Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.15 (pectinase)
 2,440 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effects of extended heat stress on polygalacturonase (PG; EC 3.2.1.15) and pectin methylesterase (PME; EC 3.1.1.11) gene expression at mRNA, protein and activity levels in ripening tomato fruits were investigated. Steady state levels of PG mRNA declined at temperatures of 27 degrees C and above, and a marked reduction in PG protein and activity was observed at temperatures of 32 degrees C and above. Exogenous ethylene treatment did not reverse heat stress-induced inhibition of PG gene expression. Transfer of heat-stressed fruits to 20 degrees C partly restored PG mRNA accumulation, but the rate of PG mRNA accumulation declined exponentially with duration of heat stress. Heat stress-induced inhibition of PME mRNA accumulation was recoverable even after 14 days of heat stress. In fruits held at 34 degrees C, both PG and PME protein and activity continued to accumulate for about 4 days, but thereafter PG protein and activity declined while little change was observed in PME protein and activity. In spite of increases in mRNA levels of both PG and PME during the recovery of heat-stressed fruit at 20 degrees C, levels of PG protein and activity declined in fruits heat-stressed for four or more days while PME protein and activity levels remained unchanged. Collectively, these data suggest that PG gene expression is being gradually and irreversibly shut off during heat stress, while PME gene expression is much less sensitive to heat stress.
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PMID:Differential regulation of polygalacturonase and pectin methylesterase gene expression during and after heat stress in ripening tomato (Lycopersicon esculentum Mill.) fruits. 861 11

Increased binding of ruthenium red to pectin as the number of methyl esters attached to the pectin decreases was used as the basis for a gel diffusion assay for pectin methylesterase (PME, EC 3.1.1.11) activity. The stained zone diameters resulting from the hydrolysis of 0.1% (w/v) 90% esterified pectin in an agarose gel by diffused, commercial PME were log-linear over 4 orders of magnitude, with a minimum detection limit of 3.6 pkatals. Pectin deesterification as the cause for a stained zone after PME incubation was confirmed when only 1 N NaOH, which will chemically deesterify the pectin, and not methanol or acid, the two products formed when PME acts on a methyl ester, resulted in the characteristic stained zone. The stained zone diameters decreased with increasing percentage of substrate esterification, were independent of pH, and were insensitive to simultaneous incubation with two forms of pectin lyase (EC 4.2.2.10), polygalacturonase (EC 3.2.1.15), or all combinations. PME extracted from tomato seeds, cotton fibers, and melon fruit showed pH optima of 6, 6, and 8, respectively. Using individual tomato seed parts, the assay was adapted to quantify diffusate activity and to localize activity in tissue prints. The sensitivity, specificity, and simplicity of this PME assay are superior to all others.
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PMID:A gel diffusion assay for quantification of pectin methylesterase activity. 986 76

A series of pectins with different distribution patterns of methyl ester groups was produced by treatment with either plant (p-PME) or fungal pectin methyl esterases (f-PME) and compared with those obtained by base catalysed de-esterification. The products generated by digestion of these pectins with either endopectin lyase (PL) or endopolygalacturonase II (PG II) from Aspergillus niger were analysed using matrix assisted laser desorption ionisation mass spectrometry (MALDIMS) and high-performance anion-exchange chromatography with pulsed amperometric or UV detection (HPAEC-PAD/UV). Time course analysis using MALDIMS was used to identify the most preferred substrate for each enzyme. For PL, this was shown to be fully methyl esterified HG whereas for PG II, long regions of HG without any methyl esterification, as produced by p-PME was the optimal substrate. The blockwise de-esterification caused by p-PME treatment gave a decrease of partly methylated oligomers in PL fingerprints, which did not effect the relative composition of partly methylated oligomers. PG II fingerprints showed a constant increase of monomers and oligomers without any methyl ester groups with decreasing degree of esterification (DE), but almost no change in the concentration of partly methylated compounds. PL fingerprints of f-PME and chemically treated pectins showed decreasing amounts of partly methyl esterified oligomers with decreasing DE, together with a relative shift towards longer oligomers. PG II fingerprints were characterised by an increase of partly methylated and not methylated oligomers with decreasing DE. But differences were also seen between these two forms of homogenous de-esterification. Introduction of a certain pattern of methyl ester distribution caused by selective removal of certain methyl ester groups by f-PME is the most reasonable explanation for the detected differences.
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PMID:Analysis of different de-esterification mechanisms for pectin by enzymatic fingerprinting using endopectin lyase and endopolygalacturonase II from A. niger. 1094 78

A method to determine the amount of galacturonic acid in blocksequence (BS) in pectin homogalacturonan (HG) is described. The method is based on a combination of endopolygalacturonase II (endo-PG II) and exopolygalacturonase (exo-PG) digestion followed by quantification of the liberated galacturonic acid monomer. The amount of monomers released is directly related to the amount of non-esterified galacturonic acid units located between two other non-esterified galacturonic acids units on the HG chain. The amount released for exo-PG digestion only corresponds to the BS located at the non-reducing end of the polymer. The difference between total- and exo-BS was calculated to be the amount of endo-BS located either within or on the reducing end of the HG. Three series of model pectins obtained by de-esterification of a high-ester pectin with either plant pectin methyl-esterase (p-PME, P-series), fungal pectin methyl-esterase (f-PME, F-series) and chemical de-esterification using base (B-series) were analysed and compared with a fully de-esterified pectic acid sample obtained from the same raw material. Clear differences for the increase of the amounts of blocksequence could be seen between de-esterification of the P- and F-series samples supporting a blockwise and a homogenous de-esterification mechanism, respectively. f-PME and base treatment showed only minor differences in the increase of galacturonic acid units in BS, despite differences seen in their methyl-esterification pattern. Differences between the amounts of galacturonic acid located in exo- and endo-BS, provided evidence for the need of a certain start side or blocklength for p-PME to de-esterify blockwise.
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PMID:Quantification of the amount of galacturonic acid residues in blocksequences in pectin homogalacturonan by enzymatic fingerprinting with exo- and endo-polygalacturonase II from Aspergillus niger. 1094 80

Pectin methylesterase (PME, EC 3.1.11) demethoxylates pectins and is believed to be involved in degradation of pectic cell wall components by polygalacturonase in ripening tomato fruit. We have introduced antisense and sense chimeric PME genes into tomato to elucidate the role of PME in fruit development and ripening. Fruits from transgenic plants expressing high levels of antisense PME RNA showed <10% of wild-type PME enzyme activity and undetectable levels of PME protein and mRNA. Lower PME enzyme activity in fruits from transgenic plants was associated with an increased molecular weight and methylesterification of pectins and decreased levels of total and chelator soluble polyuronides in cell walls. The fruits of transgenic plants also contained higher levels of soluble solids than wild-type fruits. This trait was maintained in subsequent generations and segregated in normal Mendelian fashion with the antisense PME gene. These results indicate that reduction in PME enzyme activity in ripening tomato fruits had a marked influence on fruit pectin metabolism and increased the soluble solids content of fruits, but did not interfere with the ripening process.
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PMID:An Antisense Pectin Methylesterase Gene Alters Pectin Chemistry and Soluble Solids in Tomato Fruit. 1229 58

Pectinmethylesterase (PME, EC 3.2.1.11) and polygalacturonase (PG, EC 3.2.1.15) are known to operate in tandem to degrade methylesterified polyuronides. In this study, PGs purified from tomato and avocado fruit were compared in terms of their capacity to hydrolyze water-soluble polyuronides from avocado before and following enzymic or chemical de-esterification. When assayed using polygalacturonic acid or polyuronides from avocado fruit, the activity of PG from tomato fruit was 3-4 times higher than that from avocado fruit. High molecular mass, low methylesterified (33%) water-soluble polyuronides (WSP) from pre-ripe avocado fruit (day 0) were partially depolymerized upon incubation with purified avocado and tomato PGs. In contrast, middle molecular mass, highly methylesterified (74%) WSP from day 2 fruit were largely resistant to the action of both PGs. PME or weak alkali treatment of highly methylesterified WSP decreased the methylesterification values to 11 and 4.5%, respectively. Treatment of de-esterified WSP with either avocado or tomato PGs caused extensive molecular mass downshifts, paralleling those observed during avocado fruit ripening. Although PME and PG are found in many fruits, the pattern of depolymerization of native polyuronides indicates that the degree of cooperativity between these enzymes in vivo differs dramatically among fruits. The contribution of PME to patterns of polyuronide depolymerization observed during ripening compared with physically compromised fruit tissues is discussed.
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PMID:Methyl de-esterification as a major factor regulating the extent of pectin depolymerization during fruit ripening: a comparison of the action of avocado (Persea americana) and tomato (Lycopersicon esculentum) polygalacturonases. 1287 89

Watermelon fruit exhibit acute softening and placental-tissue water soaking following short exposure to exogenous ethylene. Experiments were performed to address transcript abundance and activities of cell wall and membrane hydrolases in placental tissue in response to treatment of watermelon fruit with ethylene. Watermelon fruit were harvested at immature and full-ripe stages and exposed to 50 microL L(-1) ethylene for 6 days at 20 degrees C. Ethylene affected the abundance of transcripts for PME (EC 3.2.1.11), and alpha-(EC 3.2.1.22) and beta-GAL (EC 3.2.1.23) but these effects were dependent on fruit maturity and appeared not to be associated with the water-soaking syndrome. PG (EC 3.2.1.15) and EXP mRNAs accumulated significantly in response to ethylene exposure. Additionally, the levels of mRNA and activities of LOX (EC 1.13.11.12), PLC (EC 3.1.4.3) and PLD (EC 3.1.4.4) were elevated in fruit of both maturity classes exposed to ethylene and were temporally associated with the visible symptoms of water soaking. The activity trends and transcript abundance in ethylene- compared with air-treated fruit indicate that PG, EXP, LOX, PLC and PLD levels increase with the onset and development of the water-soaking disorder and support the view that catabolic reactions targeting the membranes and cell-walls contribute to the disorder.
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PMID:Ethylene-induced gene expression, enzyme activities, and water soaking in immature and ripe watermelon (Citrullus lanatus) fruit. 1512 25

One of the main forms of tomato pectin methylesterase (PME; EC 3.1.1.1.1) that is applicable to the food industry was isolated from fresh tomato fruit. The extraction of the PME isoenzymes involved washing the fresh tomato flesh with water in order to remove sugars and than solubilizing the enzymes with a diluted HCl solution at pH 1.6. The extract was then neutralized to pH 7.4 using buffer solution. After filtration, the solution was directly fractioned using Convective Interaction Media (CIM) short monolithic disk column bearing sulfonyl (SO3) groups and using a linear gradient from 0 to 700 mM NaCl. The injection volume was 3 ml and the diameter of the column was 12 mm and length 3 mm. The isolated fractions were monitored for protein content and PME activity. The fraction with the targeted enzyme, which showed NaCl independent activity, was further purified and concentrated by ultrafiltration and finally purified by a second semi-preparative cation-exchange chromatography step using a CIM carboxymethyl (CM) disk monolithic column consisting of two disks and applying a step gradient. From 1 kg of fresh tomato fruits, 7.5 mg of purified PME with molecular mass estimated to be 26 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was obtained. A fraction with mixed PME and polygalacturonase activity was also obtained. Compared to the published procedures for the isolation and purification of PME from plant materials, this new procedure is much faster and more efficient. The potential application of CIM disk short monolithic columns in the analysis and semi-preparative extraction and isolation of the PME isoenzyme is presented.
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PMID:Separation of pectin methylesterase isoenzymes from tomato fruits using short monolithic columns. 1578 58

The most abundant isoforms of tomato pectin methylesterase (PME; EC 3.1.1.11; M(r) 26 kDa), polygalacturonase (PG; EC 3.2.1.15; PG1 with M(r) 82 kDa) and a basic protein with M(r) 42 kDa and unknown function were isolated from fresh tomato fruit by a fast chromatographic procedure on a Convective Interaction Media (CIM) short monolithic disk column bearing carboxymethyl (CM) groups. The extraction of the targeted enzymes with 1.2M NaCl solution was followed by precipitation with ammonium sulfate at 60% of saturation, solubilisation of the pellet in 0.5M NaCl and fractionation using a linear gradient from 0 to 700 mM NaCl. Among six fractions five had PME activity and four had PG activity, while one fraction containing a pure protein with M(r) 42 kDa with neither of these activities. Two concentrated fractions, one with PG and one with PME were further purified. A linear gradient from 0 to 500 mM NaCl with 20% CH(3)CN in the mobile phase was used for the PG fraction and two CM disks and a linear gradient from 0 to 200 mM NaCl were used for the PME fraction as a greater capacity was necessary in this case. From 4 kg of fresh tomato flesh we obtained 22 mg of purified PME, 1.8 mg of purified, active PG1, 13.5mg of additional basic protein and a fraction with PG2 contaminated by a PME isoform. Carboxymethyl CIM disk short monolithic columns are convenient for semi-preparative and analytical work with tomato fruit pectolytic enzymes.
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PMID:Separation of pectin methylesterases and polygalacturonases on monolithic columns. 1700 Jan 40

An improved cation-exchange chromatographic procedure on Convective Interaction Media (CIM, BIA Separations, Ljubljana, Slovenia) short monolithic methacrylate disk columns was used for the isolation of salt-independent pectin methylesterase (PME; EC 3.1.1.11) isoform and endo-polygalacturonase PG1 (PG, EC 3.2.1.15) from ripe tomato fruit extract after studying the chromatographic conditions including type of disk, binding buffer, pH, eluent composition and different gradients. Between 10 and 20 microg of proteins gave reliable chromatograms. Both carboxymethyl (CM) and sulfonyl (SO3) disks were equally suitable for the fractionation of tomato extract using the new gradient, but only CM disk was appropriate for further purification of the PME and PG fractions, and provided fast and sharp separation of proteins. The isolation of pure PG1 could be achieved only by addition of 20% of acetonitrile to the mobile phase. About 200 microg of proteins were loaded at one chromatographic run at the fractionation and purification. Determination of the molecular weights of the separated proteins showed that dimer of salt-independent PME isoform was formed in concentrated solutions of the enzyme but dissociated upon dilution of the solution. From 6 kg of fresh tomato flesh, 28 mg of purified salt-independent PME, 12.5mg of purified and active PG1 and 4 mg of PG2 fraction contaminated with salt-dependent PME isoform were obtained by means of semi-preparative chromatography on CIM disks.
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PMID:Isolation of tomato pectin methylesterase and polygalacturonase on monolithic columns. 1701 33


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