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.     

绿豆下胚轴中的雌二醇结合位点

越来越多的证据表明,动物的甾类激素广泛存在于植物体内(Hewitt等1980,周永春和曹宗巽1982,王焕民和曹宗巽1986,Jones和Roddick 1988,Simons和Grinwich 1989,Zhang等1991a,b)。而这些激素对植物的生长发育也具有多方面的作用,如雌二醇可以促进多种植物开花,增加菠菜、女蒌菜等雌花比例;雌酮和雌二醇在适当浓度下可以促进豌豆     

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)     

Phytochrome-induced Increase of Fluorescein Translocation in Mung Bean Hypocotyls

Moderate doses of red (660 nanometer) irradiation cause a rapid increase in the translocation of fluorescein in dark-grown mung bean hypocotyl (Vigna radiata L.) segments. The increase fails to appear following large doses of red (660 nanometers) irradiation. The red induced increase is prevented by a subsequent far red (730 nanometer) irradiation. Reversibility suggests the participation of phytochrome in the process. The increase in translocation is attributed to the generation of a positive electrostatic charge in the plasma membrane by some action of phytochrome on membrane molecules.     

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)     

Roles of Sucrose-Metabolizing Enzymes in Growth of Seedlings. Purification of Acid Invertase from Growing Hypocotyls of Mung Bean Seedlings

Changes in activities of acid invertase and sucrose synthaseduring growth of mung bean seedlings were examined and the correlationbetween the activity of acid invertase and growth was confirmed.Acid invertase was purified from hypocotyls of etiolated seedlingsand separated into two fractions (A and B) by chromatographyon hydroxylapatite. Acid invertase in fraction B consisted oftwo polypeptides of 30 kDa and 38 kDa, but that in fractionA was 70 kDa in size. Antibodies raised against the 30-kDa polypeptideimmunoprecipitated enzymatic activity but those raised againstthe 38-kDa polypeptide did not. The concanavalin A-binding siteof acid invertase was contained in the 38-kDa polypeptide andnot in the 30-kDa polypeptide. However, when acid invertasewas bound to and eluted from concanavalin A-Sepharose, the 30-kDapolypeptide was found together with the 38-kDa polypeptide inthe eluate. Acid invertase in hypocotyls of mung bean seedlingsappears to be present in two forms: a monomer of 70 kDa anda hetero-dimer of 30-kDa and 38-kDa polypeptides. The monomerwas not converted to the heterodimer during incubation of acrude extract and was present together with the heterodimerin very young hypocotyls. In older hypocotyls, the heterodimerwas present but the monomer was barely detectable. We concludethat the two forms of acid invertase are present within cells,but the relationship between the two forms is unknown at present. (Received July 18, 1991; Accepted October 9, 1991)     

Alterations in Protein Synthesis in vivo in Chilling Sensitive Mung Bean Hypocotyls Caused by Chilling Stress

The effects of chilling on protein synthesis in vivo in etiolatedhypocotyls of Vigna radiata L. were investigated. After exposureof the tissues to 0?C for various periods of time, proteinswere labeled with [³⁵S]-methionine at 26?C. The total amountof ³⁵S incorporated into soluble and membrane proteins was reversiblyreduced by chilling for 24 h, during which time the tissuessuffered no injury. Further prolonged chilling produced an irreversibledecline both in the incorporation of radioactivity and in cellviability as assessed by the extent of leakage of electrolyte.The ³⁵S-labeled proteins in the soluble and the total membranefractions were analyzed quantitatively. Chilling of etiolatedhypocotyls for one or two days induced the syntheses of twosoluble proteins (82 and 74 kDa) and one membrane protein (80kDa). Moreover, three heat-shock proteins (HSPs) that were inducedby heat stress (41 ?C, 4h) had the same electrophoretic mobilitiesas those of the proteins induced directly or indirectly by thechilling treatment. ¹Contribution No. 3170 from the Institute of Low TemperatureScience. (Received April 22, 1988; Accepted September 30, 1988)     

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.     

Development of Vacuolar Membranes during Elongation of Cells in Mung Bean Hypocotyls

The development of vacuolar membrane in the elongating hypocotylsof the mung bean was investigated. The hypocotyls from 3-day-oldseedlings were dissected into the dividing, elongating and matureregions. The diameter of protoplasts prepared from the matureregions was about 3-fold greater than the diameter of thosefrom the dividing region. The activity of inorganic pyrophosphatase,an enzyme associated with the vacuolar membrane, was detectableeven in the dividing region. The level of pyrophosphatase wasquantified by slot-blot analysis with the pyrophosphatase-specificantibody. The relative amount of pyrophosphatase per cell, calculatedon the basis of DNA content, increased about 4-fold during cellmaturation. When the densities of vacuolar membranes were comparedby sucrose density gradient centrifugation, there was no markeddifference among the preparations from three regions. Furthermore,most of the major proteins were common to the three purifiedpreparations of vacuolar membranes. From the results, it appearsthat most components of vacuolar membrane may be synthesizedde novo and added to the existing membrane during cell elongation.Furthermore, it is proposed that the H⁺-pyrophosphatase mayactively hydrolyze its substrate to maintain the internal acidityof expanding vacuoles, because pyrophosphate was present ata concentration of more than 70 µM in the dividing andelongating regions. (Received August 7, 1989; Accepted January 11, 1990)     

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.     

Chill-Induced Changes in the Activity and Abundance of the Vacuolar Proton-Pumping Pyrophosphatase from Mung Bean Hypocotyls

Changes in the properties of extractable vacuolar H+-pumping pyrophosphatase (V-PPase) and vacuolar ATPase activities in chilling-sensitive seedlings of mung bean (Vigna radiata) were investigated. Following chilling at 4[deg]C for 48 h, both hydrolytic and proton-pumping activities of the V-PPase increased 1.5- to 2-fold over controls and remained elevated even after 72 h at low temperatures. Vacuolar ATPase levels did not change significantly throughout the chilling regime. However a large increase in alcohol dehydrogenase activity during chilling suggests a shift toward fermentative metabolism, which can be expected to decrease ATPase activity in situ. Western blotting of vacuolar membrane-enriched fractions from control and treated plants has confirmed that the changes in V-PPase activity are mirrored by increases in the amount of pump protein. Results suggest a specific role for the V-PPase in protecting chill-sensitive plants from the injurious effects of low temperatures via the maintenance of the proton gradient across the vacuolar membrane.     

Accumulation of a Glycoprotein That is Homologous to a Seed Storage Protein in Mung Bean Hypocotyls at the Late Stage of Tissue Elongation

Physiological changes were examined in the amount of a 50-kDaglycoprotein (gp50) that was recovered in a nuclear fractionfrom hypocotyls of mung bean (Vigna radiata) seedlings. Immunoblotanalysis indicated that the glycoprotein was present in hypocotylsand epicotyls from 4- and 5-day-old seedlings but not in hypocotylsfrom 2-day-old seedlings. The glycoprotein was not detectedin leaves or roots. When we divided hypocotyls of 3-day-oldseedlings into the elongating region (0 to 1.5 cm below thecotyledon) and the mature region, we found gp50 in the matureregion only. The results suggest that the 50-kDa glycoproteinis synthesized de novo and accumulates at the late stage duringelongation of cells in the hypocotyl. Furthermore, an antibodyspecific to gp50 reacted with a major 50-kDa protein in cotyledons,which is known as a storage protein in mung bean cotyledon.Eighteen amino acid residues among 22 amino-terminal residuesof gp50 were identical to those of the storage protein fromcotyledon. A peptide map of the glycoprotein after digestionwith V8 protease was similar to that of the storage protein.Overall, our findings suggest that the glycoprotein recoveredin the nuclear fraction is an isoform of the seed storage proteinthat is expressed only in the mature cells of hypocotyls andepicotyls. ⁴ Present address: Bioscience and Chemistry Division, HokkaidoNational Industrial Research Institute, Agency of IndustrialScience and Technology, Sapporo, 062 Japan     

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)     

Reconstitution of Tonoplast H+-ATPase from Mung Bean (Vigna radiata L.) Hypocotyls in Liposomes

Reconstituted proteoliposomes of tonoplast ATPase are formedon solubilization of tonoplast membranes from mung bean (Vignaradiata L.) with deoxycholate (DOC) in the presence of a mixtureof soybean phospholipids (asolectin), after removal of DOC bypassage through a PD-10 column (Pharmacia). This method is idealbecause of its simplicity and rapidity. Selective insertionof sets of tonoplast H⁺-ATPase polypeptides (68 kDa, 60 kDa,16 kDa and several minor polypeptides) into liposomes usingthis method was confirmed by SDS-PAGE and immuno-blotting withantibodies raised against 68-kDa and 60-kDa polypeptides. Pumping of protons across the membranes of the proteoliposomeswas demonstrated by quinacrine-fluorescence quenching in thepresence of ATP-Mg²⁺. ATP-Mg²⁺ was shown to be the preferredsubstrate in both reconstituted and native tonoplast vesicles,and its optimum concentration was 0.75 to 3.0 mM. Quenchingwas completely abolished by a channel-forming ionophore, gramicidinD, and an inhibitor of tonoplast H⁺-ATPase, KNO₃. Antibodiesto 68-kDa and 60-kDa peptides partially inhibited the pumpingof protons. The rate of pumping of protons increased with thenumber of proteoliposomes, the maximal concentration of whichwas equivalent to 250 µg of protein per reaction mixture.The optimum pH for pumping was 6.5 when inside of proteoliposomeswere loaded pH at 7.2. The rate of pumping of protons was reducedwhen proteoliposomes were made using asolectin and cholesterolat 3 : 1 (w/w), as compared with those made with asolectin alone. The ATPase activity in reconstituted proteoliposomes was inhibitedby KNO₃, with half-maximal inhibition at approximately 7 mM.The enzyme actively hydrolyzed ATP in preference to GTP, CTP,UTP, and ADP, but it did not hydrolyze pNPP or AMP. Antibodiesagainst the 60-kDa polypeptide strongly inhibited ATPase activityas compared to antibodies against the 68-kDa polypeptide. Theresults obtained in this study demonstrate directly that functionaltonoplast H⁺-ATPase can be inserted selectively into liposomes. (Received August 31, 1990; Accepted April 18, 1991)     

Properties of Two N-Linked Glycoproteins Associated with the Nuclear Envelope in Mung Bean Hypocotyls

Nuclei from mung bean (Vigna radiata) hypocotyls contained twoglycoproteins of 50 and 49 kDa, respectively, that reacted withconcanavalin A. The glycoproteins were released from the nuclearenvelope by treatment with 2 M KCl but not with nucleases. Theglycoproteins, tentatively named gp50 and gp49, were isolatedand characterized. Gel-permeation chromatography suggested thatgp50 and gp49 seem to exist as a complex with other components.The glycoproteins could be detected only in the nuclear fractionby immunoblot analysis with specific antibodies, and they werenot detected in endoplasmic reticulum, plasma membrane, vacuolarmembrane or mitochondria. Agglutinin I from Ulex europeaus,peanut agglutinin, soybean agglutinin and wheat germ agglutininall failed to bind to the glycoproteins. Treatment with glycopeptidaseF removed all oligosaccharides from the glycoproteins and decreasedtheir molecular masses by about one thousand daltons each. Theseresults suggest that the glycoproteins contained N-linked, high-mannose-typeoligosaccharides with six or seven hexose residues. gp50 andgp49 seemed to be isoforms of a single glycoprotein becausethe two proteins had some common properties. Nuclear fractionsfrom azuki bean (Phaseolus angularis) and soybean (Glycine max)contained proteins that were immunologically similar to gp50and gp49. (Received March 18, 1995; Accepted May 24, 1995)     

The Relationship between Growth and Proline Incorporation after Auxin Treatment of Mung Bean Hypocotyls

Indoleacetic acid (IAA) stimulates the incorporation of ¹⁴C-proline into both the cyloplasmic and the cell wall fractions of the hypocotyl of mung bean (Phaseolus aureus Roxb. cv. Black). It neither stimulates the transfer of ¹⁴C-proline from the cyloplasmic fraction into the cell wall fraction, nor the retention of ¹⁴C-proline in the wall or cytoplasmic fractions. Moreover, the stimulation of growth caused by IAA parallels the stimulation of the incorporation of proline into the cytoplasmic fraction, but does not parallel the stimulation into the cell wall fractions. The stimulation of the incorporation into the cyloplasmic fraction seems to appear within 30 minutes after auxin treatment, at about the same time the increase in the growth is observed in response to IAA, suggesting a connection between these effects. On the other hand, the stimulation of the proline incorporation into the cell wall fraction seems to require more than 90 minutes after auxin treatment, suggesting no close connection between growth and proline incorporation into the cell wall fraction.     

In Vitro and In Situ Properties of Cell Wall Pectinmethylesterases From Mung Bean Hypocotyls

Pectinmethylesterases (EC 3.1.1.11 [EC] ) have been solubilized fromyoung and mature tissues of mung bean hypocotyls. Whatever theplastic potential of the tissues, most of the pectinmethylesteraseactivity was located in the cell walls. Several active fractionswere obtained after chromatography on CM Sépharose. Equilibriumsedimentation in an analytical ultracentrifuge indicated theMW of the isolated isoforms to be close to 75 000 whereas SDS-PAGEelectrophoresis gave a MW around 32 000, suggesting the possibilityof dimeric structures. Mung bean pectinmethylesterase (PME)showed cross reactivity with soybean antiserum. Experiments carried out with p-nitrophenylacetate and Citruspectin revealed that PME and esterase activities might correspondto different isoforms. It was also noted that the stimulationinduced by cations was stronger when the enzymes were boundto the cell walls. The high ionic sensitivity suggested that,in situ, the ionic environment regulates pectinmethylesteraseactivity principally by modifying the pectin molecules, whichenhances the affinity of the enzymes for their substrate. Thesedata indicate the importance of the calcium content of the cellwalls and might explain the decrease in methylated pectins alongthe mung bean hypocotyl and, in turn, the loss of plasticity. Key words: Cell wall, hypocotyl, pectinmethylesterase, Vigna radiata     

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.     

Isolation and Partial Characterization of a Subtilisin Inhibitor from the Mung Bean (Vigna radiata)

The subtilisin inhibitor (MBSI-A) from the mung bean (Vigna radiata (L.) Wilczek) seed has been purified to homogeneity. MBSI-A consists of a single polypeptide chain of 119 residues, with a high content of glutamic acid/glutamine, aspartic acid/asparagine, valine, threonine, and proline (19, 12, 10, 9, and 8 residue percent, respectively). MBSI-A is a potent inhibitor of subtilisin Carlsberg, but is inactive toward bovine trypsin and α-chymotrypsin and the plant cysteinyl proteinase papain. The MBSI is located exclusively in the cytosol of the seed cotyledon cell, unlike the mung bean trypsin inhibitor (MBTI), which is located primarily in the protein bodies. Both MBSI and MBTI accumulate in the seed during the most active period of reserve protein accumulation, 12 to 18 days after flowering. During germination MBSI, like MBTI, is broken down beginning 2 to 3 days after seed imbibition. The disappearance of MBSI-A is accompanied by the transient appearance of a new inhibitor species, MBSI-D. The amino acid composition of MBSI-D suggests that it may be produced by the loss of approximately 20 amino acid residues from MBSI-A.     

H2O2在黄瓜和绿豆下胚轴不定根形成中的作用

以黄瓜和绿豆下胚轴插条为试验材料,研究了H2O2对不定根形成的诱导作用,以及在此过程中与IAA、NO的关系。结果表明,H2O2能促进不定根的形成,最佳浓度是100μmol/L;H2O2的清除剂———过氧化氢酶(CAT)抑制了IAA诱导的不定根形成,说明H2O2可能介导了IAA诱导不定根形成的过程;NO清除剂———PTIO能抑制H2O2诱导的不定根形成,表明NO在H2O2诱导不定根形成的过程中起介导作用。综合上述结果,推测在不定根形成的过程中可能存在以下关系:IAA首先诱导H2O2合成,H2O2升高后,再诱导NO产生,最终导致不定根生成。