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Activity of pectin esterase and cellulase in the abscission zone of citrus leaf explants.

The activity of pectin esterase and cellulase in abscission of citrus explants was studied. No relation was established between pectin esterase and abscission, while cellulase activity was markedly increased before abscission and for a certain period after excision. IAA and cycloheximide delay abscission and cellulase activity, while ethylene and, to a lesser extent, GA(3) accelerate them. Application of cycloheximide during the lag period and before cellulase activity can be measured, inhibits to a certain extent the formation of cellulase. An escape from the inhibitory effect of cycloheximide is detected when inhibitor is supplied at the end of the lag period.

Journal Article↗

Structural insights into the target specificity of plant invertase and pectin methylesterase inhibitory proteins.

Pectin methylesterase (PME) and invertase are key enzymes in plant carbohydrate metabolism. Inhibitors of both enzymes constitute a sequence family of extracellular proteins. Members of this family are selectively targeted toward either PME or invertase. In a comparative structural approach we have studied how this target specificity is implemented on homologous sequences. By extending crystallographic work on the invertase inhibitor Nt-CIF to a pectin methylesterase inhibitor (PMEI) from Arabidopsis thaliana, we show an alpha-helical hairpin motif to be an independent and mobile structural entity in PMEI. Removal of this hairpin fully inactivates the inhibitor. A chimera composed of the alpha-hairpin of PMEI and the four-helix bundle of Nt-CIF is still active against PME. By contrast, combining the corresponding segment of Nt-CIF with the four-helix bundle of PMEI renders the protein inactive toward either PME or invertase. Our experiments provide insight in how these homologous inhibitors can make differential use of similar structural modules to achieve distinct functions. Integrating our results with previous findings, we present a model for the PME-PMEI complex with important implications.

Amino Acid Motifs↗

Structural basis for the interaction between pectin methylesterase and a specific inhibitor protein.

Pectin, one of the main components of the plant cell wall, is secreted in a highly methyl-esterified form and subsequently deesterified in muro by pectin methylesterases (PMEs). In many developmental processes, PMEs are regulated by either differential expression or posttranslational control by protein inhibitors (PMEIs). PMEIs are typically active against plant PMEs and ineffective against microbial enzymes. Here, we describe the three-dimensional structure of the complex between the most abundant PME isoform from tomato fruit (Lycopersicon esculentum) and PMEI from kiwi (Actinidia deliciosa) at 1.9-A resolution. The enzyme folds into a right-handed parallel beta-helical structure typical of pectic enzymes. The inhibitor is almost all helical, with four long alpha-helices aligned in an antiparallel manner in a classical up-and-down four-helical bundle. The two proteins form a stoichiometric 1:1 complex in which the inhibitor covers the shallow cleft of the enzyme where the putative active site is located. The four-helix bundle of the inhibitor packs roughly perpendicular to the main axis of the parallel beta-helix of PME, and three helices of the bundle interact with the enzyme. The interaction interface displays a polar character, typical of nonobligate complexes formed by soluble proteins. The structure of the complex gives an insight into the specificity of the inhibitor toward plant PMEs and the mechanism of regulation of these enzymes.

Actinidia↗

Effect of pectin methylesterase gene expression on pea root development.

Expression of an inducible gene with sequences common to genes encoding pectin methylesterase (PME) was found to be tightly correlated, both spatially and temporally, with border cell separation in pea root caps. Partial inhibition of the gene's expression by antisense mRNA in transgenic pea hairy roots prevented the normal separation of root border cells from the root tip into the external environment. This phenotype was correlated with an increase in extracellular pH, reduced root elongation, and altered cellular morphology. The translation product of the gene exhibited PME activity in vitro. These results are consistent with the long-standing hypothesis that the demethylation of pectin by PME plays a key role in cell wall metabolism.

Amino Acid Sequence↗

Molecular cloning and nucleotide sequence of the pectin methyl esterase gene of Erwinia chrysanthemi B374.

The gene encoding pectin methyl esterase (pme) has been cloned from Erwinia chrysanthemi B374. Expression of pme in Escherichia coli allowed the enzyme to be characterized. Pectin methyl esterase (PME) was found to have an apparent molecular weight of 36,000 Daltons and an isoelectric point of approximately 9.9. The structural gene was sequenced and consists of a 1098-bp open reading frame encoding a polypeptide of 39,318 Daltons, which includes an amino-terminal signal peptide. The isolation of the Erwinia gene provides a simple method for the production of PME free from depolymerizing pectinases thereby extending its potential uses.

Amino Acid Sequence↗

Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells. 3. Interplay between limited cell-wall autolysis, pectin methyl esterase activity and electrostatic effects in soybean cell walls.

Soybean cell walls display a process of autolysis which results in the release of reducing sugars from the walls. Loosening and autolysis of cell wall are involved in the cell-wall growth process, for autolysis is maximum during both cell extension and cell-wall synthesis. Autolysis goes to completion within about 50 h and is an enzymatic process that results from the activity of cell wall exo- and endo-glycosyltransferases. The optimum pH of autolysis is about 5. Increasing the ionic strength of the bulk phase where cell-wall fragments are suspended, results in a shift of the pH profile towards low pH. This is consistent with the view that at 'low' ionic strength, the local pH in the cell wall is lower than in the bulk phase. One of the main ideas of the model proposed in a preceding paper, is that pectin methyl esterase reaction, by building up a high fixed charge density, results in proton attraction in the wall. Low pH must then activate the wall loosening enzymes involved in autolysis and cell growth. This view may be directly confirmed experimentally. The pH of a cell-wall suspension, initially equal to 5, was brought to 8 for 20 min, then back to 5. Under these conditions, the rate of cell-wall autolysis was enhanced with respect to the rate of autolysis obtained with cell-wall fragments kept at pH 5. The pH response of the multienzyme plant cell-wall system basically relies on opposite pH sensitivities of the two types of enzymes involved in the growth process. Pectin methyl esterase, which generates the cell-wall Donnan potential, is inhibited by protons, whereas the wall-loosening enzymes involved in cell growth are activated by protons.

Autolysis↗

Effect of Metal Cations on the Viscosity of a Pectin-Like Capsular Polysaccharide from the Cyanobacterium Microcystis flos-aquae C3-40.

The properties of purified capsular polysaccharide from the cyanobacterium Microcystis flos-aquae C3-40 were examined by capillary viscometry. Capsule suspensions exhibited similar viscosities between pH 6 and 10 but were more viscous at pH <=4 than at pH 6 to 11. At pH 7, a biphasic effect of metal ion concentration on capsule viscosity was observed: (i) capsule viscosity increased with increasing metal ion concentration until a maximal viscosity occurred at a specific concentration that was a reproducible characteristic of each metal ion, and (ii) the viscosity decreased with further addition of that ion. Because the latter part of the biphasic curve was complicated by additional factors (especially the precipitation or gelation of capsule by divalent metal ions), the effects of various metal chlorides were compared for the former phase in which capsule viscosity increased in the presence of metal ions. Equivalent increases in capsule viscosity were observed with micromolar concentrations of divalent metal ions but only with 10 to 20 times greater concentrations of Na(sup+). The relative abilities of various metal salts to increase capsule viscosity were as follows: CdCl(inf2), Pb(NO(inf3))(inf2), FeCl(inf2) > MnCl(inf2) > CuCl(inf2), CaCl(inf2) >> NaCl. This pattern of metal efficacy resembles known cation influences on the structural integrity of capsule in naturally occurring and cultured M. flos-aquae colonies. The data are the first direct demonstration of an interaction between metal ions and purified M. flos-aquae capsule, which has previously been proposed to play a role in the environmental cycling of certain multivalent metals, especially manganese. The M. flos-aquae capsule and the plant polysaccharide pectin have similar sugar compositions but differ in their relative responses to various metals, suggesting that capsular polysaccharide could be a preferable alternative to pectin for certain biotechnological applications.

Journal Article↗

Morphological significance of the pectineal ligament of the eye.

We have studied the arrangement of the pectineal ligament or its equivalent, the uveal trabecula, in herbivores, carnivores, primates and humans. From our investigations, the pectineal ligament, the uveal trabecula and the so-called processes of the iris form a morphological unit that is made up of the tendinous fibres of the longitudinal portion of the ciliary muscle, that are inserted into the periphery of Descemet's membrane and send out ahead prolongations that extend to the anterior face of the iris. The so-called processes of the iris cannot be considered as independent structures since they represent the innermost fibres of the trabecular or uveal meshwork; in some species these have a thicker appearance, an arrangement that can occasionally be found in the human eye.

Acinonyx↗

Dysplasia of the pectinate ligament and primary glaucoma in the Bouvier des Flandres dog.

The eyes of 80 Bouvier des Flandres dogs without a history of clinical eye disease and 43 glaucomatous eyes out of 35 Bouvier des Flandres dogs were examined for changes in the pectinate ligament. Slight to severe dysplasia of the pectinate ligament occurred in 75% of the normotensive eyes. In glaucomatous eyes, dysplasia was severe and similar to the severity of dysplasia in the most affected normotensive eyes. A high percentage of the Bouvier des Flandres dogs in predisposed to develop glaucoma.

Animals↗

A second pectin lyase gene (pel2) from Aspergillus oryzae KBN616: its sequence analysis and overexpression, and characterization of the gene products.

A second pectin lyase gene, designated pel2, was isolated from a shoyu koji mold Aspergillus oryzae KBN616 and characterized. The structural gene comprised 1306 bp with three introns. The ORF encoded 375 amino acids with a signal peptide of 19 amino acids. The deduced amino acid sequence showed high similarity to those of A. oryzae Pel1, Aspergillus niger pectin lyases and Glomerella cingulata Pn1A. The pel2 gene was overexpressed under the control of the promoter of the A. oryzae TEF1 gene for purification and enzymatic characterization of its gene product. The gene product exhibited two molecular masses of 48 and 44 kDa due to different degrees of glycosylation. Both proteins had the same pH optimum of 6.0 and temperature optimum of 50 degrees C.

Journal Article↗

Purification and general properties of pectin methyl esterase from Curvularia inaequalis NRRL 13884 in solid state culture using orange peels as an inducer.

Pectin methyl esterase (PME) [E.C.3. 1.1.11] production by Curvularia inaequalis (Shear) Boedijn NRRL 13884 was investigated using solid-state culture. The highest level of extracellular pectin methyl esterase was detected with orange peels as an inducing substrate and as a sole carbon source. The enzyme was partially purified using Sephadex G-100 and DEAE-Cellulose column chromatography. It was purified about 40 fold with optimum activity at pH 4.4 and 45 degrees C. The enzyme was activated by Co++, Mg++, Na+, whereas it was slightly activated in the presence of Cu++, K+, Mn++, Zn++. On the other hand Ag++, Ca++ and Hg++ inhibited the activity of the enzyme. The Km was calculated to be 0.52 mM.

Ascomycota↗

[Demonstration of pectin-lyase in Bacillus subtilis].

After a screening performed on pectinolytic micro-organisms, a strain of B. subtilis was isolated. This strain has a pectic enzyme capable of beta-elimination on highly methylated pectin (degree of esterification = 85%), without the action of a pectinesterase. This activity corresponds to that of a pectin-lyase.

Bacillus subtilis↗

The canine eye: pectinate ligaments and aqueous outflow resistance.

The contribution of the pectinate ligaments to total aqueous outflow resistance was studied in 30 excised canine eye pairs by means of constant-pressure aqueous perfusion. Incision of these ligaments over one and two quadrants of the angle circumference increased the facility of aqueous outflow significantly more than could be attributed to normal "washout" alone. Neither mechanical nor enzymatic disruption of angle glycosaminoglycans could fully account for this phenomenon. The pectinate ligaments may therefore contribute to the canine aqueous outflow barrier by compartmentalizing the glycosaminoglycans in the spaces of Fontana. In addition, these ligaments, analogous to iris processes in the human, prevent widening of the canine angle and hold the filtration structures in a relatively compressed state, a situation that can be reversed by ligament incision.

Animals↗

[Pathological changes in the tendons of the pectineal muscles in a case of canine hip dysplasia (author's transl)].

This report gives radiological and pathological evidence of bilateral ossification of the aponeurotic tendons of insertion of the pectineal muscles in a dog. This was associated with a severe grade 3 hip dysplasia of which the pathological changes are described. The ossification of the tendons is considered to have resulted from excessive and continous tension of the pectineus muscles. Various aetiological factors are discussed and some substantiation provided for the theory that pathology of the pectineal muscles and their tendons of insertion may be a contributing factor in the pathogenesis of hip dysplasia.

Animals↗

A presumably astroglial cell in the retino-pectineal junction in Gallus domesticus, demonstrated by non-specific esterase staining.

Enzyme-histochemical localization of nonspecific esterase permits identification of a characteristic neuroglial cell profile in the optic disc and in the nerve fiber layer of the retinal along the retinopectineal junction in the chick. The cells showing intense enzyme activity are disposed radially in the stretch of the nerve fiber layer subjacent to the pectineal base and the adjoining optic disc. Outer and inner processes can be traced from their spindle shaped perikarya up to the inner margin of the inner plexiform layer, and to the base of the pectineal pleats or the inner limiting membrane lying in between. Structural organization and location of these cells suggest their inclusion as fibrous astrocytes. The possible role of these cells in effecting firm anchorage of the intraocular conglomerate of blood vessels with the optic disc and the retina, as well as their possible source of recruitment during embryogenesis have been discussed.

Animals↗

A new technique of uterine suspension to pectineal ligaments in the management of uterovaginal prolapse.

The incidence of uterovaginal prolapse in young women is high in developing countries. Preservation of the uterus is of prime importance in the surgical management of these patients. A new technique of uterine suspension to the pectineal ligaments is presented as an alternative to traditional procedures. Through a Cherney incision, the uterus is suspended to the pectineal ligaments on both sides with mersilene tape. A simultaneous Burch colposuspension can be useful in selected cases. The operation has been done in 20 women, who averaged 27.5 years of age. There was no early or late morbidity during the follow-up period of 6-30 months. Of nine women desiring further childbearing, seven conceived within 6 months of surgery and thus far five have had an uneventful vaginal delivery at term; the other two have continuing normal gestations. There was no recurrence of prolapse at 6 weeks postpartum in any of the women.

Adult↗

Solution conformations of pectin polysaccharides: determination of chain characteristics by small angle neutron scattering, viscometry, and molecular modeling.

The solution behavior of pectin polysaccharides has been investigated by small angle neutron scattering (SANS), viscosimetric, and molecular modeling studies. The samples used in the experimental study were obtained from apple and citrus and had degrees of methylation ranging from 28 to 73%, with a rhamnose content lying between 0.6 and 2.2%. Persistence lengths, derived from intrinsic viscosity measurements, ranged from 59 to 126 A, whereas those derived by SANS were between 45 and 75 A. These values correspond to 10-17 monomer units. The modeling simulations were performed for both homogalacturonan itself and homogalacturonan carrying various degrees of rhamnose inserts (rhamnogalacturonan). This required the evaluation of the accessible conformational space for the eight disaccharides that represent the constituent repeating segments of the homogalacturonan and rhamnogalacturonan polysaccharides. For each dimer, complete conformational analysis was accomplished using the flexible residue method of the MM3 molecular mechanics procedure and the results used to access the configurational statistics of representative pectic polysaccharide chains. For homogalacturonan, an extended chain conformation having a persistence length of 135 A (corresponding to 30 monomers) was predicted. The inclusion of varying amounts of rhamnose units (5-25%) in the model in strict alternating sequence with galacturonate residues (equivalent to the rhamnogalacturonan "hairy region" chains) only slightly reduced the calculated persistence length. The extended overall chain conformation remained relatively unchanged as a consequence of the self-cancellation of the kinking effects of successive paired rhamnose units.

Carbohydrate Conformation↗

Differential fragmentation patterns of pectin oligogalacturonides observed by nanoelectrospray quadrupole ion-trap mass spectrometry using automated spectra interpretation.

Oligogalacturonides of different degrees of polymerization (DP) and methyl esterification (DE) were structurally analyzed by nanoESI quadrupole ion-trap mass spectrometry. The fragmentation patterns of the oligogalacturonides were compared using the program 'Virtual Expert Mass Spectrometrist' (VEMS) for structural annotation. In the analyzed oligogalacturonides of lower DP, the generation of C/Y ions, i.e. ions retaining the glycosidic oxygen, was higher than that of B/Z ions. In general, with oligogalacturonides of higher DP, the B/Z ions were generated more abundantly. Oligogalacturonides with free carboxylic acid groups underwent higher water loss compared to fully methyl-esterified oligogalacturonides under the same fragmentation conditions. Cross-ring cleavage, in which fragmentation occurs across the ring system of the galacturonate residue and signified by unique mass losses, was observed to be higher in fully methyl-esterified oligogalacturonides than in non-methyl-esterified ones. This study demonstrates the different fragmentation patterns of oligogalacturonides as influenced by the presence or absence of methyl ester groups. For a detailed analysis of unknown oligogalacturonides, cross-ring fragmentation gives more structural information than glycosidic bond cleavage. One implication of this is that more structural information is obtained when analyzing methyl-esterified oligogalacturonides than non-methyl-esterified ones in an ion-trap instrument. This is of particular importance in pectin chemistry, where mass spectrometry has become the technique of choice for structural determination. Although this study was not designed to explain the mechanisms of oligogalacturonide fragmentation, possible explanations for why non-methyl-esterified oligogalacturonides undergo more water loss than methyl-esterified ones will be postulated. In addition, the VEMS program was extended to automatically interpret and assign the fragment ions peaks generated in this study.

Nanotechnology↗