Partial Purification of a Soluble Gibberellin-Binding Protein from Mung Bean Hypocotyls

A soluble binding protein specific for GA₄, GA₇ and GA₉ waspartially purified from mung bean hypocotyls, and its characteristicswere examined. Affinity chromatography using immobilized GA₃coupled to Sepharose 4B via the C-7 carboxyl group was veryeffective for purification of the protein. The molecular weightof the protein in its native state was estimated to be 150–200kDa by gel-permeation chromatography. This protein may be aheterooligomer consisting of two subunits (23 kDa and 35 kDa).The optimum pH for binding of GA₄ to the protein was around6.0 and the apparent dissociation constant (K_d) was 310^-7 M. (Received April 24, 1992; Accepted December 16, 1992)     

Purification of a Soluble Cytockinin-binding Protein from Etiolated Mung Bean Seedlings

A cytokinin-binding protein (CBP) was purified from a crude extract of etiolated mung bean seedlings by a protocol involving affinity chromatography on benzyladenine-linked Sepharose 4B, ion exchange chromatography on DEAE-Sephadex A50, and gel filtration on Sphacryl S-400. The molecular weight was estimatd to be about 200,000 by gel filtration. CBP appeared as two bands corresponding to molecular weights of about 45,000 and 48,000 on SDS-polyacrylamide gel electrophoresis. The dissociation constant for benzyladenine was 7.5 x 10^-7 M. ¹⁴C-Benzyladenine-binding to CBP was reversible and could be inhibited by the addition of kinetin or trans-zeatin. Adenine, AMP, and ADP had no inhibitory effect on the binding of ¹⁴C-benzyladenine to CBP but the addition of ATP to the assay mixture enhanced the binding.     

Isolation and Characterization of a Soluble NADPH-Dependent Fe(III) Reductase from Geobacter sulfurreducens

NADPH is an intermediate in the oxidation of organic compounds coupled to Fe(III) reduction in Geobacter species, but Fe(III) reduction with NADPH as the electron donor has not been studied in these organisms. Crude extracts of Geobacter sulfurreducens catalyzed the NADPH-dependent reduction of Fe(III)-nitrilotriacetic acid (NTA). The responsible enzyme, which was recovered in the soluble protein fraction, was purified to apparent homogeneity in a four-step procedure. Its specific activity for Fe(III) reduction was 65 micromol. min(-1). mg(-1). The soluble Fe(III) reductase was specific for NADPH and did not utilize NADH as an electron donor. Although the enzyme reduced several forms of Fe(III), Fe(III)-NTA was the preferred electron acceptor. The protein possessed methyl viologen:NADP(+) oxidoreductase activity and catalyzed the reduction of NADP(+) with reduced methyl viologen as electron donor at a rate of 385 U/mg. The enzyme consisted of two subunits with molecular masses of 87 and 78 kDa and had a native molecular mass of 320 kDa, as determined by gel filtration. The purified enzyme contained 28.9 mol of Fe, 17.4 mol of acid-labile sulfur, and 0.7 mol of flavin adenine dinucleotide per mol of protein. The genes encoding the two subunits were identified in the complete sequence of the G. sulfurreducens genome from the N-terminal amino acid sequences derived from the subunits of the purified protein. The sequences of the two subunits had about 30% amino acid identity to the respective subunits of the formate dehydrogenase from Moorella thermoacetica, but the soluble Fe(III) reductase did not possess formate dehydrogenase activity. This soluble Fe(III) reductase differs significantly from previously characterized dissimilatory and assimilatory Fe(III) reductases in its molecular composition and cofactor content.     

Purification and Properties of a Glycoprotein Processing alpha-Mannosidase from Mung Bean Seedlings

The microsomal fraction of mung bean seedlings contains mannosidase activities capable of hydrolyzing [³H]mannose from the [³H]Man₉GlcNAc as well as for releasing mannose from p-nitrophenyl-α-d-mannopyranoside. The glycoprotein processing mannosidase was solubilized from the microsomes with 1.5% Triton X-100 and was purified 130-fold by conventional methods and also by affinity chromatography on mannan-Sepharose and mannosamine-Sepharose. The final enzyme preparation contained a trace of aryl-mannosidase, but this activity was inhibited by swainsonine whereas the processing enzyme was not. The pH optimum for the processing enzyme was 5.5 to 6.0, and activity was optimum in the presence of 0.1% Triton X-100. The enzyme was inhibited by ethylenediaminetetraacetate while Ca²⁺ was the most effective cation for reversing this inhibition. Mn²⁺ was considerably less effective than Ca²⁺ and Mg²⁺ was without effect. The processing mannosidase was inhibited by α1,2- and α1,3-linked mannose oligosaccharides (50% inhibition at 3 millimolar), whereas free mannose and α1,6-linked mannose oligosaccharides were ineffective. Mannosamine was also an inhibitor of this enzyme. The aryl-mannosidase and the processing mannosidase could also be distinguished by their susceptibility to various processing inhibitors. The aryl-mannosidase was inhibited by swainsonine and 1,4-dideoxy-1,4-imino-d-mannitol but not by deoxymannojirimycin or other inhibitors, while the processing mannosidase was only inhibited by deoxymannojirimycin. The processing mannosidase was incubated for long periods with [³H]Man₉GlcNAc and the products were identified by gel filtration. Even after a 24 hour incubation, the only two radioactive products were Man₅GlcNAc and free mannose. Thus, this enzyme appears to be similar to the animal processing enzyme, mannosidase I, and is apparently a specific α1,2-mannosidase.     

Purification and Characterization of Four NADPH-Dependent Aldehyde Reductases from Pig Brain

By a procedure involving ammonium sulfate precipitation, gel filtration, and affinity chromatography, four aldehyde reductases (ALRs) were purified to enzymatic homogeneity from pig brain. These enzymes, designated ALR1, ALR2, ALR3, and succinic semialdehyde reductase were chemically and physically identical with, respectively, the high-Km aldehyde reductase, the low-Km aldehyde reductase, carbonyl reductase, and succinic semialdehyde reductase of other tissues and species. The purification procedure allows the purification of these enzymes from the same tissue homogenate in amounts sufficient for characterization and other enzymatic studies. This methodology should be applicable to the simultaneous and rapid purification of aldehyde reductases from other tissues.     

Purification and Characterization of a Cytochrome P450 (trans-Cinnamic Acid 4-Hydroxylase) from Etiolated Mung Bean Seedlings

A Cyt P450 (P450_C4H) possessing trans-cinnamate 4-hydroxylase(C4H) activity was purified to apparent homogeneity from microsomesof etiolated mung bean seedlings. Upon SDS-polyacrylamide gelelectrophoresis, the purified preparation gave a single proteinband with a molecular mass of 58-kDa. Its specific P450 contentwas 12.6 nmol (mg protein)^–1. Using NADPH as electrondonor, purified P450_C4H aerobically converted trans-cinnamicacid to p-coumaric acid with a specific activity of 68 nmolmin^–1 nmol^–1 P450 in a reconstituted system containingNADPH-Cyt P450 reductase purified from the seedlings or rabbitliver microsomes, dilauroyl phosphatidylcholine, and cholate.This specific activity is by far the highest for reconstitutedC4H systems so far reported and provides direct evidence thatC4H activity is actually associated with a P450 protein. Inthe oxidized state P450_C4H showed a typical low-spin type absorptionspectrum with a Soret peak at 419 nm. A partial spectral shiftto the high spin state was observed when trans-cinnamic acidwas added to oxidized P450_C4H. By spectral titration, the dissociationconstant of the cinnamic acid-P450_C4H complex was determinedto be 2.8 µM. This value is similar to the K_m value (1.8µM) for trans-cinnamic acid determined in the reconstitutedsystem. (Received November 20, 1992; Accepted February 17, 1993)     

Characterization of a Low-Molecular-Weight Glutenin Subunit Gene from Bread Wheat and the Corresponding Protein That Represents a Major Subunit of the Glutenin Polymer

Both high- and low-molecular-weight glutenin subunits (LMW-GS) play the major role in determining the viscoelastic properties of wheat (Triticum aestivum L.) flour. To date there has been no clear correspondence between the amino acid sequences of LMW-GS derived from DNA sequencing and those of actual LMW-GS present in the endosperm. We have characterized a particular LMW-GS from hexaploid bread wheat, a major component of the glutenin polymer, which we call the 42K LMW-GS, and have isolated and sequenced the putative corresponding gene. Extensive amino acid sequences obtained directly for this 42K LMW-GS indicate correspondence between this protein and the putative corresponding gene. This subunit did not show a cysteine (Cys) at position 5, in contrast to what has frequently been reported for nucleotide-based sequences of LMW-GS. This Cys has been replaced by one occurring in the repeated-sequence domain, leaving the total number of Cys residues in the molecule the same as in various other LMW-GS. On the basis of the deduced amino acid sequence and literature-based assignment of disulfide linkages, a computer-generated molecular model of the 42K subunit was constructed.     

Purification and Characterization of Dehydroascorbate Reductase from Rice

Dehydroascorbate reductase (DHAR; EC 1.8.5.1[EC]) is an enzyme thatis critical for maintenance of an appropriate level of ascorbatein plant cells. This report describes the purification and characterizationof a GSH-dependent DHAR from rice (Oryza saliva) bran and isthe first, to our knowledge, of such an analysis of DHAR froma monocot. The enzyme was a monomeric thiol enzyme, resemblingDHARs purified from dicots, but it was different from them interms of heat stability and antigenicity. The amino-terminalamino acid sequence of the DHAR from rice did not show any obvioussimilarity to those of known proteins with DHAR activity, suchas, glutaredoxin (thioltrans-ferase), protein disulfide isomerase,and trypsin inhibitor. Immunoprecipitation analysis showed thatthis enzyme was a major DHAR in etiolated seedlings. Westernblot analysis indicated that this enzyme was distributed ubiquitouslyin rice tissues. A similar protein was found in barley but notin dicots. (Received July 18, 1996; Accepted December 4, 1996)     

Isolation and Identification of Eutypa lata from Pistacia vera in Greece

The fungus Eutypa lata (syn. E. armeniacae), known as the causal agent of the death of many different woody plants, was found on dead branches of pistachio (Pistacia vera L.) in Greece. Isolations from diseased branches yielded consistently typical colonies of the asexual stage of the fungus (Libertella blepharis, syn. Cytosporina sp.), which proved to be undistinguishable from other cultures of the pathogen obtained from 15 different hosts. Furthermore, all isolates from pistachio tested for pathogenicity on apricot were pathogenic and yielded characteristic cankers.     

嗜酸氧化亚铁硫杆菌APS还原酶的表达、纯化及其性质鉴定

嗜酸氧化亚铁硫杆菌(Acidithiobacillus ferrooxidans)中APS还原酶是硫同化途径的一个关键酶,其对硫酸盐的还原及硫化物的氧化具有重要调节作用.本文以A.ferrooxidans ATCC23270基因组为模板.通过PCR扩增得到编码APS还原酶的cysH基因,与原核表达载体pLM l构建重组体,转化入大肠杆茵(Escherichia coli,E.coli)DH5a中,测序正确后,加IPTG诱导表达,用一步亲和层析法纯化出浓度和纯度都较高的APS还原酶.由蛋白颜色和紫外分析,确定其含有一个[Fe4S4]簇作为活性中心.表达产物进行SDS-PAGE分析,证实分子量为28 kD.酶活测定表明其具有将APS还原为亚硫酸盐跟AMP的功能.     

Characterization of Proteins of Nuclear Envelopes Prepared from Mung Bean Hypocotyls

The polypeptide composition of nuclear envelopes prepared fromhypocotyls of mung bean (Vigna radiata) was investigated. Thetissue was homogenized in the presence of Triton X-100 and nucleiwere isolated by differential and discontinuous Percoll gradientcentrifugation. The nuclei were subjected to sonication in 2M KC1 or 50 mM lithium diiodosalicylate and then the nuclearenvelopes were collected by centrifugation. Proteins in theenvelope fraction were analyzed by sodium dodecylsulfate-polyacrylamidegel electrophoresis and blotting techniques. When the envelopefraction was incubated with [-³²P]ATP, 10 to 15 polypeptideswere labeled and the intensity of labeling of some of thesepolypeptides was enhanced by the addition of calcium ions. Theresults suggest the presence of a protein-phosphorylation systemin nuclear envelopes. Three polypeptides of 100, 42, and 40kDa stained blue with the cationic carbocyanine dye "Stains-all",and they were labeled with ⁴⁵Ca²⁺ on a transfer membrane. Thelectin concanavalin A recognized glycoproteins that migratedas polypeptides of 50, 49, 47, 43, 35 and 32 kDa, respectively.Of these polypeptides the two larger ones were prominent andwere solubilized by treatment of the envelope fraction withKCl at 2 M but not at less than 100 mM. These results suggestthat the mung bean nuclear envelope contains some calcium-bindingproteins and glycoproteins. These newly identified proteinsmay become useful as characteristic markers of the nuclear envelope. (Received July 16, 1993; Accepted December 15, 1993)     

Purification and Characterization of a Novel Erythrose Reductase from Candida magnoliae

Erythritol biosynthesis is catalyzed by erythrose reductase, which converts erythrose to erythritol. Erythrose reductase, however, has never been characterized in terms of amino acid sequence and kinetics. In this study, NAD(P)H-dependent erythrose reductase was purified to homogeneity from Candida magnoliae KFCC 11023 by ion exchange, gel filtration, affinity chromatography, and preparative electrophoresis. The molecular weights of erythrose reductase determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography were 38,800 and 79,000, respectively, suggesting that the enzyme is homodimeric. Partial amino acid sequence analysis indicates that the enzyme is closely related to other yeast aldose reductases. C. magnoliae erythrose reductase catalyzes the reduction of various aldehydes. Among aldoses, erythrose was the preferred substrate (K_m = 7.9 mM; k_cat/K_m = 0.73 mM⁻¹ s⁻¹). This enzyme had a dual coenzyme specificity with greater catalytic efficiency with NADH (k_cat/K_m = 450 mM⁻¹ s⁻¹) than with NADPH (k_cat/K_m = 5.5 mM⁻¹ s⁻¹), unlike previously characterized aldose reductases, and is specific for transferring the 4-pro-R hydrogen of NADH, which is typical of members of the aldo/keto reductase superfamily. Initial velocity and product inhibition studies are consistent with the hypothesis that the reduction proceeds via a sequential ordered mechanism. The enzyme required sulfhydryl compounds for optimal activity and was strongly inhibited by Cu²⁺ and quercetin, a strong aldose reductase inhibitor, but was not inhibited by aldehyde reductase inhibitors and did not catalyze the reduction of the substrates for carbonyl reductase. These data indicate that the C. magnoliae erythrose reductase is an NAD(P)H-dependent homodimeric aldose reductase with an unusual dual coenzyme specificity.     

Heterologous Expression, Purification, and Characterization of a Highly Active Xylose Reductase from Neurospora crassa

A xylose reductase (XR) gene was identified from the Neurospora crassa whole-genome sequence, expressed heterologously in Escherichia coli, and purified as a His₆-tagged fusion in high yield. This enzyme is one of the most active XRs thus far characterized and may be used for the in vitro production of xylitol.     

Purification of an Auxin-Binding Protein from Etiolated Mung Bean Seedlings by Affinity Chromatography

An auxin-binding protein with high affinity for 2,4-D and IAAwas purified from the extract of etiolated mung bean seedlingsby affinity chromatography on 2,4-D-linked Sepharose 4B andby gel nitration on Sepharose 4B. Its molecular weight was estimatedto be about 390,000 by gel nitration on Sepharose 4B and itconsisted of two different subunits with molecular weights ofabout 47,000 and 15,000. This protein had no ribulose-l,5-bisphosphatecarboxylase activity. Its dissociation constants for 2,4-D andIAA were 9.3 x 10^–6 M and 3.2 x 10^–6 M, respectively,as determined by Scatchard's method. (Received December 21, 1982; Accepted March 23, 1983)     

Molecular Characterization of an NADPH-Dependent Acetoin Reductase/2,3-Butanediol Dehydrogenase from Clostridium beijerinckii NCIMB 8052

Acetoin reductase is an important enzyme for the fermentative production of 2,3-butanediol, a chemical compound with a very broad industrial use. Here, we report on the discovery and characterization of an acetoin reductase from Clostridium beijerinckii NCIMB 8052. An in silico screen of the C. beijerinckii genome revealed eight potential acetoin reductases. One of them (CBEI_1464) showed substantial acetoin reductase activity after expression in Escherichia coli. The purified enzyme (C. beijerinckii acetoin reductase [Cb-ACR]) was found to exist predominantly as a homodimer. In addition to acetoin (or 2,3-butanediol), other secondary alcohols and corresponding ketones were converted as well, provided that another electronegative group was attached to the adjacent C-3 carbon. Optimal activity was at pH 6.5 (reduction) and 9.5 (oxidation) and around 68°C. Cb-ACR accepts both NADH and NADPH as electron donors; however, unlike closely related enzymes, NADPH is preferred (K_m, 32 μM). Cb-ACR was compared to characterized close homologs, all belonging to the “threonine dehydrogenase and related Zn-dependent dehydrogenases” (COG1063). Metal analysis confirmed the presence of 2 Zn²⁺ atoms. To gain insight into the substrate and cofactor specificity, a structural model was constructed. The catalytic zinc atom is likely coordinated by Cys₃₇, His₇₀, and Glu₇₁, while the structural zinc site is probably composed of Cys₁₀₀, Cys₁₀₃, Cys₁₀₆, and Cys₁₁₄. Residues determining NADP specificity were predicted as well. The physiological role of Cb-ACR in C. beijerinckii is discussed.     

Characterization and Localization of a Phenoloxidase in Mung Bean Hypocotyl Cell Walls

The occurrence of proteins able to oxidize polyphenols even in the absence of H2O2 was recently reported in mung bean (Vigna radiata L.) hypocotyl cell wall extracts (R. Goldberg, A. Chabanet, A.M. Catesson [1993] In K.G. Welinder, S.K. Rasmussen, C. Penel, H. Greppin, eds, Plant Peroxidases: Biochemistry and Physiology, pp. 296-300). Therefore, the possible presence of a laccase in the extracts was investigated using immunocytological and biochemical approaches. An enzyme catalyzing phenol oxidation in the presence of molecular O2 was extracted and purified from the cell walls. This 38-kD cationic protein, like o-diphenoloxidases, was unable to oxidize p-diphenols or p-diamines. However, it crossreacted with an anti-laccase antiserum and, like laccases, its activity was inhibited by N-cetyl-N,N,N-trimethylammonium bromide but not by ferulic acid salts. Immunolabeling data showed that the 38-kD oxidase was absent from all cellulosic cell walls. It was localized only in lignifying and lignified cell walls. This restricted localization suggests that this laccase-like phenoloxidase could participate in the lignification process but not in the primary wall stiffening, which develops in the epidermal and cortical tissues along the mung bean hypocotyl.     

Partial Purification, Photoaffinity Labeling, and Properties of Mung Bean UDP-Glucose:Dolicholphosphate Glucosyltransferase

UDP-glucose:dolichylphosphate glucosyltransferase has been purified 734-fold from Triton X-100 solubilized mung bean (Phaseolus aureus) microsomes. The partially purified enzyme has broad pH optima of activity from 6.0 to 7.0 and is maximally stimulated with 10 millimolar MgCl₂. The K_m for UDP-glucose was determined as 27 micromolar, and the K_m for dolichol-P was 2 micromolar. Using the UDP-glucose photoaffinity analog, 5-azido-UDP-glucose, a polypeptide of 39 kilodaltons on sodium dodecyl sulfate-polyacrylamide gels was identified as the catalytic subunit of the enzyme. Photoinsertion into this 39-kilodalton polypeptide with [³²P]5-azido-UDP-glucose was saturable, and was maximally protected with the native substrate UDP-glucose. 5-Azido-UDP-glucose behaves competitively with UDP-glucose in enzyme assays, and upon photolysis inhibits activity in proportion to its concentration. This study represents the first subunit identification of a plant glycosyltransferase involved in the biosynthesis of the lipid-linked oligosaccharides that are precursors of N-linked glycoproteins.     

Characterization of LeMir, a Root-Knot Nematode-Induced Gene in Tomato with an Encoded Product Secreted from the Root

A tomato gene that is induced early after infection of tomato (Lycopersicon esculentum Mill.) with root-knot nematodes (Meloidogyne javanica) encodes a protein with 54% amino acid identity to miraculin, a flavorless protein that causes sour substances to be perceived as sweet. This gene was therefore named LeMir (L. esculentum miraculin). Sequence similarity places the encoded protein in the soybean trypsin-inhibitor family (Kunitz). LeMir mRNA is found in root, hypocotyl, and flower tissues, with the highest expression in the root. Rapid induction of expression upon nematode infection is localized to root tips. In situ hybridization shows that LeMir is expressed constitutively in the root-cap and root-tip epidermis. The LeMir protein product (LeMir) was produced in the yeast Pichia pastoris for generation of antibodies. Western-blot analysis showed that LeMir expression is up-regulated by nematode infection and by wounding. LeMir is also expressed in tomato callus tissue. Immunoprint analysis revealed that LeMir is expressed throughout the seedling root, but that levels are highest at the root/shoot junction. Analysis of seedling root exudates revealed that LeMir is secreted from the root into the surrounding environment, suggesting that it may interact with soil-borne microorganisms.Root-knot nematodes (Meloidogyne spp.) are endoparasites of the roots of most cultivated crops and cause significant economic losses worldwide (Sasser, 1980). This group of nematodes has a complex life cycle (Williamson and Hussey, 1996). The infective stage, the second-stage juvenile, is attracted to root tips, where it penetrates the zone of elongation and then migrates intercellularly, first toward the root tip and then up to the developing vascular tissue (Wyss et al., 1992). There the nematode initiates a feeding site, causing the formation of large, multinucleate, metabolically active giant cells (Jones, 1978; Huang, 1985). Nearby cells of the cortex, pericycle, and vascular parenchyma enlarge and divide, forming a root-knot or gall. Initiation of the gall can be seen under the microscope between 12 and 24 h after inoculation. Although tomato (Lycopersicon esculentum Mill.) is an excellent host for these nematodes, some varieties are resistant because of the presence of the dominant gene Mi. The presence of Mi is correlated with the development of a localized necrosis of host cells at the feeding site within 24 h of infection (Dropkin et al., 1969; Paulson and Webster, 1972).Because of the economic importance of the root-knot nematode, the molecular biology of the formation and maintenance of the feeding site has been studied by a number of authors (for review, see Williamson and Hussey, 1996). Several genes have been characterized that are up-regulated in the giant cell and the gall (Goddijn et al., 1993; Niebel et al., 1993, 1995, 1996; Bird and Wilson, 1994; Opperman et al., 1994). However, very little is understood regarding molecular changes that occur in the root early after infection, before giant-cell initiation or induction of the hypersensitive response. Genes induced early after nematode infection could potentially have a role in the defense against nematodes or other root pathogens. Overall, root defense systems are poorly understood compared with shoot systems. Of the cases examined, proteins induced in the root during pathogenesis are similar to antimicrobial proteins found in the shoot, such as chitinase, β-1,3-glucanase, osmotin, and ribosome-inactivating protein (Maraganore et al., 1987; Benhamou et al., 1990, 1993; Neale et al., 1990; Savary and Flores, 1994; Savary et al., 1997).To identify rapidly up-regulated, nematode-induced plant genes with a possible role in defense against nematodes or other root parasites, we developed a technique to obtain synchronously infected root tips and then produced a cDNA library from them (Ho et al., 1992; Lambert and Williamson, 1993). Several genes that are increased in expression by 12 h after nematode infection were identified by differential screening (Williamson et al., 1994; Lambert, 1995). Most of these genes appeared to be equally induced in plants independent of the presence of Mi in the genome. Some have homology to known plant defense genes, including those coding for peroxidase, chitinase, lipoxygenase, and proteinase inhibitors (Lambert, 1995; B. Ferrie and V.M. Williamson, unpublished data). One nematode-induced cDNA, clone 23a, encoded the partial sequence of a protein with high similarity to that of miraculin, a protein isolated from the berries of Richadella dulcifica, a west-African shrub.Miraculin alters human taste perception, converting sour into sweet taste (Theerasilp et al., 1989). Because of its sequence similarity to miraculin, the tomato gene was named LeMir (L. esculentum miraculin). The sequence also shows similarity to the soybean trypsin-inhibitor family. Several members of this family have anti-insect/anti-pathogen activity (Ryan, 1990), suggesting that LeMir may have a role in defense against nematodes or other pathogens/pests. Furthermore, LeMir shows very high similarity to TID91, a gene of unknown function that is highly expressed in tobacco genetic tumors (Fujita et al., 1994). In the present study, we characterized the LeMir cDNA sequence and we present information regarding its expression pattern at the mRNA and protein levels.