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.     

Auxin-Binding Protein in Etiolated Mung Bean Seedlings: Purification and Properties of Auxin-Bindmg Protein-II

An auxin-binding protein (ABP-II) was purified from the extractof etiolated mung bean seedlings by affinity chromatographyon 2,4-D-linked Sepharose 4B and by gel filtration on Sepharose4B and Sephacryl S-200. The molecular weight was estimated tobe about 190,000 by gel filtration on Sephacryl S-200. ABP-IIgave a single band corresponding to a molecular weight of about48,000 on SDS-polyacrylamide gel electrophoresis. The dissociationconstants of ABP-II for 2,4-D determined by amrnonium sulfateprecipitation and equilibrium dialysis were 9.5?10^–6 Mand 1.1?10^–5 M, respectively. ¹⁴C-2,4-D-binding to ABP-IIwas reversible and inhibited by addition of IAA, naphthalene-1-aceticacid, 2,4,5-trichlorophenoxyacetic acid or p-chlorophenoxyisobutylicacid to the assay mixture. (Received September 5, 1984; Accepted November 5, 1984)     

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.     

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.     

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)     

Allantoinase from Mung Bean Seedlings

Mung bean allantoinase was purified sixty folds by calcium phosphate gel treatment, ammonium sulfate fractionation and acetone precipitation. The purified allantoinase hydro-lyzed allantoin to allantoic acid almost completely and the reaction had a broad pH optimum between 7.5 and 8.3. The accumulation of allantoic acid during the germination of mung bean was also noted. The allantoic acid content of seedlings was higher in hypocotyl than in leaf and root.     

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)     

萌发绿豆中水解大豆胰蛋白酶抑制子蛋白酶的纯化及固定化

大豆是豆类植物中最早发现存在蛋白酶抑制子的 ,由于其存在影响了豆类的利用价值 ,因此研究人员一直在寻找着解决办法。采用加热处理方法不能彻底钝化豆类蛋白的蛋白酶抑制子活性 ,且豆类蛋白的含硫氨基酸主要存在于各类蛋白酶抑制子中 ,从豆类蛋白中除去抑制子蛋白将大大降低其营养效价。本研究的目的是试图寻找一种可在常温下降解豆类胰蛋白酶抑制子的蛋白酶 ,从而钝化豆类的胰蛋白酶抑制活性。在前期工作中 ,我们发现枯草杆菌蛋白酶 (Ｓｕｂ ｔｉｌｉｓｉｎ)可在在常温下降解花生及大豆胰蛋白酶抑制剂[1] ,近期我们的研究表明 ,Ａｌｃａ…     

Retardation of Leaf Senescence by Triadimefon Treatment in Mung Bean Seedling

Treatment of triadimefon on detached leaves of mung bean (Phaseolus radiatus L. ) seedlings increased the levels of chlorophyll and soluble proteins. Declined activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate-peroxidase (AsA-POD) and contents of ascorbate (ASA) and glutathione (GSH) were observed during the senescence of detached young leaves. Triadimefon at concentration of 20 mg/L promoted the activities of POD, AsA-POD and levels of AsA and GSH, but had no effect on the activities of SOD and CAT. On the other hand, the levels of malondialdehyde (MDA) were increased and the increase of which was markedly negative correlated with the activities of POD, AsA-POD and with the contents of AsA and GSH during the senescence of leaves. MDA contents were decreased by triadimefon treatment. These resuits suggested that triadimefon retarded the senescence of leaves in mung bean seedlings in terms of enhancing the protective ability of plant tissues against membrane lipid peroxidation.     

Partial Purification and Properties of the Pyruvate-Uridine Diphospho-N-Acetylglucosamine Transferase from Staphylococcus epidermidis

A method for the partial purification of the pyruvate-uridine-diphospho-N-acetylglucosamine transferase from Staphylococcus epidermidis is presented. Some properties of the enzyme are discussed.     

Mucin synthesis. UDP-GlcNAc:GalNAc-R beta 3-N-acetylglucosaminyltransferase and UDP-GlcNAc:GlcNAc beta 1-3GalNAc-R (GlcNAc to GalNAc) beta 6-N-acetylglucosaminyltransferase from pig and rat colon mucosa

Pig and rat colon mucosal membrane preparations catalyze the in vitro transfer of N-acetyl-D-glucosamine (GlcNAc) from UDP-GlcNAc to GalNAc-ovine submaxillary mucin to form GlcNAc beta 1-3GalNAc-mucin. Rat colon also catalyzes the in vitro transfer of GlcNAc from UDP-GlcNAc to GlcNAc beta 1-3GalNAc-mucin to form GlcNAc beta 1-3(GlcNAc beta 1-6) GalNAc-mucin. This is the first demonstration of in vitro synthesis of the GlcNAc beta 1-3GalNAc disaccharide and of the GlcNAc beta 1-3-(GlcNAc beta 1-6)GalNAc trisaccharide, two of the four major core types found in mammalian glycoproteins of the mucin type, i.e., those containing oligosaccharides with GalNAc-alpha-serine (threonine) linkages. The activity catalyzing synthesis of the disaccharide has been named UDP-GlcNAc:GalNAc-R beta 3-N-acetylglucosaminyltransferase (mucin core 3 beta 3-GlcNAc-transferase), while the activity responsible for synthesizing the trisaccharide has been named UDP-GlcNAc:GlcNAc beta 1-3GalNAc-R (GlcNAc to GalNAc) beta 6-N-acetylglucosaminyltransferase (mucin core 4 beta 6-GlcNAc-transferase). The beta 3-GlcNAc-transferase from pig colon is activated by Triton X-100, has an absolute requirement for Mn2+, and transfers GlcNAc to GalNAc-alpha-phenyl, GalNAc-alpha-benzyl, and GalNAc-ovine submaxillary mucin with apparent Km values of 5, 2, and 3 mM and Vmax values of 59, 62, and 37 nmol h-1 (mg of protein)-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

菜豆幼苗EPSP合成酶的分离纯化和它的部分性质

利用硫酸铵分级沉淀,SephedexG-50凝胶柱层析,FPLCMono-Q和磷酸纤维素离子层析法从菜豆幼苗中分离提纯了EPSP合成酶。该酶被纯化2961.6倍,比活性达到6219.4nmolmg-1蛋白min-1。该酶分子量经SDS-PAGE检测为51kD,等电点为pH5.7,酶促反应最适pH7.5,最适温度45℃。6.2μmol/L的除草剂草甘膦能抑制EPSP合成酶活性的50%。     

Biosynthesis of the Macrocyclic Diterpene Casbene in Castor Bean (Ricinus communis L.) Seedlings : The Purification and Properties of Farnesyl Transferase from Elicited Seedlings

Farnesyl transferase (farnesyl pyrophosphate: isopentenyl pyrophosphate farnesyl transferase; geranylgeranyl pyrophosphate synthetase) was purified at least 400-fold from extracts of castor bean (Ricinus communis L.) seedlings that were elicited by exposure for 10 h to Rhizopus stolonifer spores. The purified enzyme was free of isopentenyl pyrophosphate isomerase and phosphatase activities which interfere with prenyl transferase assays. The purified enzyme showed a broad optimum for farnesyl transfer between pH 8 and 9. The molecular weight of the enzyme was estimated to be 72,000 ± 3,000 from its behavior on a calibrated G-100 Sephadex molecular sieving column. Mg²⁺ ion at 4 millimolar gave the greatest stimulation of activity; Mn²⁺ ion gave a small stimulation at 0.5 millimolar, but was inhibitory at higher concentrations. Farnesyl pyrophosphate (K_m = 0.5 micromolar) in combination with isopentenyl pyrophosphate (K_m = 3.5 micromolar) was the most effective substrate for the production of geranylgeranyl pyrophosphate. Geranyl pyrophosphate (K_m = 24 micromolar) could replace farnesyl pyrophosphate as the allylic pyrophosphate substrate, but dimethylallyl pyrophosphate was not utilized by the enzyme. One peak of farnesyl transferase activity (geranylgeranyl pyrophosphate synthetase) and two peaks of geranyl transferase activity (farnesyl pyrophosphate synthetases) from extracts of whole elicited seedlings were resolved by DEAE A-25 Sephadex sievorptive ion exchange chromatography. These results suggest that the pathway for geranylgeranyl pyrophosphate synthesis in elicited castor bean seedlings involves the successive actions of two enzymes—a geranyl transferase which utilizes dimethylallypyrophosphate and isopentenyl pyrophosphate as substrates and a farnesyl transferase which utilizes the farnesyl pyrophosphate produced in the first step and isopentenyl pyrophosphate as substrates.     

绿豆幼苗的超弱发光规律及盐胁迫下的变化

为了研究绿豆幼苗的超弱发光规律及盐胁迫对其的影响和改变,本研究首次采用以EMCCD(电子倍增式CCD)为主的、自行搭建的超弱光图像探测系统,以豫绿2号绿豆为实验材料,检测了绿豆幼苗的自发超弱发光以及在盐胁迫下的改变情况。结果发现:绿豆幼苗自发发光的强度远小于延迟发光的强度,不同的盐胁迫时间对延迟发光强度和自发发光强度的影响各不相同,故可以用延迟发光强度的变化来表征盐胁迫对植物的伤害程度。实验数据的统计结果表明:盐胁迫下绿豆幼苗的发光强度-时间曲线遵循指数衰减规律,说明生物光子具有一定的相干性。本文结果将为农田实时监测植物盐胁迫生理状况提供一种新的光子学手段。     

小麦谷氨酸脱羧酶的纯化及部分性质研究

谷氨酸脱羧酶（ｇｌｕｔａｍａｔｅｄｅｃａｒｂｏｘｙｌａｓｅ,ＧＡＤ,ＥＣ４．１．１．１５）催化谷氨酸脱羧生成γ－氨基丁酸（γ－ａｍｉｎｏｂｕｔｙｒａｔｅ,ＢＡ）,植物中已从南瓜［１］、马铃薯和林生山黧豆［２］纯化了ＧＡＤ．ＧＡＤ活性在禾本科作物中作为...     

Purification and Properties of Putrescine Hydroxycinnamoyl Transferase from Tobacco (Nicotiana tabacum) Cell Suspensions

The enzyme putrescine hydroxycinnamoyl transferase (PHT) was purified 400-fold in 7.1% yield from tobacco (Nicotiana tabacum L. cv Xanthi) cell suspensions to a final specific activity of 45 nanokatal per milligram protein. The purification procedure involved conventional chromatography techniques (anion exchange chromatography, gel permeation, and hydroxylapatite chromatography) followed by chromatography on caffeoyl-cysteamine-Sepharose. This procedure led to considerable enrichment of a 50 kilodalton protein that could be further purified to near homogeneity by chromatofocalization (apparent isoelectric point = 8). PHT activity was repeatedly found associated with this protein, although approximately 66% of the enzymic activity was lost during chromatofocalization. Purified PHT exhibited the same properties as in the unpurified extract. It was not specific for putrescine and used other aliphatic diamines (mainly diaminopropane and cadaverine) as substrates. The most efficient phenolic substrate was caffeoyl-CoA, but cinnamoyl-, feruloyl-, sinapoyl-, and p-coumaroyl-CoA were also conjugated to putrescine, in decreasing order of activity. PHT could also use the artificial substrate p-fluorocinnamoyl-CoA.     

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)     

Purification and Properties of Trypsin Inhibitor from Hakuhenzu Bean (Dolichos lablab)

A highly purified trypsin inhibitor was obtained from the oriental plant Hakuhenzu bean (Dolichos lablab) by column chromatography on DEAE-Sephadex and gel-filtration on Sephadex G–75. The purified Hakuhenzu bean trypsin inhibitor (HTI) was obtained as a chemically homogeneous protein, and was stable to heat and to enzymes such as pepsin. It shows no obvious maximum at 280 nm in the ultraviolet absorption spectrum, and it contains more than 20% carbohydrate as galactose and 10% hexosamine as glucosamine. The molecular weight of this inhibitor was determined to be approximately 9,500 by gel-filtration. The protein contained 59 residures of amino acids; Lys₃, His₄, Arg₁, Asp₈, Thr₃, Ser₉, Glu₆, Pro₅, Gly₁, Ala₃, l/2Cys₁₀, Val₁, Ile₁, Leu₂, Tyr₁, Phe₁, from which a molecular weight of 6,400 is obtained. No methionine and tryptophan were found in the amino acid composition of the inhibitor. This inhibitor showed inhibitory activity against α-chymotrypsin in addition to trypsin.     

绿豆上胚轴中油菜素甾酮的结合特性

绿豆上胚轴中油菜素甾酮的结合特性徐如涓，何宇炯，王玉琴，赵敏橘（中国科学院上海植物生理研究所，上海２０００３２）关键词：油菜素甾酮，绿豆，上胚轴，结合位点近十年来的研究表明，由体类化合物广泛存在于植物体内，（Ｗｅｗｉｔｔ等１９８０，横田孝雄１９８７）...     

Permeability Properties of the Inner Membrane of Mung Bean Mitochondria and Changes during Energization

The permeability properties of the inner membrane of mung bean mitochondria were studied by osmotic swelling techniques. Rapid mitochondrial swelling was observed in isotonic ammonium phosphate, which indicated that an active phosphate/hydroxyl antiporter was present. The phosphate carrier was specifically inhibited by sulfhydryl reagents. Mitochondria did not swell in isotonic ammonium salts of malate, succinate, or fumarate, either in the presence or absence of 10 millimolar phosphate. Additionally, no swelling was observed in ammonium citrate upon addition of malate plus phosphate. Consequently, no evidence was obtained with the osmotic swelling technique for a coupled exchange of phosphate for dicarboxylic acids across the membrane.