Small proteoglycan synthesis by skin fibroblasts cultured from elderly donors and patients with defined defects in types I and III collagen metabolism

The relative synthesis of two different types of small proteoglycans with potentially distinct roles in tissue function (PGI and PGII) was investigated in human skin fibroblast cultures initiated from donors of increasing age (fetal to 92 y) and from patients with defined defects in type I and type III collagen metabolism. Because these two small proteoglycans are not distinguished by the usual methods of ion-exchange and sieve chromatography, we have separated them using gel electrophoresis and confirmed this by specific immunoprecipitation. Small proteoglycans of the PGII type were the predominant species found in the medium of all cultures from normal donors, regardless of age. Most of the mutant cell lines showed a profile of small proteoglycan synthesis like that of the normal cells (i.e., predominantly PGII) although an increased ratio of PGI/PGII was seen for two cell strains from patients with Ehlers-Danlos syndrome type IV characterized by intracellular accumulation of type III procollagen. We conclude that mutations affecting collagen primary structure and secretion appear to have little effect on the cells' synthesis and secretion of small proteoglycans. These findings fail to support an hypothesis suggesting that the metabolism of normal cellular synthetic products (proteoglycans) is altered by abnormal cellular processing of a defective product (collagen).     

Proteoglycans from pig aorta. Comparative study of their interactions with lipoproteins.

Different proteoglycans (PGs) were isolated from pig aorta for aggregation studies with hyaluronic acid and human low-density lipoproteins (LDL). Extraction of the intima-media with 4M-guanidinium chloride and digestion of the residue with collagenase solubilized 91% of aortic hexuronic acid content. From the guanidinium chloride extract two PGs were isolated by ion-exchange and gel-permeation chromatography: proteochondroitin sulphate (PGI) with a protein-core apparent Mr of 250 000 and proteodermatan-chondroitin sulphate (PGII) with a protein-core apparent Mr of 55 000. Only PGI forms high-Mr aggregates with hyaluronic acid. From the collagenase digest two other PGs were isolated: proteoheparan sulphate and proteochondroitin sulphate (PGIII and PGIV respectively). PGIV had a smaller hydrodynamic size than PGI. PGI and PGII formed insoluble complexes with human LDL in the presence of Ca2+. PGIII or PGIV did not form precipitates with the LDL. PGI and PGII, but neither PGIII nor PGIV, were bound to LDL-Sepharose. The main peaks of PGI and PGII were eluted from LDL-Sepharose with 60 mM- and 90 mM-NaCl respectively. The results indicate that aortic PGs have different interacting potentials with lipoproteins, depending on their Mr and their glycosaminoglycan composition.     

The polygalacturonases of Aspergillus niger are encoded by a family of diverged genes.

Aspergillus niger produces several polygalacturonases that, with other enzymes, are involved in the degradation of pectin. One of the two previously characterized genes coding for the abundant polygalacturonases I and II (PGI and PGII) found in a commercial pectinase preparation was used as a probe to isolate five more genes by screening a genomic DNA library in phage lambda EMBL4 using conditions of moderate stringency. The products of these genes were detected in the culture medium of Aspergillus nidulans transformants on the basis of activity measurements and Western-blot analysis using a polyclonal antibody raised against PGI. These transformants were, with one exception, constructed using phage DNA. A. nidulans transformants secreted high amounts of PGI and PGII in comparison to the previously characterized A. niger transformants and a novel polygalacturonase (PGC) was produced at high levels by A. nidulans transformed with the subcloned pgaC gene. This gene was sequenced and the protein-coding region was found to be interrupted by three introns; the different intron/exon organization of the three sequenced A. niger polygalacturonase genes can be explained by the gain or loss of two single introns. The pgaC gene encodes a putative 383-amino-acid prepro-protein that is cleaved after a pair of basic amino acids and shows approximately 60% amino acid sequence similarity to the other polygalacturonases in the mature protein. The N-terminal amino acid sequences of the A. niger polygalacturonases display characteristic amino acid insertions or deletions that are also observed in polygalacturonases of phytopathogenic fungi. In the upstream regions of the A. niger polygalacturonase genes, a sequence of ten conserved nucleotides comprising a CCAAT sequence was found, which is likely to represent a binding site for a regulatory protein as it shows a high similarity to the yeast CYC1 upstream activation site recognized by the HAP2/3/4 activation complex.     

Dermatan sulphate proteoglycans from sclera examined by rotary shadowing and electron microscopy.

Two dermatan sulphate-containing proteoglycans from bovine sclera were examined by rotary shadowing and electron microscopy, and the results were compared with previous biochemical findings. Both the large iduronate-poor proteoglycan (PGI) and the small iduronate-rich proteoglycan (PGII) possessed a globular proteinaceous region. Whereas PGI had a branched extension from the globular region, with five to eight side chains attached to it, PGII had only a single tail, which was of glycosaminoglycuronan. PGII aggregated via globular-region interactions, which were much diminished by reduction and alkylation. PGI aggregated via side chains and globular-region interactions. Although a few PGI aggregates were large, and similar to the hyaluronan-cartilage proteoglycan aggregates [Weidemann, Paulsson, Timpl, Engel & Heinegård (1984) Biochem. J. 224, 331-333], hyaluronan did not cause enhanced aggregation. PGII is very similar in shape to the small cartilage chondroitin sulphate proteoglycan, whereas PGI somewhat resembles the large cartilage chondroitin sulphate proteoglycan, although with many fewer glycosaminoglycan side chains, and probably only one globular region as opposed to two in the cartilage proteoglycan.     

Biochemical characterization of an extracellular polygalacturonase from Trichoderma harzianum

An extracellular polygalacturonase (PGII) from Trichoderma harzianum was purified to homogeneity by two chromatography steps using DEAE-Sepharose and Sephacryl S-200. The molecular weight of T. harzianum PGII was 31,000 Da by gel filtration and SDS-PAGE. PGII had isoelectric point of 4.5 and optimum pH of 5.0. PGII was very stable at the pH 5.0. The extent of hydrolysis of different pectins by enzyme was decreased with increasing of degree of esterification (DE). PGII had very low activity toward non-pectic polysaccharides. The apparent K(m) value and K(cat) value for hydrolyzing polygalacturonic acid (PGA) were 3.4 mg/ml and 592 s(-1), respectively. PGII was found to have temperature optimum at 40 degrees C and was approximately stable up to 30 degrees C for 60 min of incubation. All the examined metal cations showed inhibitory effects on the enzyme activity. A 1,10-phenanthroline, Tween 20, Tween 80, Triton X-100 and SDS had no effect on the enzyme activity. The rate of enzyme catalyzed reduction of viscosity of solutions of PGA or pectin was higher three times than the rate of release of reducing sugars indicating that the enzyme had an endo-action. The storage stability of the enzyme in liquid and powder forms was studied, where the activity of the powder form was stable up to 1 year. These properties of T. harzianum PGII with appreciable activity would be potentially novel source of enzyme for food processing.     

Pectin Modification in Cell Walls of Ripening Tomatoes Occurs in Distinct Domains

The class of cell wall polysaccharides that undergoes the most extensive modification during tomato (Lycopersicon esculentum) fruit ripening is pectin. De-esterification of the polygalacturonic acid backbone by pectin methylesterase facilitates the depolymerization of pectins by polygalacturonase II (PGII). To investigate the spatial aspects of the de-esterification of cell wall pectins and the subsequent deposition of PGII, we have used antibodies to relatively methylesterified and nonesterified pectic epitopes and to the PGII protein on thin sections of pericarp tissue at different developmental stages. De-esterification of pectins and deposition of PGII protein occur in block-like domains within the cell wall. The boundaries of these domains are distinct and persistent, implying strict, spatial regulation of enzymic activities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins strongly associated with cell walls of pericarp tissue at each stage of fruit development show ripening-related changes in this protein population. Western blots of these gels with anti-PGII antiserum demonstrate that PGII expression is ripening-related. The PGII co-extracts with specific pectic fractions extracted with imidazole or with Na2CO3 at 0[deg]C from the walls of red-ripe pericarp tissue, indicating that the strong association between PGII and the cell wall involves binding to particular pectic polysaccharides.     

Accuracy and cut-off values of pepsinogens I, II and gastrin 17 for diagnosis of gastric fundic atrophy: influence of gastritis

Background

To establish optimal cutoff values for serologic diagnosis of fundic atrophy in a high-risk area for oesophageal squamous cell carcinoma and gastric cancer with high prevalence of Helicobacter pylori (H. pylori) in Northern Iran, we performed an endoscopy-room-based validation study.

Methods

We measured serum pepsinogens I (PGI) and II (PGII), gastrin 17 (G-17), and antibodies against whole H. pylori, or cytotoxin-associated gene A (CagA) antigen among 309 consecutive patients in two major endoscopy clinics in northeastern Iran. Updated Sydney System was used as histology gold standard. Areas under curves (AUCs), optimal cutoff and predictive values were calculated for serum biomarkers against the histology.

Results

309 persons were recruited (mean age: 63.5 years old, 59.5% female). 84.5% were H. pylori positive and 77.5% were CagA positive. 21 fundic atrophy and 101 nonatrophic pangastritis were diagnosed. The best cutoff values in fundic atrophy assessment were calculated at PGI<56 µg/l (sensitivity: 61.9%, specificity: 94.8%) and PGI/PGII ratio<5 (sensitivity: 75.0%, specificity: 91.0%). A serum G-17<2.6 pmol/l or G-17>40 pmol/l was 81% sensitive and 73.3% specific for diagnosing fundic atrophy. At cutoff concentration of 11.8 µg/l, PGII showed 84.2% sensitivity and 45.4% specificity to distinguish nonatrophic pangastritis. Exclusion of nonatrophic pangastritis enhanced diagnostic ability of PGI/PGII ratio (from AUC = 0.66 to 0.90) but did not affect AUC of PGI. After restricting study samples to those with PGII<11.8, the sensitivity of using PGI<56 to define fundic atrophy increased to 83.3% (95%CI 51.6–97.9) and its specificity decreased to 88.8% (95%CI 80.8–94.3).

Conclusions

Among endoscopy clinic patients, PGII is a sensitive marker for extension of nonatrophic gastritis toward the corpus. PGI is a stable biomarker in assessment of fundic atrophy and has similar accuracy to PGI/PGII ratio among populations with prevalent nonatrophic pangastritis.     

Composition of proteoglycans from rabbit aorta in the experimentally induced atherosclerosis.

Proteoglycans (PG) from normal and atherosclerotic rabbits aortas were extracted with 4 M guanidine hydrochloride and digested with collagenase in the presence of protease inhibitors. The contents of uronic acid and hexosamine from PG fractions purified by isopycnic CsCl gradient ultracentrifugation under associative and dissociative conditions were significantly higher in the atherosclerotic aortas (up to 40%) than in the control tissue. The uronic acid/protein ratio increased from 0.7 to 1.3 in the monomers PG fraction of atherosclerotic aortas. Chromatographic separation and electrophoretic analysis of PG monomers indicated the presence of three different subfractions PGI, PGII and PGIII in both groups of animals. The uronic acid/protein ratio in PGI from experimental aorta was increased whereas this ratio in PGIII was decreased compared to contrast tissue. The observed increase of sugar component in the core proteins suggests their over glycosylation.     

Production and properties of the linamarase and amygdalase activities of Penicillium aurantiogriseum P35.

The effects of medium composition on the production of beta-glucosidase (amygdalase and linamarase) by Penicillium aurantiogriseum P35 were studied and the medium optimized as follows (g/l of deionized water): pectin, 10.0; (NH4)2SO4, 8.0; KH2PO4, 8.0; Na2HPO4, 2.8; MgSO4.7H2O, 0.5; yeast extract, 4.0; initial pH 6.0. When grown in a bench fermenter on this medium, the fungus produced 50.5 mU of amygdalase and 9.4 mU of linamarase per ml of culture broth. Two beta-glucosidases (PGI and PGII), each having amygdalase and linamarase activities, were recovered from the culture broth and purified; their relative molecular weights, as native enzymes, were estimated to be about 247,000 and 147,000, respectively. Both enzymes showed the same optimum pH (6.0) but different optimum temperatures (55 and 60 degrees C for PGI and PGII, respectively). Thermostability (10 min at 60 degrees C) and half-life of enzyme activity (7 hours at 60 degrees C) of PGII were higher than those of PGI (10 min at 50 degrees C and 2 hours at 55 degrees C, respectively). A wide range of cyanogenic glycosides (such as tetraphyllin B, epivolkenin, gynocardin, passibiflorin, prunasin, taxiphyllin, amygdalin, lucumin, sambunigrin, dhurrin, linamarin and cardiospermin sulfate) were hydrolyzed by both enzymes.     

Chemically methylated and reduced pectins: preparation,characterisation by 1H NMR spectroscopy,enzymatic degradation,and gelling properties

The gelling properties of pectins are known to be closely related to the degree of methylation (DM) and the distribution of the ester groups. In order to investigate this dependency, a natural citrus pectin (DM 64%) has been methylated to pectins with higher DM or saponified to achieve pectins with lower DM. A simple method for determination of DM by 1H NMR spectroscopy is presented. New modified pectins have been prepared by treatment of pectins having different DM with NaBH(4) to reduce selectively the methyl esters to primary alcohols in the presence of free acids. The degree of reduction (DR) and the DM of the remaining carboxylic acids could likewise be determined by 1H NMR spectroscopy. The new reduced pectins are recognized by the pectin degrading enzymes polygalacturonase PGI and PGII as well as by pectin lyase, all from Aspergillus niger, but the enzymes exhibit lower specific activities as compared with unmodified pectin. The new reduced pectins exhibit high gelling properties.     

Production of oxalic acid by some fungi infected tubers

Oxalic acid (as oxalate) was detected in four tubers commonly used for food in Nigeria-Dioscorea rotundata (White yam), Solanum tuberosum (Irish potato), Ipomoea batatas (Sweet potato), and Manihot esculenta (cassava). Whereas healthy I. batata had the highest oxalic acid content, healthy M. esculenta contained the lowest. When all tubers were artifically inoculated with four fungi-Penicillium oxalicum CURIE and THOM, Aspergillus niger VAN TIEGH, A. flavus and A. tamarii KITA, there was an increase in oxalate content/g of tuber tissue. The greatest amount of oxalate was produced by P. oxalicum in D. rotundata tuber. Consistently higher amounts of oxalate were produced by the four fungi in infected sweet potato tuber than in any other tuber and consistently lower amounts of oxalate were produced by the four fungi in Irish potato tuber. Differences in the carbohydrate type present in the tubers and in the biosynthesis pathway are thought to be responsible for variation in the production of oxalate in the different tubers by the four fungi used.     

血清胃蛋白酶原联合G-17对萎缩性胃炎及胃癌早期诊断价值

目的:探索检测血清胃蛋白酶原Ⅰ(PGⅠ)、胃蛋白酶原Ⅱ(PGⅡ)、胃泌素-17(G-17)在萎缩性胃炎及胃癌中的诊断价值。方法:收集医院2015年2月至12月门诊及住院的慢性非萎缩性胃炎44例(非萎缩性胃炎组),慢性萎缩性胃炎47例(萎缩性胃炎组),早期胃癌42例(胃癌组)。采用酶联免疫吸附试验(ELISA)测定各组血清PGⅠ、PGⅡ、G-17的水平,同时计算PGⅠ/PGⅡ的比值(PGR),比较各组指标间的差异,同时绘制各指标筛查萎缩性胃炎及胃癌的受试者工作曲线(ROC)曲线,分别评价其诊断价值。结果:胃癌组及萎缩性胃炎组的血清PGⅠ、PGR水平较非萎缩性胃炎组明显下降,且胃癌组下降更明显,差异均具有统计学意义(P0.05),萎缩性胃炎组血清PGⅡ显著低于非萎缩性胃炎组,差异均具有统计学意义(P0.05);胃癌组的血清G-17水平较非萎缩性胃炎组及萎缩性胃炎组均升高,差异有统计学意义(P0.05)。血清PGⅠ筛查萎缩性胃炎的最佳界值为PGⅠ90 ng/m L,其灵敏度和特异度分别为71.5%和51.0%,血清PGR筛查萎缩性胃炎的最佳界值为PGR8,其灵敏度和特异度分别为71.9%和54.0%,血清G-17筛查萎缩性胃炎的最佳界值为G-175 pmol/L,其灵敏度和特异度分别为66.1%和64.0%。血清PGⅠ筛查胃癌的最佳界值为PGⅠ73 ng/m L,其灵敏度和特异度分别为86.0%和74.9%;血清PGR筛查胃癌的最佳界值为PGR3,其灵敏度和特异度分别为90.2%和62.5%;血清G-17筛查胃癌的最佳界值为G-174 pmol/L,其灵敏度和特异度分别为62.5%和61.3%。结论:胃癌及萎缩性胃炎患者血清PGⅠ、PGR水平下降明显,且胃癌患者的血清G-17异常升高,血清PG联合GS-17测定可用于萎缩性胃炎及胃癌的早期筛查。     

胃癌前病变患者胃蛋白酶原、促胃液素-17水平变化及幽门螺杆菌感染情况

目的研究胃癌前病变患者胃蛋白酶原、促胃液素-17及幽门螺杆菌(H.pylori)感染情况变化。方法试验组患者选取2017年8月至2018年8月间于我院进行治疗的100例胃癌前病变患者,对照组选取来我院体检中心进行体检的健康人员60例,对比两组人群胃蛋白酶原、促胃液素-17及H.pylori感染情况变化。结果试验组PGI(57.45±11.52)、PGII(8.65±1.75)、PGR(5.89±1.26)和促胃液素-17(8.05±1.45)水平显著低于对照组(均P<0.05);试验组H.pylori感染率81.00%显著高于对照组感染率41.67%,差异有统计学意义(P<0.05)。结论胃癌前病变患者PGI、PGII、PGR和促胃液素-17水平较低,并且H.pylori感染率高,患病与H.pylori感染有一定关系,胃蛋白酶原、促胃液素-17和H.pylori感染率对胃癌前病变临床诊断有重要作用,能够作为胃癌前病变患者筛查参考指标。     

Correlation between Gliotoxin Production and Virulence of Aspergillus fumigatus in Galleria mellonella

Thanatephorus cucumeris is a ubiquitous fungus responsible for many types of plant diseases worldwide. All isolates from infected Hevea brasiliensis trees secreted pectolytic enzymes; polygalacturonase (PG), pectin lyase (PL) and cellulolytic enzymes; beta-glucosidase and cellobiase in culture. The extracts of the rubber tree leaf tissues, inoculated with T. cucumeris did not show any PG activity. However, PL activity was detected in tissue with the establishment of the infection. The levels of beta-glucosidase, an inherent enzyme in Hevea spp. increased rapidly following infection. However, cellobiase was detected only with the initiation of infection. Molecular weights of PG in all isolates were similar and in the range of 53,000 to 58,000. PL also followed the same pattern showing a molecular weight around 39,000.     

Genetic and biochemical characterization of an exopolygalacturonase and a pectate lyase from Yersinia enterocolitica.

Yersinia enterocolitica, an invasive foodborne human pathogen, degrades polypectate by producing two depolymerizing enzymes, pectate lyase (PL) and polygalacturonase (PG). The gene encoding the PG activity, designated pehY, was located in a 3-kb genomic fragment of Y. enterocolitica ATCC 49397. The complete nucleotide sequence of this 3-kb fragment was determined and an open reading frame consisting of 1803 bp was predicted to encode a PG protein with an estimated M(r) of 66 kDa and pI of 6.3. The amino acid sequence of prePG showed 59 and 43% identity to that of the exopolygalacturonase (exoPG) of Erwinia chrysanthemi and Ralstonia solanacearum, respectively. The Y. enterocolitica PG overproduced in Escherichia coli was purified to near homogeneity using perfusion cation exchange chromatography. Analysis of the PG depolymerization products by high performance anion-exchange chromatography and pulsed amperometric detection (HPAEC-PAD) revealed the exolytic nature of this enzyme. The Y. enterocolitica PL overproduced in E. coli was also partially purified and the M(r) and pI were estimated to be 55 kDa and 5.2, respectively. HPAEC-PAD analysis of the PL depolymerization products indicated the endolytic nature of this enzyme. Southern hybridization analyses revealed that pehY and pel genes of Y. enterocolitica are possibly encoded in the chromosome rather than in the plasmid. Purified exopolygalacturonase (over 10 activity units) was unable to macerate plant tissues.     

Endopolygalacturonases reveal molecular features for processivity pattern and tolerance towards acetylated pectin

Endopolygalacturonases (EndoPGs) hydrolyse the 1-4 linkages between two alpha-d-galacturonic acids (GalA) of the smooth homogalacturonan regions of pectin. GalA may be methyl-esterified on the carboxylic group and acetyl-esterified on the hydroxylic groups. EndoPG activity most often decreases with such increasing degree of substitution. In this paper, we used bioinformatics and molecular modelling technics to explain the tolerance profile at the molecular scale and processivity scheme of three endoPGs with respect to acetylated pectin substrate; the first two enzymes originate from Aspergillus niger (AnPGI and AnPGII) and the third from Fusarium moniliforme (FmPG). Partly acetylated and methylated homogalacturonan fragments in complex with the three PGs were successively modelled in silico. The amino acid residues involved in substrate binding were identified for each enzyme. Similarly, the docking pattern of the differently decorated oligomers in the catalytic groove was individually characterized for each enzyme. This work shows full agreement with our previous extensive mass spectrometry analysis of the hydrolytic products that established distinct tolerance profiles for the three endoPGs and earlier work that ascertained processivity, specifically for AnPGI. In our previous work, AnPGI was shown to be the most powerful enzyme among the three enzymes with an enhanced tolerance towards O2- and O3-acetylated substrates. We report here amino acids of AnPGI that are unique in binding the pectin backbone and that are identified as possibly crucial for its specificity, namely S191(An)(PGI)/D240(An)(PGI). Similarly, topologically equivalent residues in AnPGII and FmPG were identified that could impede such binding; S234(An)(PGII)/S91(An)(PGII) and S245(Fm)(PG)/V89(Fm)(PG). In addition, we report here, from normal mode analysis computed on AnPG1, a shear bending motion of 15 A of amplitude that fully accredits the processive action pattern for this enzyme, with D240(An)(PGI) and R96(An)(PGI) working as crampons to favour the sliding of the substrate. Conversely, the same method clearly evidences a hinge binding motion for AnPGII and FmPG that should only authorize one hydrolytic event per enzyme/substrate encounter.     

Purification and characterization of iduronic acid-rich and glucuronic acid-rich proteoglycans implicated in human post-burn keloid scar

Small proteoglycans (PGs), extracted from human keloid scar tissue with 4M guanidinium chloride and fractionated by DEAE-cellulose chromatography, were separated by ethanol precipitation into one L-iduronic acid-rich and one D-glucuronic acid-rich fraction. The size of the L-iduronic acid-rich PG was 102 kDa with a 27 kDa glycosaminoglycan chain, that of the D-glucuronic acid-rich PG was 90 kDa with a 26 kDa glycosaminoglycan chain, and the protein core of both PGs was 14.5 kDa. The two PGs carried sulfate groups mostly attached at C-4 of the 2-amino-2-deoxy-D-galactose units. The N-terminal amino acid sequence of both was similar to human bone PGII (decorin), normal and hypertrophic scar, and human dermal tissue PG.     

Analysis of conductance responses during depolymerization of pectate by soft rot Erwinia spp. and other pectolytic bacteria isolated from potato tubers

Different bacteria isolated from potato tubers were screened for their pectolytic properties by examining pitting in polypectate agar, recording conductance responses in polypectate medium and performing potato tuber soft rot tests. For bacteria found positive in conductimetry, the role of polygalacturonase (PG) and pectate lyase (PL) in the generation of conductance changes in a polygalacturonic acid (PGA) medium was further analysed using enzyme activity staining after gel electrophoresis and high-performance anion exchange chromatography. The extent of the conductance changes during depolymerization of PGA was dependent on the amounts of galacturonate monomers and oligomers accumulated in the medium. In comparison with an unidentified saprophyte and a Klebsiella strain, both mainly having PL activity, soft rot Erwinia spp. rapidly produced larger conductance responses, due to a combined action of multiple forms of PG and PL. The responses of Erwinia spp. were initially associated with the accumulation of large amounts of monomers and saturated dimers to heptamers, due to PG activity. Subsequently, as well as monomers and saturated dimers, large amounts of unsaturated dimers were also detected, due to PL activity. The role of PG as an important conductimetric factor was also demonstrated for a pectinase preparation derived from Aspergillus niger . Besides detection, automated conductimetric assays in pectate media may also be useful for monitoring of pectolytic activity in pectinase preparations and for screening of pectolytic activity of micro-organisms under different media and growth conditions.     

Purified tomato polygalacturonase activity during thermal and high-pressure treatment

Extracted tomato polygalacturonase was purified by cation-exchange chromatography (and gel filtration) and characterized for molar mass, isoelectric point, as well as optimal pH for polygalacturonase activity. The enzymatic reaction of purified tomato polygalacturonase on polygalacturonic acid as substrate was investigated during a combined high-pressure/temperature treatment in a temperature range of 25 degrees to 80 degrees C and in a pressure range of 0.1 to 500 MPa at pH 4.4 (the pH of tomato-based products). The optimal temperature for initial tomato polygalacturonase activity in the presence of polygalacturonic acid at atmospheric pressure is about 55 degrees to 60 degrees C. The optimal temperature for initial tomato polygalacturonase activity during processing shifted to lower values at elevated pressure as compared with atmospheric pressure, and the catalytic activity of pure tomato polygalacturonase decreased with increasing pressure, which was mostly pronounced at higher temperatures. The elution profiles of the degradation products on high-performance anion-exchange chromatography indicated that for both thermal and high-pressure treatment all oligomers were present in very small amounts in the initial stage of polygalacturonase activity. The amounts of monomer and small oligomers increased with increasing incubation times, whereas the amount of larger oligomers decreased due to further degradation.