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At least 451 records · Page 25Linked to original sources
PubMed2005

Pectin methylesterase, a regulator of pollen tube growth.

The apical wall of growing pollen tubes must be strong enough to withstand the internal turgor pressure, but plastic enough to allow the incorporation of new membrane and cell wall material to support polarized tip growth. These essential rheological properties appear to be controlled by pectins, which constitute the principal component of the apical cell wall. Pectins are secreted as methylesters and subsequently deesterified by the enzyme pectin methylesterase (PME) in a process that exposes acidic residues. These carboxyls can be cross-linked by calcium, which structurally rigidifies the cell wall. Here, we examine the role of PME in cell elongation and the regulation of its secretion and enzymatic activity. Application of an exogenous PME induces thickening of the apical cell wall and inhibits pollen tube growth. Screening a Nicotiana tabacum pollen cDNA library yielded a pollen-specific PME, NtPPME1, containing a pre-region and a pro-region. Expression studies with green fluorescent protein fusion proteins show that the pro-region participates in the correct targeting of the mature PME. Results from in vitro growth analysis and immunolocalization studies using antipectin antibodies (JIM5 and JIM7) provide support for the idea that the pro-region acts as an intracellular inhibitor of PME activity, thereby preventing premature deesterification of pectins. In addition to providing experimental data that help resolve the significance and function of the pro-region, our results give insight into the mechanism by which PME and its pro-region regulate the cell wall dynamics of growing pollen tubes.

Arabidopsis↗
PubMed1994

Cell Wall Metabolism in Ripening Fruit (VII. Biologically Active Pectin Oligomers in Ripening Tomato (Lycopersicon esculentum Mill.) Fruits).

A water-soluble, ethanol-insoluble extract of autolytically inactive tomato (Lycopersicon esculentum Mill.) pericarp tissue contains a series of galacturonic acid-containing (pectic) oligosaccharides that will elicit a transient increase in ethylene biosynthesis when applied to pericarp discs cut from mature green fruit. The concentration of these oligosaccharides in extracts (2.2 [mu]g/g fresh weight) is in excess of that required to promote ethylene synthesis. Oligomers in extracts of ripening fruits were partially purified by preparative high-performance liquid chromatography, and their compositions are described. Pectins were extracted from cell walls prepared from mature green fruit using chelator and Na2CO3 solutions. These pectins are not active in eliciting ethylene synthesis. However, treatment of the Na2CO3-soluble, but not the chelator-soluble, pectin with pure tomato polygalacturonase 1 generates oligomers that are similar to those extracted from ripening fruit (according to high-performance liquid chromatography analysis) and are active as elicitors. The possibility that pectin-derived oligomers are endogenous regulators of ripening is discussed.

Journal Article↗
PubMed1992

Influence of the degree of polymerization of oligogalacturonates and of esterification pattern of pectin on their recognition by monoclonal antibodies.

The ability of galacturonic and oligogalacturonic acids with degrees of polymerization (DP) from 2 to 10 to inhibit the recognition of homopolygalacturonic acid by a monoclonal antibody specific for dimers of pectin (F Liners, J-J Letesson, C Didembourg, P Van Cutsem [1989] Plant Physiol 91: 1419-1424) has been tested by enzyme-linked immunosorbent assays. Oligomers of DP9 and above preincubated with the antibodies clearly inhibited the association between the antibodies and immobilized pectin. A minimum DP of nine consecutive galacturonic residues is thus necessary to be associated through calcium cations to form dimers. Randomly deesterified pectin was recognized by the antibody if its degree of methylesterification was <30%, whereas blockwise deesterified pectin was recognized up to 40% of methylesterification. The replacement of calcium ions by magnesium prevented the recognition of polygalacturonic acid by the antibody.

Journal Article↗
PubMed1988

Identification and sequence determination of a cDNA clone for tomato pectin esterase.

Cell wall softening during tomato fruit ripening is brought about through the action of a number of pectolytic enzymes. We have reported previously the cloning and characterisation of the cDNA for the major cell wall softening (degrading) enzyme, polygalacturonase [Grierson, D., Tucker, G. A., Keen, J., Ray, J., Bird, C. R. and Schuch, W. (1986) Nucleic Acids Res. 14, 8595-8603]. We have now isolated a cDNA clone for tomato pectin esterase, an enzyme also implicated in cell wall softening. Here we report the structure of this cDNA and compare it with the structure of pectin esterase derived from amino acid sequence experiments [Markovic, O. and Jornvall, H. (1986) Eur. J. Biochem. 158, 455-462]. We have used the pectin esterase cDNA clone to analyse pectin esterase gene expression during development and ripening of normal and mutant fruit.

Amino Acid Sequence↗
PubMed1995

A glycoprotein inhibitor of pectin methylesterase in kiwi fruit. Purification by affinity chromatography and evidence of a ripening-related precursor.

The pectin methylesterase inhibitor from kiwi fruit (Actinidia chinensis) was purified by a single-step procedure based on affinity chromatography. Partially purified tomato pectin methylesterase was covalently bound to Sepharose. The affinity resin strongly and selectively binds the inhibitor, which could be eluted in high yield as a single, homogeneous and sharp peak by high salt concentration at pH 9.5 without loss of inhibitory activity. The purified protein possesses a molecular mass of 18 kDa, as estimated by SDS/PAGE, whereas by gel filtration under native conditions, its molecular mass appears to be 25 kDa. The inhibitor interacts with pectin methylesterase, forming a 1:1 complex, as demonstrated by gel-filtration experiments. The inhibitor was glycosylated. Its glycidic portion can be removed by digestion with N-glycosidase F after protein denaturation and, to a minor extent, by digestion with N-glycosidase H. No glycidic residue could be removed by digesting the native protein with those N-glycosidases. Antibodies against pectin methylesterase inhibitor were raised in rabbits and used to evidence protein expression during fruit ripening. The results showed that the inhibitor is present in the unripe fruit as an inactive precursor with a higher molecular mass (30 kDa) and is transformed into the active protein, most likely by proteinase action, during the course of the ripening process.

Carboxylic Ester Hydrolases↗
PubMed1993

Involvement of an Intracellular Oligogalacturonate Hydrolase in Metabolism of Pectin by Clostridium thermosaccharolyticum.

The enzymes pectin methylesterase and polygalacturonate hydrolase, which are responsible for the initial steps of pectin degradation by Clostridium thermosaccharolyticum, were shown to be induced on the polymeric substrates pectin and pectate, as well as on oligogalacturonates, and to be repressed in the presence of glucose. The digalacturonate and trigalacturonate produced by the extracellular pectin methylesterase-polygalacturonate hydrolase complex were transported across the cytoplasmic membrane and hydrolyzed by an inducible oligogalacturonate hydrolase to galacturonate. The oligogalacturonate hydrolase was separated from the polygalacturonate hydrolase and characterized. Its temperature optimum was 65 degrees C, and its pH optimum was 6. The native molecular size was 90 kDa, and the enzyme was stable for more than 1 h at 65 degrees C. The maximum reaction rate on oligomers decreased with the increasing degree of polymerization. Galacturonate was released by hydrolysis from the nonreducing end of the oligomer. The amounts of pectinolytic enzymes produced were all strictly correlated to the amount of biomass formed. Galacturonate was metabolized via a modified Entner-Doudoroff route.

Journal Article↗
PubMed1994

Expression of a pectin lyase gene in Escherichia coli from Pseudomonas marginalis N6301.

We constructed deletion mutant clones of a pectin lyase gene, and measured their pectin lyase activities in Escherichia coli. Pectin lyase activities were detected only in a recA+ strain but not in a recA- strain of E. coli. We also cloned and sequenced recA from Pseudomonas marginalis N6301. The recA from P. marginalis N6301 can complement recA- to form recA+ in the phenotype of E. coli. Highly conserved sequences of recA are observed among E. coli, P. fluorescens, and P. marginalis. From these results, we presume that recA is required for the expression of the pectin lyase gene in P. marginalis N6301.

Amino Acid Sequence↗
PubMedDate not supplied

[Use of a preparation from fungal pectin lyase in the food industry].

A new enzyme preparation of fungal pectin lyase (EC 4.2.2.10) was shown to be useful for the production of cranberry juice and clarification of apple juice in the food industry. A comparative study showed that the preparation of pectin lyase is competitive with commercial pectinase products. The molecular weight of homogeneous pectin lyase was 38 kDa. Properties of the homogeneous enzyme were studied. This enzyme was most efficient in removing highly esterified pectin.

Beverages↗
PubMed1996

Determination of residual pectin methylesterase activity in food products.

A method for the determination of a low level of pectin methylesterase activity from vegetable products is described. The method is based on an affinity chromatography technique that employs a resin-bound pectin methylesterase inhibitor, purified from kiwi fruit, which selectively binds the pectin methylesterase. The resin has the capacity to concentrate the enzyme, allowing measurement of enzyme activities too low to be determined by commonly employed techniques and commensurate with those found in pasteurized food products. The enzyme is eluted from the resin at alkaline pH (9.5) and assayed by a pH-stat method. Depulped orange juices containing different amounts of pectin methylesterase were prepared and used to determine enzyme recovery. The results show a recovery of 90% with a standard deviation of 6.8%.

Carboxylic Ester Hydrolases↗
PubMed1987

A comparison of some methods for the determination of methoxyl groups in commercial pectin preparations.

The results of methoxyl groups determination in pectin preparations were compared. The methods of direct (I, II) and indirect (III, IV) methanol determination connected with enzymatic (II, IV) or alkaline (I, III) pectin demethylation were applied. Higher values were obtained using indirect methanol methods than in the case of a direct methanol determination. The difference between these groups of methods was statistically significant. The precision of all the methods was high. The degree of methylation (DM) was calculated based on methanol content; moreover in the methods III and IV it was also determined from the ratio of methylated carboxyl groups to a total of acidic groups. From the difference (%) between these two ways of calculating DM the purity of pectin can be determined. The method based on enzymatic demethylation and direct methanol analysis is recommended as a precise and selective one.

Dealkylation↗
PubMed1981

In vitro interaction of quinidine with kaolin and pectin.

The adsorption of quinidine onto kaolin was studied as a function of pH in aqueous solutions in which the ionic strength was adjusted to 0.1. The interaction of quinidine with pectin also was investigated in water and in phosphate buffer; the buffer pH and ionic strength were adjusted to 6.5 and 0.1, respectively. The in vitro results indicated that quinidine was adsorbed onto kaolin. At the highest concentration studied, the extent of adsorption increased from 3.64 mg of quinidine adsorbed/g of adsorbent at pH 2.4 to an average of 5.81 mg/g in the pH 5.5-7.5 range. In the presence of electrolytes, the interaction of quinidine with pectin was relatively small (3-13% bound) as compared to studies performed in water (66-90% bound). The data indicate that some quinidine may be adsorbed when this drug is administered concurrently with kaolin-pectin preparations.

Adsorption↗
PubMed2004

Computer modeling of the rhamnogalacturonase-"hairy" pectin complex.

The structure of a pectin-bound complex of rhamnogalacturonase was modeled to identify the amino acid residues involved in catalysis and substrate binding. The "hairy" region of pectin, represented by six repeating stretches of (1-->4)-D-galacturonate-(1-->2)-L-rhamnose dimer, was flexibly docked into the putative binding site of rhamnogalacturonase from Aspergillus aculeatus whose X-ray structure is known. A search of the complex configurational space was performed using AutoDock for the dimeric and tetrameric sugar units in which the -1 galacturonate residue has various ring conformations. Then the plausible AutoDock solutions were manually extended to the dodecameric pectin models. Subsequently, the resulting complex models were subjected to solvated molecular dynamics using AMBER. In the best model, the substrate has an extended pseudo-threefold helix with the -1 ring in a 4H3 half-chair that approaches the transition state conformation. The catalytic machinery is clearly defined: Asp197 is a general acid and the activated water bound between Asp177 and Glu198 is a nucleophile. The active site is similar, with a small yet significant difference, to that of polygalacturonase that degrades the pectic "smooth" region of linear homopolymer of D-(1-->4)-linked galacturonic acid. Rhamnogalacturonase has ten binding subsites ranging from -3 to +7, while polygalacturonase has eight subsites from -5 to +3. The model suggests that the eight amino acids including three arginine and three lysine residues, all of which are invariantly conserved in the rhamnogalacturonase family of proteins, are important in substrate binding. The present study may aid in designing mutational studies to characterize rhamnogalacturonase.

Amino Acid Sequence↗
PubMed1995

Enzymatic synthesis and purification of uridine diphosphate [14C]galacturonic acid: a substrate for pectin biosynthesis.

Pectins are complex polysaccharides that contain 1,4-linked alpha-D-galactosyluronic acid residues found in the primary wall of all higher plant cells. The pectic polysaccharides play critical roles in cell wall structure and in plant growth and development. As a first step in studying pectin biosynthesis a method was developed to routinely generate and purify UDP-[U-14C]galacturonic acid (UDP-[14C]GalA), the nucleotide sugar substrate for homogalacturonan biosynthesis. UDP-[14C]GalA was enzymatically synthesized by 4-epimerization of commercially available UDP-[U-14C]glucuronic acid (UDP-[14C]GlcA) using a particulate preparation from radish roots. The resulting mixture of UDP-[14C]GalA and UDP-[14C]GlcA was separated by high-performance anion-exchange chromatography using a Dionex CarboPac PA1 anion-exchange column. The UDP-sugars were detected by their absorbance at 262 nm or by pulsed amperometric detection following postcolumn addition of NaOH. The yield of UDP-[14C]GalA obtained using this procedure was 16% of the starting UDP-[14C]GlcA. Establishment of a reliable method to synthesize and purify UDP-[14C]GalA will facilitate the identification and purification of the galacturonosyltransferase(s) involved in pectin biosynthesis.

Anions↗
PubMed1997

A spectrophotometric assay for the enzymatic demethoxylation of pectins and the determination of pectinesterase activity.

A rapid spectrophotometric method for the determination of pectinesterase activity is presented. In this assay, methanol released from pectin by pectinesterase is oxidized with alcohol oxidase to form hydrogen peroxide and formaldehyde. Hydrogen peroxide is then quantitated with peroxidase and the chromogen 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). Since both reactions exhibit the same pH optimum it was possible to couple the methanol assay directly to the action of pectinesterase for the real-time determination of this enzyme. The assay is reliable and sensitive, being capable of quantitating a minimum pectinesterase activity of 0.0625 unit (1 unit = 1 microM methanol released per minute). It is also capable of detecting the enzymatic demethoxylation of galactopyranosyl uronic acid methyl esters of pectin down to a minimum concentration of 1.56 nM of methanol per milliliter using a pectin substrate with a methoxy content of 10% (w/w) at a concentration of 0.5 microgram/ml.

Alcohol Oxidoreductases↗
PubMed1993

Pectin transeliminase complex from Aspergillus flavus.

Aspergillus flavus grown in a liquid medium containing pectin as the sole carbon source produced extracellular enzymes which degraded the 1,4-alpha-D-glycosidic bonds of pectin. The products of degradation were characteristic of substances produced by transeliminase. Synthesis of this enzyme was repressed by the addition of sucrose, glucose, fructose and maltose. The crude enzyme was partially purified by a combination of ultrafiltration and ammonium sulfate precipitation. The partially purified enzyme was separated by molecular exclusion chromatography into three components A, B and C, with molar masses ranging from 13.2 to 64 kDa. Only fraction B exhibited enzymic activity and further fractionated by ion-exchange chromatography into four components I-IV. Among these components, only fractions I and II possessed transeliminase activity. Both fractions had an optimum activity at pH 8.5 and 35 degrees C, and were stimulated by Ca2+, Mg2+, Na+ and K+ but inhibited by EDTA and DNP. The apparent Km for the degradation of pectin by fractions I and II were 6.2 and 8.0 g/L, respectively.

2,4-Dinitrophenol↗
PubMed2001

Altered pectin composition in primary cell walls of korrigan, a dwarf mutant of Arabidopsis deficient in a membrane-bound endo-1,4-beta-glucanase.

Korrigan (kor) is a dwarf mutant of Arabidopsis thaliana (L.) Heynh. that is deficient in a membrane-bound endo-1,4-beta-glucanase. The effect of the mutation on the pectin network has been studied in kor by microscopical techniques associated with various probes specific for different classes of pectic polysaccharides. The localisation of native crystalline cellulose was also examined using the cellobiohydrolase I-gold probe. The investigations were focused on the external cell walls of the epidermis, a cell layer that, in a number of plant species, has been shown to be growth limiting. Anionic sites associated with pectic polymers were quantified using the cationic gold probe. Homogalacturonans were quantified using polyclonal anti-polygalacturonic acid/rhamnogalacturonan I antibodies recognising polygalacturonic acid, and monoclonal JIM7 and JIM5 antibodies recognising homogalacturonans with a high or low degree of methyl-esterification, respectively. Rhamnogalacturonans were quantified with two monoclonal antibodies, LM5, recognising beta-1,4 galactan side chains of rhamnogalacturonan I, and CCRCM2. Our results show a marked increase in homogalacturonan epitopes and a decrease in rhamnogalacturonan epitopes in kor compared to the wild type. A substantial decrease in cellobiohydrolase I-gold labelling was also observed in the mutant cell walls. These findings demonstrate that a deficiency in an endo-1,4-beta-glucanase, which is in principle not directly implicated in pectin metabolism, can induce important changes in pectin composition in the primary cell wall. The changes indicate the existence of feedback mechanisms controlling the synthesis and/or deposition of pectic polysaccharides in primary cell walls.

Antibodies, Monoclonal↗
PubMed2006

Immunodetection of pectin and arabinogalactan protein epitopes during pollen exine formation of Beta vulgaris L.

We present the results of ultrastructural and immunocytochemical studies of sugar beet microsporocytes during the developmental phase that begins with the first meiotic metaphase and ends with the formation of young tetrads. The most prominent feature noted during this period of microsporogenesis was the presence of numerous cisternae of endoplasmic reticulum which frequently lie perpendicular to the surface of the plasma membrane and eventually fuse to it. Microscopic observations have been combined with the detection of several carbohydrate epitopes representing pectins and arabinogalactan proteins in the primexine and incipient exine. Pectin domains that possess both low and highly methylesterified epitopes, as well as pectin side chains enriched in (1-->4)-beta-D-galactose residues, are deposited in this young microspore wall. The epitopes of arabinogalactan protein that bind to JIM13, JIM8, and LM2 antibodies are localised within the callose wall surrounding posttelophase tetrads. The possibility of endoplasmic-reticulum involvement in the synthesis, transport, or metabolism of several microspore wall compounds is discussed.

Beta vulgaris↗
PubMed2006

The structure and biochemistry of charophycean cell walls: I. Pectins of Penium margaritaceum.

Plant cell walls are essential for proper growth, development, and interaction with the environment. It is generally accepted that land plants arose from aquatic ancestors which are sister groups to the charophycean algae (i.e., Streptophyta), and study of wall evolution during this transition promises insight into structure-function relationships of wall components. In this paper, we explore wall evolutionary history by studying the incorporation of pectin polymers into cell walls of the model organism Penium margaritaceum, a simple single-cell desmid. This organism produces only a primary wall consisting of three fibrillar or fibrous layers, with the outermost stratum terminating in distinct, calcified projections. Extraction of isolated cell walls with trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid yielded a homogalacturonan (HGA) that was partially methyl esterified and equivalent to that found in land plants. Other pectins common to land plants were not detected, although selected components of some of these polymers were present. Labeling with specific monoclonal antibodies raised against higher-plant HGA epitopes (e.g., JIM5, JIM7, LM7, 2F4, and PAM1) demonstrated that the wall complex and outer layer projections were composed of the HGA which was significantly calcium complexed. JIM5 and JIM7 labeling suggested that highly methyl esterified HGA was secreted into the isthmus zone of dividing cells, the site of active wall secretion. As the HGA was displaced to more polar regions, de-esterification in a non-blockwise fashion occurred. This, in turn, allowed for calcium binding and the formation of the rigid outer wall layer. The patterning of HGA deposition provides interesting insights into the complex process of pectin involvement in the development of the plant cell wall.

Cell Wall↗