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1-Aminocyclopropane-1-carboxylate (ACC) N-malonyltransferase converts ACC, an immediate precursor of ethylene, to the presumably inactive product malonyl-ACC (MACC). This enzyme plays a role in ethylene production by reducing the level of free ACC in plant tissue. In this study, ACC N-malonyltransferase was purified 3660-fold from etiolated mung bean (Vigna radiata) hypocotyls, with a 6% overall recovery. The final specific activity was about 83,000 nmol of MACC formed mg−1 protein h−1. The five-step purification protocol consisted of polyethylene glycol fractionation, Cibacron blue 3GA-agarose chromatography using salt gradient elution, Sephadex G-100 gel filtration, MonoQ anion-exchange chromatography, and Cibacron blue 3GA-agarose chromatography using malonyl-CoA plus ACC for elution. The molecular mass of the native enzyme determined by Sephadex G-100 chromatography was 50 ± 3 kD. Protein from the final purification step showed one major band at 55 kD after sodium dodecyl sulfate polyacrylamide gel electrophoresis, indicating that ACC N-malonyltransferase is a monomer. The mung bean ACC N-malonyltransferase has a pH optimum of 8.0, an apparent Km of 0.5 mm for ACC and 0.2 mm for malonyl-coenzyme A, and an Arrhenius activation energy of 70.29 kJ mol−1 degree−1. 相似文献
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Jose R. Botella Carl D. Schlagnhaufer Richard N. Arteca Allen T. Phillips 《Plant molecular biology》1992,18(4):793-797
The polymerase chain reaction (PCR) was used to produce 3 putative clones for ACC synthase from etiolated mung bean (Vigna radiata Rwilcz cv. Berken) hypocotyls. This was accomplished by utilizing genomic DNA from mung bean and degenerate primers made from information derived from highly conserved regions of ACC synthase from different plant tissues. The total length of pMAC-1, pMAC-2 and pMAC-3 are 308, 321, and 326 bp, respectively, all of which code for 68 amino acids. The introns for pMAC-1, pMAC-2 and pMAC-3 are 92, 105, and 110 bp, respectively. The degrees of homology at the DNA level for each of these clones is ca. 80% in their coding region and ca. 50% in their respective introns. This is the first report providing evidence that there are at least 3 genes for ACC synthase in etiolated mung bean. 相似文献
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Purification and characterization of 1-aminocyclopropane-1-carboxylate synthase from apple fruits 总被引:2,自引:2,他引:0
1-Aminocyclopropane-1-carboxylate (ACC) synthase, a key enzyme in ethylene biosynthesis, was isolated and partially purified from apple (Malus sylvestris Mill.) fruits. Unlike ACC synthase isolated from other sources, apple ACC synthase is associated with the pellet fraction and can be solubilized in active form with Triton X-100. Following five purification steps, the solubilized enzyme was purified over 5000-fold to a specific activity of 100 micromoles per milligram protein per hour, and its purity was estimated to be 20 to 30%. Using this preparation, specific monoclonal antibodies were raised. Monoclonal antibodies against ACC synthase immunoglobulin were coupled to protein-A agarose to make an immunoaffinity column, which effectively purified the enzyme from a relatively crude enzyme preparation (100 units per milligram protein). As with the tomato enzyme, apple ACC synthase was inactivated and radiolabeled by its substrate S-adenosyl-l-methionine. Apple ACC synthase was identified to be a 48-kilodalton protein based on the observation that it was specifically bound to immunoaffinity column and it was specifically radiolabeled by its substrate S-adenosyl-l-methionine. 相似文献
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Purification and partial characterization of 1-aminocyclopropane-1-carboxylate synthase from tomato pericarp 总被引:5,自引:0,他引:5
D Van der Straeten L Van Wiemeersch H M Goodman M Van Montagu 《European journal of biochemistry》1989,182(3):639-647
1-Aminocyclopropane-1-carboxylate synthase was purified 5000-fold from LiCl-induced tomato fruit slices by conventional and high-performance liquid chromatography. The final preparation was estimated to be between 25% and 50% pure. Two-dimensional gel electrophoresis indicates that 1-aminocyclopropane-1-carboxylate synthase activity is associated with a 45-kDa polypeptide, with a pI of 5.8 +/- 0.2. The enzyme is inactivated both by its substrate, S-adenosyl-L-methionine (AdoMet) and by one of its products, 1-aminocyclopropane-1-carboxylate. Due to the extremely low abundance of the protein it was necessary to scale up the extraction in order to obtain reasonable amounts for sequence analysis. Therefore, 200 kg tomatoes were extracted on semi-industrial scale and 1-aminocyclopropane-1-carboxylate synthase purified. This yielded approximately 150 micrograms enzyme. 相似文献
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Purification and characterization of tonoplast ATPase from etiolated mung bean seedlings 总被引:3,自引:3,他引:0
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The tonoplast ATPase from etiolated seedlings of Vigna radiata L. (mung bean) was isolated using a two-step detergent solubilization modified from Mandala and Taiz (S Mandala, L Taiz [1985] Plant Physiol 78: 327-333). After ultracentrifugation on 10 to 28% sucrose gradient, the ATPase showed a 31.6-fold purification over the initial specific activity of the starting tonoplast-enriched membranes. The purified ATPase used Mg2+-ATP as the preferred substrate. The tonoplast ATPase was isolated in a form with characteristics similar to that on its native membrane environment. Analysis by SDS-PAGE revealed two prominent bands with molecular weights of 78,000 (α subunit) and 64,000 (β subunit). The intensity of Coomassie blue staining showed a 1:1 stoichiometry for α and β subunits. The amino acid composition of α and β subunits also confirmed the suggested stoichiometry of the subunit composition of the tonoplast ATPase. Moreover, radiation inactivation analysis yielded a functional size of 414 ± 24 and 405 ± 25 kilodaltons for soluble and membrane bound tonoplast ATPases, respectively. It is possible that the functioning tonoplast ATPase may be in a form of αβ-heteromultimer. 相似文献
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Immunopurification and characterization of a 40-kD 1-aminocyclopropane-1-carboxylic acid N-malonyltransferase from mung bean seedling hypocotyls.
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1-Aminocyclopropane-1-carboxylic acid (ACC) N-malonyltransferase catalyzes the transfer of the malonyl group from malonyl coenzyme A to ACC to form malonyl ACC. Using partially purified ACC N-malonyltransferase from the hypocotyls of mung bean (Vigna radiata) seedlings, we produced two mouse monoclonal antibodies (1H5 and 2G3) to this enzyme. These antibodies bind to sites other than the active site of the enzyme because monoclonal antibody-bound ACC N-malonyltransferase still exhibits full catalytic activity. A monoclonal antibody column was constructed using 1H5 and protein G Sepharose. The ACC N-malonyltransferase purified from this monoclonal antibody column has a molecular mass of 40 kD, which is different from that reported previously. The enzyme has a higher electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence of the reducing agent dithiothreitol. The optimum temperature of this 40-kD ACC N-malonyltransferase is 45 degrees C and the apparent Kms for ACC and malonyl coenzyme A are 66.7 and 40 microns, respectively. 相似文献
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Mohamed Benichou Gracia Martinez-Reina Felix Romojaro Jean-Claude Pech Alain Latché 《Physiologia plantarum》1995,94(4):629-634
A 36-kDa 1-aminocyclopropane-1-carboxylate (ACC) N-malonyltransferase, which converts the ethylene precursor ACC into the conjugated derivative malonyl-ACC (MACC), has been isolated from etiolated mung bean ( Vigna radiata ) hypocotyls, and partially purified in a four-step procedure. The enzyme is stimulated about 7-fold by 100 m M K+ salts or 0.5 m M Co2+ salts, and is inhibited competitively by D-phenylalanine (Ki = 1.3 m M ) and non competitively by CoASH (0.3 m M ). Beside malonyl-CoA, it is capable to use succinyl-CoA as an acyl donor. The 36-kDa enzyme described here exhibits a lower optimum temperature (40°C) and a 7- or 3-fold lower apparent Km for ACC (68 μ M ) and malonyl-CoA (74 μ M ), respectively, when compared with its 55 kDa isoform already isolated from the same plant material. This data support the idea that several isoforms of ACC N-malonyltransferase exist in plants. These isoforms may play a differential role in regulating the availability of ACC, and consequently the rate of ethylene production, as well as detoxifying cells from D-amino acids. 相似文献
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Liu PF Chang WC Wang YK Munisamy SB Hsu SH Chang HY Wu SH Pan RL 《Biochimica et biophysica acta》2007,1769(7-8):443-454
Plant Ku genes were identified very recently in Arabidopsis thaliana, and their roles in repair of double-stranded break DNA and maintenance of telomere integrity were scrutinized. In this study, the cDNAs encoding Ku70 (VrKu70) and Ku80 (VrKu80) were isolated from mung bean (Vigna radiata L.) hypocotyls. Both genes were expressed widely among different tissues of mung bean with the highest levels in hypocotyls and leaves. The VrKu gene expression was stimulated by exogenous auxins in a concentration- and time-dependent manner. The stimulation could be abolished by auxin transport inhibitors, N-(1-naphthyl) phthalamic acid and 2,3,5-triiodobenzoic acid implicating that exogenous auxins triggered the effects following their uptake by the cells. Further analysis using specific inhibitors of auxin signaling showed that the stimulation of VrKu expression by 2,4-dichlorophenoxyacetic acid (2,4-D) was suppressed by intracellular Ca(2+) chelators, calmodulin antagonists, and calcium/calmodulin dependent protein kinase inhibitors, suggesting the involvement of calmodulin in the signaling pathway. On the other hand, exogenous indole-3-acetic acid (IAA) and alpha-naphthalene acetic acid (NAA) stimulated VrKu expression through the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway. Altogether, it is thus proposed that 2,4-D and IAA (or NAA) regulate the expression of VrKu through two distinct pathways. 相似文献
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The extracellular invertase (β-D-fructofuranoside fructohydrolase, EC 3.2.1.26) was isolated and characterized from the hypocotyls
of mung bean (Phaseolus radiatus L.). The enzyme was purified to apparent homogeneity by ammonium sulfate fractionation and sequential chromatography over
diethylaminoethyl (DEAE)-cellulose anion exchange, Concanavalin (Con) A-Sepharose 4B affinity and Sephadex G-200. The overall
purification was about 77-fold with a recovery of about 11%. The finally purified enzyme exhibited a specific activity of
about 113 μmol of glucose produced mg-1 protein min-1 at pH 5.0 and appeared to be a single protein by nondenaturing polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl
sulfate (SDS)-PAGE. The enzyme had the native molecular mass of 134 kD and subunit molecular weight of 67 kD as estimated
by Sephadex G-200 chromatography and SDS-PAGE, respectively, suggesting that the enzyme was composed of homodimeric proteins.
On the other hand, the enzyme appeared to be a glycoprotein containing mannosyl residues on the basis of its ability to interact
specifically with the immobilized Con A and the separability of invertase-Con A complex by methyl-α-D-mannopyranoside. The
enzyme had a Km for sucrose of 3.4 mM and its pH optimum of 4.0. The enzyme showed highest enzyme activity with sucrose as substrate. Raffinose
and cellobiose were hydrolyzed at a low rate, maltose and lactose were not cleaved by the enzyme. These results indicate the
extracellular invertase is a β-fructofuranosidase. 相似文献
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Brassinosteroid (BR) stimulation of auxin-induced ethylene production and the particular step at which BR acts to promote such synthesis were studied in mung bean ( Vigna radiata L. Rwilcz cv. Berken) hypocotyl segments. Increasing concentrations of methionine alone and in combination with 3 μ M BR and 10 μ M IAA had a minimal effect on ethylene production. With increasing concentrations of 1-aminocyclopro-pane-1-carboxylic acid (ACC), however, ethylene production increased. BR or IAA further enhanced ethylene production with maximum rates occurring when these compounds were added together with ACC. The addition of 10 μ M CoCl2 in conjunction with BR and/or IAA resulted in 85–97% inhibition of ethylene production. When 20 μ M cycloheximide was used in conjunction with BR and/or IAA there was a complete inhibition of ethylene production. Total inhibition also resulted when 1.0 μ M aminoethoxy-vinylglycine (AVG) was used in combination with BR and/or IAA. AVG alone had no effect on ACC conversion to ethylene. 相似文献
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The mechanistic fate of pyridoxal phosphate (PLP)-dependent enzymes diverges after the quinonoid intermediate. 1-Aminocyclopropane-1-carboxylate (ACC) synthase, a member of the alpha family of PLP-dependent enzymes, is optimized to direct electrons from the quinonoid intermediate to the gamma-carbon of its substrate, S-adenosyl-L-methionine (SAM), to yield ACC and 5'-methylthioadenosine. The data presented show that this quinonoid may also accept a proton at C(4)' of the cofactor to yield alpha-keto acids and the pyridoxamine phosphate (PMP) form of the enzyme when other amino acids are presented as alternative substrates. Addition of excess pyruvate converts the PMP form of the enzyme back to the PLP form. C(alpha)-deprotonation from L-Ala is shown by NMR-monitored solvent exchange to be reversible with a rate that is less than 25-fold slower than that of deprotonation of SAM. The rate-determining step for transamination follows the formation of the quinonoid intermediate. The rate-determining step for alpha, gamma-elimination from enzyme-bound SAM is likewise shown to occur after C(alpha)-deprotonation, and the quinonoid intermediate accumulates during this reaction. BLAST searches, sequence alignments, and structural comparisons indicate that ACC synthases are evolutionarily related to the aminotransferases. In agreement with previously published reports, an absence of homology was found between the alpha and beta families of the PLP-dependent enzyme superfamily. 相似文献