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1.
Tomassen MM Barrett DM van der Valk HC Woltering EJ 《Journal of experimental botany》2007,58(5):1151-1160
An important aspect of the ripening process of tomato fruit is softening. Softening is accompanied by hydrolysis of the pectin in the cell wall by pectinases, causing loss of cell adhesion in the middle lamella. One of the most significant pectin-degrading enzymes is polygalacturonase (PG). Previous reports have shown that PG in tomato may exist in different forms (PG1, PG2a, PG2b, and PGx) commonly referred to as PG isoenzymes. The gene product PG2 is differentially glycosylated and is thought to associate with other proteins to form PG1 and PGx. This association is thought to modulate its pectin-degrading activity in planta. An 8 kDa protein that is part of the tomato PG1 multiprotein complex has been isolated, purified, and functionally characterized. This protein, designated 'activator' (ACT), belongs to the class of non-specific lipid transfer proteins (nsLTPs). ACT is capable of 'converting' the gene product PG2 into a more active and heat-stable form, which increases PG-mediated pectin degradation in vitro and stimulates PG-mediated tissue breakdown in planta. This finding suggests a new, not previously identified, function for nsLTPs in the modification of hydrolytic enzyme activity. It is proposed that ACT plays a role in the modulation of PG activity during tomato fruit softening. 相似文献
2.
黑曲霉(AspergilluS niger)AS 3.3883所产果胶酶经DEAE Sephadex A50及Sephadex G100柱层析分离出电泳纯的两种聚半乳糖醛酸酶(PG1,PG2),并对它们的性质及结构进行了比较研究。结果证明两种酶作用的最适条件、动力学性质、分子量、氨基酸组成及金属离子对酶活力影响等方面有很大差异,但二者的每个摩尔的活力及酶的构象很相似。 相似文献
3.
Action of the active metabolites of tiazofurin and ribavirin on purified IMP dehydrogenase 总被引:1,自引:0,他引:1
The inhibitory mechanisms of ribavirin 5'-monophosphate (RMP) and thiazole-4-carboxamide adenine dinucleotide (TAD), the active forms of the antimetabolites ribavirin and tiazofurin, were investigated in IMP dehydrogenase purified to homogeneity from rat hepatoma 3924A. The hepatoma IMP dehydrogenase has a tetrameric structure with a subunit molecular weight of 60,000. For the substrates IMP and NAD+, Km's were 23 and 65 microM, respectively. Product-inhibition patterns showed an ordered Bi-Bi mechanism for the enzyme reaction where IMP binds to the enzyme first, followed by NAD+; NADH dissociates from the ternary complex first and then XMP is released. XMP interacts with the free enzyme and competes for the ligand site with IMP, while NADH binds to the enzyme-XMP complex. RMP exerted the same inhibitory mechanisms as XMP, and the inhibition by TAD was similar to that by NADH. However, the Ki values for RMP (0.8 microM) and TAD (0.13 microM) were orders of magnitude lower than those of XMP (136 microM) and NADH (210 microM). Thus, the drugs interact with IMP dehydrogenase with higher affinities than the natural substrates and products, RMP with the IMP-XMP site and TAD with the NADH site. Preincubation of the purified enzyme with RMP enhanced its inhibitory effect in a time-dependent manner. The enzyme was protected from this inactivation by IMP or XMP. These results provide a biochemical basis for combination chemotherapy with tiazofurin and ribavirin targeted against the two different ligand sites of IMP dehydrogenase. 相似文献
4.
Danielle Biscaro Pedrolli Eleonora Cano Carmona 《Journal of industrial microbiology & biotechnology》2010,37(6):567-573
Polygalacturonases are pectinolytic enzymes that catalyze the hydrolysis of the plant cell-wall pectin backbone. They are
widely used in the food industry for juice extraction and clarification. Aspergillus giganteus produces one polygalacturonase (PG) on liquid Vogel medium with citrus pectin as the only carbon source. In specific applications,
such as those used in the food and medicine industries, the PG must be free of substances that could affect the characteristics
of the product and the process, such as color, flavor, toxicity, and inhibitors. We present here an efficient, simple, and
inexpensive method for purifying the A. giganteus PG and describe the characteristics of the purified enzyme. Purified PG was obtained after two simple steps: (1) protein
precipitation with 70% ammonium sulfate saturation and (2) anion-exchange chromatography on a DEAE-Sephadex A-50 column. The
final enzyme solution retained 86.4% of its initial PG activity. The purified PG had a molecular weight of 69.7 kDa, exhibited
maximal activity at pH 6.0 and 55–60°C, and was stable in neutral and alkaline media. It had a half-life of 115, 18, and 6 min
at 40, 50 and 55°C, respectively. Purified PG showed its highest hydrolytic activity with low-esterified and nonesterified
substrates, releasing monogalacturonic acid from substrate, indicating that it is an exopolygalacturonase. PG activity was
enhanced in the presence of β-mercaptoethanol, dithiothreitol, Co2+, Mn2+, Mg2+, NH4
+, and Na+ and was resistant to inhibition by Pb2+. 相似文献
5.
Partial purification, properties and regulation of inosine 5''phosphate dehydrogenase in normal and malignant rat tissues.
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IMP dehydrogenase (EC 1.2.1.14) was purified 180-fold from rat liver and from the transplantable rat hepatoma 3924A. The enzymes from the two sources were apparently identical; they exhibited hyperbolic saturation kinetics and an ordered, sequential mechanism, and were subject to inhibition by a number of purine nucleotides. Km values for the substrates, IMP and NAD+, were 12 and 24 micrometer respectively. IMP dehydrogenase activity in a spectrum of rat hepatomas was increased, relative to normal liver, by 2.5--13-fold; these increases correlated with tumour growth rate. Activity in two rat kidney tumours was increased 3-fold relative to that in normal renal cortex; control of activity of this enzyme is apparently altered in neoplastic cells. After partial hepatectomy, IMP dehydrogenase activity began to rise 6 h after operation, reaching a peak of 580% of normal activity by 18 h. Activity in neonatal liver, however, was only slightly higher than that in the adult. Organ-distribution studies showed highest enzyme activities in spleen and thymus. In livers of rats starved for 3 days, where all enzymes, except those involved in gluconeogenesis, showed decreased activity IMP dehydrogenase activity was increased; this change was accompanied by a rise in hepatic GTP concentrations. It is concluded that IMP dehydrogenase is a key enzyme in the regulation of GTP production, and thus involved in regulation of nucleic acid biosynthesis. The increased activity of IMP dehydrogenase in liver of starved rats may be related to the requirements for GTP for gluconeogenesis. 相似文献
6.
Savary BJ 《Preparative biochemistry & biotechnology》2001,31(3):241-258
A rapid and simple method was developed, using perfusion chromatography media, to separate the fruit-specific pectin methylesterase (PME) isoform from the depolymerizing enzyme polygalacturonase (PG) and other contaminating pectinases present in a commercial tomato enzyme preparation. Pectinase activities were adsorbed onto a Poros HS (a strong cation exchanger) column in 20 M HEPES buffer at pH 7.5. The fruit-specific PME was eluted from the column with 80 mM NaCl, followed by a step to 300 mM NaCl to elute PG activity. Rechromatography of the PME activity peak with a linear gradient further resolved two PME isoenzymes and removed residual traces of PG activity. The PG activity peak was further treated with lectin affinity chromatography to provide purified PG enzyme, which was separated from a salt-dependent PME (tentatively identified as a "ubiquitous-type" isoform), and a pectin acetylesterase. The later enzyme has not been reported previously in tomato. This method provides monocomponent enzymes that will be useful for studying enzyme mechanisms and for modifying pectin structure and functional properties. 相似文献
7.
Tiazofurin was demonstrated to be an effective inhibitor of the growth of human cultured blast cells, and the high specific activities of IMP dehydrogenase (EC 1.1.1.205) were observed in all the cell extracts tested. IMP dehydrogenase has been purified to homogeneity from MOLT 4F human T-lymphoblast, and the Km values for IMP and NAD were 29 and 54 microM, respectively. The inhibitory mechanisms of thiazole-4-carboxamide adenine dinucleotide (TAD) and ribavirin 5'-monophosphate (RMP), the active forms of the antimetabolites tiazofurin and ribavirin, were investigated on the purified enzyme. RMP inhibits competitively with respect to IMP as well as XMP, and the inhibition by TAD was similar to that by NADH, which was uncompetitive with NAD. However, the Ki values of RMP (0.58 microM) and TAD (0.075 microM) were several orders of magnitude lower than those of XMP (85 microM) and NADH (94 microM). Thus, the drugs interact with the two distinct sites of IMP dehydrogenase with much higher affinities than the natural substrates and products. Preincubation of the purified enzyme with RMP enhanced its inhibitory effect in a time-dependent manner, and the enhancement was further increased by the addition of TAD. The combination of tiazofurin and ribavirin exerted a synergistic effect on the growth inhibition in MOLT 4F cells. 相似文献
8.
Proteinase activities in rat thioglycollate elicited peritoneal cells and the cell-free supernatant (lavage fluid) were measured by using the following substrates: Suc-Ala-Ala-Pro-Phe-Methyl-Coumarin-Amide (for cathepsin G or chymase), Suc-Ala-Ala-Ala-AMC (for elastase or elastase-like), Z-Arg-Arg-AMC (for cathepsin B), haemoglobin (for cathepsin D) and Ala-AMC (for alanine-aminopeptidase: AAP). The enzyme activities were correlated to the quantitative distribution of various cell types in the exudate from 0 to 192 nd h. In the supernatant all the examined activities showed a higher value at 72nd h. In the cells activity of chymase and AAP proved to be very high at 0 h but after four h the activities were dropped. From this time all enzyme activities started to elevate till the 24th h. At the 96th h only the activity of cathepsin B and AAP had a high value. We conclude that the intracellular activation and secretion of proteolytic enzymes characteristic for the various peritoneal cell types involved in the acute and chronic inflammatory reaction can be followed by activity measurements using enzyme-specific substrates and inhibitors. 相似文献
9.
Influence of beta-subunit on thermal and high-pressure process stability of tomato polygalacturonase
Peeters L Fachin D Smout C van Loey A Hendrickx ME 《Biotechnology and bioengineering》2004,86(5):543-549
Polygalacturonase (PG; E.C. 3.2.1.15) was extracted from tomato fruit and purified by cation-exchange chromatography. Two peaks containing PG activity were detected: the first denotes a thermolabile PG fraction (PG2) and the second a thermostable fraction (PG1). PG1 is a dimer of PG2 and a heat-stable protein called the beta-subunit. In contrast to its resistance to heat, PG is easily inactivated at elevated pressure. Although the thermal stability of purified tomato PG1 and PG2 is distinctly different, they show an identical pressure stability. To gain further insight into the thermal and pressure stability of both PG isoenzymes, the in vitro recombination of PG2 and beta-subunit was studied. After severe heat (up to 140 degrees C for 5 min) and pressure (up to 800 MPa for 15 min) treatments, the residual fractions containing the beta-subunit were able to convert PG2 into the heat-stable PG1, showing the extreme thermal and pressure stability of the beta-subunit. PG1 was detected in heat-treated tomato juice and, to a lesser extent, in tomato pieces. In contrast, as was the case for purified PG, no pressure-stable fraction was observed when tomato juice and pieces were treated under pressure. These data clearly show the differing behavior of the PG1-PG2-beta-subunit system under thermal and high-pressure treatments and offer the possibility of inactivating tomato PG using high pressure without the need for high temperatures. 相似文献
10.
Vovk I Simonovska B 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2007,849(1-2):337-343
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. 相似文献
11.
Jurick WM Vico I Gaskins VL Whitaker BD Garrett WM Janisiewicz WJ Conway WS 《Mycologia》2012,104(3):604-612
A polygalacturonase (PG) isozyme was isolated from Penicillium solitum-decayed Anjou pear fruit and purified to homogeneity with a multistep process. Both gel filtration and cation exchange chromatography revealed a single PG activity peak, and analysis of the purified protein showed a single band with a molecular mass of 43 kDa, which is of fungal origin. The purified enzyme was active from pH 3.5-6, with an optimum at pH 4.5. PG activity was detectable 0-70 C with 50 C maximum. The purified isozyme was inhibited by the divalent cations Ca(2+), Mg(2+), Mn(2+) and Fe(2+) and analysis of enzymatic hydrolysis products revealed polygalacturonic acid monomers and oligomers. The purified enzyme has an isoelectric point of 5.3 and is not associated with a glycosylated protein. The PG isozyme macerated fruit tissue plugs in vitro and produced ~1.2-fold more soluble polyuronides from pear than from apple tissue, which further substantiates the role of PG in postharvest decay. Data from this study show for the first time that the purified PG produced in decayed Anjou pear by P. solitum, a weakly virulent fungus, is different from that PG produced by the same fungus in decayed apple. 相似文献
12.
Inosine 5''-monophosphate dehydrogenase of Escherichia coli. Purification by affinity chromatography, subunit structure and inhibition by guanosine 5''-monophosphate. 总被引:3,自引:0,他引:3
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Escherichia coli IMP dehydrogenase (EC 1.2.1.14) was purified by affinity chromatography on immobilized nucleotides. The enzyme binds to agarose-bound 8-(6-aminohexyl)-AMP, N6-(6-aminohexyl)-AMP and 8-(8-amino-octyl)-IMP but not to immobilized NAD+ or Cibacron Blue F3G-A. AMP proved to be an effective eluent. A large-scale purification scheme in which 8-(6-aminohexyl)-AMP-agarose was used resulted in a homogeneous preparation of IMP dehydrogenase. The enzyme was also purified by immunoprecipitation with monospecific antisera. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, N-terminal amino acid analysis and tryptic 'finger-printing' demonstrated that IMP dehydrogenase comprises identical subunits of mol.wt. 58000. Trypsin and Pronase cleave the 58000-mol.wt. subunit into peptides of mol.wts. 42000 and 14000, with a concomitant decrease in enzyme activity. These observations rationalize much of the contradictory data on the subunit composition of the enzyme found in the literature. GMP appears to be a competitive inhibitor with respect to IMP, with no evidence for regulatory behaviour being found. The two purification procedures were also used to purify inactive mutant enzymes from guaB mutant strains of E. coli. 相似文献
13.
IMP dehydrogenase from the intracellular parasitic protozoan Eimeria tenella and its inhibition by mycophenolic acid 总被引:3,自引:0,他引:3
Mycophenolic acid (MA) was demonstrated to be an effective inhibitor of the growth of the intracellular parasitic protozoan Eimeria tenella in tissue culture and guanine was shown to reverse this inhibition as expected for an inhibitor of IMP dehydrogenase (IMP:NAD+ oxidoreductase, EC 1.1.1.205). A high performance liquid chromatography study of the intracellular nucleotide pools labeled with [3H]hypoxanthine was carried out in host cells lacking hypoxanthine-guanine phosphoribosyltransferase, and the depletion of guanine nucleotides demonstrated that the intracellular parasite enzyme was being inhibited by the drug. Kinetic studies carried out on the enzyme derived from E. tenella oocysts demonstrated substrate inhibition by NAD and mycophenolic acid inhibition similar to that found for mammalian enzymes, but different from that for bacterial enzymes. The inhibition by mycophenolic acid was not time-dependent and was immediately reversed upon dilution. As found previously for other IMP dehydrogenases, an Ordered Bi-Bi mechanism prevails with IMP on first followed by NAD, NADH off first, and then XMP. The kinetic patterns are consistent with substrate inhibition at high concentrations of NAD due to the formation of an E X XMP X NAD complex. Uncompetitive inhibition by MA versus IMP, NAD, and K+ was found and this was interpreted as evidence for the formation of an E X XMP X MA complex. A speculative mechanism for the inhibition of the enzyme is offered which is consistent with the fact that E X XMP X MA readily forms, whereas E X IMP X MA does not. 相似文献
14.
G G Heinert J E Scherberich W A Mondorf W Weber 《Analytical and quantitative cytology》1981,3(2):103-111
Tissue sections of kidneys from 172 patients with various pathologic conditions, such as hydronephrosis, interstitial nephropathies, ischemia, chronic graft rejection and renal cancer, were evaluated by an image analysis technique. Structurally defined kidney alterations were monitored for enzymatic, immunologic and other histochemical changes. Indicator enzymes of the proximal tubule, alanine-aminopeptidase (AAP), alkaline phosphatase (AP), beta-glucoronidase (beta-Gl) and gamma-glutamyltranspeptidase (GGTP), were used as parameters for screening. Enzyme concentrations were found to be significantly decreased in kidney sections of patients with various renal diseases (AP less than 15%, AAP less than 55% and beta-Gl less than 60%) as compared to normal kidney tissues (100%). AAP concentration was measured quantitatively by specific immunofluorescence using an antienzyme antibody. Immunofluorescence of AAP was comparable to that of AAP calculated by the colorimetric technique (substrate: DL-alanine-beta-naphthylamide-HCl) and decreased to less than 50% in altered kidney tissues. Furthermore, kidney cancer (less than 20%) and kidney tissue adjacent to tumours (less than 65%) displayed significantly decreased levels of kidney marker enzyme activity. This study suggests that (1) the diseased kidney is characterized by a defined change in key enzymes of the cell surface and (2) renal cancer exhibits partial depletion of these constituents. Image analysis of the pattern of enzyme activity appears to be a useful tool in the analysis of renal pathology. 相似文献
15.
Erat M 《Protein expression and purification》2004,34(2):257-260
The enzymes of glucose 6-phosphate dehydrogenase and glutathione reductase were purified from human erythrocytes in one chromatographic step consisting of the use of the commercially available resin 2',5'-ADP Sepharose 4B by using different washing buffers. Ammonium sulfate (30-70%) precipitation was performed on the hemolysate before applying to the affinity column. Using this procedure, G6PG, having the specific activity of 22.9 EU/mg proteins, was purified with a yield of 43% and 9150-fold; GR, having the specific activity of 20.7 EU/mg proteins, was purified with a yield of 26% and 8600-fold. The purity of the enzymes was checked on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and each purified enzyme showed a single band on the gel. This procedure has advantages of preventing of enzyme denaturation, short experimental duration, and use of less chemical materials for purification of the enzymes. 相似文献
16.
Lucas Dal Magro Jakub F. Kornecki Manuela P. Klein Rafael C. Rodrigues Roberto Fernandez-Lafuente 《Biotechnology progress》2019,35(6):e2877
Rohapect 10L is an enzyme cocktail commercialized for juice clarification. Here, we characterized the activity and stability of five enzymatic activities present in this cocktail: total pectinase (PE), polygalacturonase (PG), pectin lyase (PL), pectin methyl esterase (PME), and total cellulase (CE) activities. All these enzyme activities have the maximum activity and stability at pH 4, conditions near those found in most fruit juices. However, if the enzymes need to be handled under different conditions (e.g., to immobilize them), their stability becomes extremely low in some cases, just at pH values slightly higher than the optimal one. For example, at pH 10 only CE was reasonably stable at 25°C, while many other enzyme activities were rapidly almost inactivated, even at 4°C. For these cases, different additives were evaluated, and we found that polyethylene glycol was positive or very positive for all enzyme stabilities, allowing keeping reasonable activities after several hours at pH 10 and 25°C. Another additive, that is, dextran, has a small positive effect for PE, PG, and CE, and a very positive effect for PL, albeit significantly destabilizing PME. Thus, the handling and use of this extract requires some care when is performed out of optimal conditions. 相似文献
17.
Summary
Tubercularia vulgaris produces extracellular pectolytic activity in solid state culture using citrus pulp pellets as a carbon source. Five peaks containing hydrolase activities were separated by ion exchange chromatography (DEAE-Sephacel). Two enzymatic forms, PG II and PG III, were purified about 8 and 10 fold, respectively. The molecular weight of PG II was similar to that of PG III (24.000 Da). The two enzymes had similar endo activities on polygalacturonic acid, as determined by comparison of viscosity reduction and reducing groups release. 相似文献
18.
Isabel Fernaud-Espinosa Manuel Nieto-Sampedro Paola Bovolenta 《Developmental neurobiology》1998,36(1):16-29
The expression of proteoglycans (PGs) in the mammalian central nervous system (CNS) appears to be strictly regulated both during development and after damage to the mammalian CNS. Recently, we have isolated from membranes of injured adult brain a neurite outgrowth-inhibitory proteoglycan (IMP), the activity of which could be specifically counteracted by a monoclonal antibody (mAB) against the PG. We described in this report the characterization of perinatal membrane proteoglycan (PMP), a heparan-sulfate/chondroitin-sulfate-containing PG expressed during brain development. Its maximal expression was observed around postnatal day 3, decreasing strongly in normal adult tissue. This PG was purified and characterized using mABs generated against IMP. The comparison of PMP and IMP properties indicates that the two PGs are highly related and share expression patterns, biochemical characteristics, and the ability to inhibit neurite initiation in culture. However, IMP and PMP displayed a distinct effect on neurite elongation, which may be explained by their differences in glycosilation pattern. The data presented in this report support the idea that proteoglycans expressed during CNS development are re-expressed following injury. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 16–29, 1998. 相似文献
19.
Glyceraldehyde-3-phosphate dehydrogenase [ED 1.2.1.12] was purified from the horseshoe crab, a living fossil, and its properties were examined. 1 The purified enzyme was homogeneous as judged by various tests. The enzyme, like enzymes from other sources, was a tetramer with a subunit molecular weight of 36,000. The kinetic parameters and pH optimum were also similar to those of other enzymes, though the enzyme was more stable against heat and pH denaturations. 2 Analysis of SH groups showed that there were 4 SH groups per subunit, one of which was essential for the enzyme activity and was highly reactive. 3. CD spectra of the enzyme suggested that the enzyme had a very high content of beta-structure (ca. 45 per cent). 4. The horseshoe crab enzyme could form a hybrid in vitro with the rabbit muscle enzymes in concentrated salt solution at acidic pH. 5. There results indicate that the enzyme has overall structural similarity to other enzymes and that the enzyme is highly conserved during a long period of evolution. Some discussions on the structure and activity of the horseshoe crab enzyme are made in comparison with the enzymes from other sources. 相似文献
20.
The enzyme responsible for observed IMP cyclohydrolase activity in Methanococcus jannaschii was purified and sequenced: its genetic locus was found to correspond to gene MJ0626. The MJ0626 gene was cloned, and its protein product was expressed in Escherichia coli and shown to catalyze the cyclization of 5-formylamidoimidazole-4-carboxamide ribonucleotide to IMP. The enzyme has no sequence similarity to known enzymes, and its catalytic properties appear distinct from any characterized IMP cyclohydrolase. The purO gene for the enzyme is currently found only in the domain ARCHAEA: 相似文献