, anion-exchange, and gel filtration chromatography. Further purification to homogeneity resulted from gel elution of single bands from native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was identified as a homotetramer with a total molecular mass of 360 kD and subunits of 92 to 93 kD. Antibodies were raised to both native and denatured protein. The identity of the polypeptide was confirmed in western blots using antibodies raised against soybean nodule sucrose synthase. The enzyme has a pH optimum of 6.4 (cleavage direction) and an isoelectric point of 5.5. The affinity of the enzyme for sucrose (K_m) was estimated at 169 mm, and for UDP at 0.2 mm. With uridine diphosphate as the nucleoside diphosphate, the V_max is 4-fold higher than with adenosine diphosphate. Fructose acts as a competitive inhibitor with an inhibitor constant (K_i) of 2.48 mm.     

Purification and Characterization of Sucrose Synthase from Peach (Prunus persica) Fruit

Sucrose synthase (EC 2.4.1.13 [EC] ) was purified from peach fruit(Prunus persica) to a single band of protein on SDS-PAGE byammonium sulfate fractionation, DEAE-cellulose (DE-52) chromatography,Sepharose CL-6B gel filtration, PBA-60 affinity chromatographyand Sephadex G-200 gel filtration. The molecular weight wasestimated to be 360,000 by gel filtration. The enzyme was foundto be a tetramer of identical 87-kDa subunits. The maximum activityfor the synthesis and cleavage of sucrose was observed at pH8.5 and pH 7.0, respectively. The enzymatic reaction followedtypical Michaelis-Menten kinetics in both directions, with thefollowing parameters: K_m(fructose), 4.8 mmM; K_m(UDPglucose),0.033 mM; K_m(sucrose), 62.5 mM; K_m(UDP), 0.080 mM. Other properties,such as substrate specificity and the effects of divalent cations,were also investigated. The relationship between the enzymeand the accumulation of sucrose in peach fruit is discussed. Present address: Laboratory of Horticulture, Faculty of Agriculture,Nagoya University, Chikusa, Nagoya 464, Japan. (Received May 2, 1988; Accepted September 14, 1988)     

Purification and Characterization of a Ferredoxin-NADP Oxidoreductase-Like Enzyme from Radish Root Tissues

An enzyme able to reduce cytochrome c via ferredoxin in the presence of NADPH, was isolated, purified from radish (Raphanus sativus var acanthiformis cultivar miyashige) roots and characterized. The enzyme was purified by DEAE-cellulose, Blue-Cellulofine, Ferredoxin-Sepharose 4B, and Sephadex G-100 column chromatography. Molecular mass of the enzyme was estimated to be 33,000 and 35,000 daltons by Sephadex G-100 gel filtration and SDS-PAGE, respectively. Its absorption spectrum suggested that the enzyme contains flavin as a prosthetic group. The K_m values for NADPH and ferredoxin were calculated to be 9.2 and 1.2 micromolar, respectively. The enzyme required NADPH and did not use NADH as an electron donor. The optimal pH was 8.4. The enzyme also catalyzed the photoreduction of NADP⁺ in the spinach leaf thylakoid membranes depleted of ferredoxin and ferredoxin-NADP⁺ oxidoreductase. The effect of NaCl and MgCl₂ concentration on the activity and amino acid composition of the enzyme were demonstrated. The results suggest that the enzyme is similar to ferredoxin-NADP⁺ oxidoreductase from chloroplasts and cyanobacteria and is the key enzyme catalyzing the electron transport between NADPH, generated by the pentose phosphate pathway, and ferredoxin in plastids of plant heterotrophic tissues.     

Isolation,Purification, and Characterization of Acetolactate Synthase from a Lactococcus lactis Culture

Acetolactate synthase catalyzing the synthesis of -acetolactate was isolated from lactic acid bacteria Lactococcus lactissubsp. lactisbiovar. diacetylactis4 and purified. Acetolactate synthase was shown to be an allosteric enzyme with low affinity for the substrate: the K _Mfor pyruvate was 70 mM. The curve relating the dependence of enzyme activity to pyruvate concentration had a sigmoid shape. The enzyme activity persisted for 24 h in the presence of stabilizers, pyruvate, and thiamine pyrophosphate. Acetolactate synthase had pH optimums of 5.8 and 6.5–7.0 in acetate and phosphate buffers, respectively. The temperature optimum for this enzyme was 38–40°C at pH 6.5. The molecular weight of acetolactate synthase was 150 kDa. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the enzyme consisted of three identical subunits with a molecular weight of 55 kDa.     

Production and Characterization of Monoclonal Antibodies against Aspartate Aminotransferase-P(2) from Lupin Root Nodules

Twenty-one monoclonal antibodies were raised against the aspartate aminotransferase-P₂ isoenzyme from root nodules of Lupinus angustifolius [L.] cv Uniharvest. Induction of this isoenzyme is positively correlated with the onset of N₂ fixation in effective root nodules and is associated with the assimilation of ammonia by the plant in the Rhizobium-legume symbiosis. The monoclonal antibodies produced were all of the IgG class, recognized five different epitopes on the protein, and represented greater than 90% of the available epitopes. These epitopes were not unique to lupin nodule aspartate aminotransferase-P₂ but were shown to be present on the enzyme from tobacco leaves and potato. Four of the epitopes were conformational with a fifth epitope recognized by the appropriate monoclonals in both its native and denatured forms. None of the monoclonal antibodies produced reacted with Rhizobium Iupini NZP2257 extracts. Antibodies against two epitopes showed some cross-reaction with the constitutive aspartate aminotransferase-P₁ isoenzyme also found in lupin root nodules. However, affinity of these monoclonals for AAT-P₁ was three orders of magnitude lower than for AAT-P₂. Monoclonals against the other epitopes appeared to be specific for aspartate aminotransferase-P₂.