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1.
Kathleen A. Killick 《Analytical biochemistry》1980,105(1):291-298
Continuous and discontinuous coupled fluorometric assays which couple trehalose hydrolysis to peroxidation of the fluorogenic compounds eugenol (4-allyl-2-methoxyphenol) or p-hydroxyphenylacetic acid using glucose oxidase (EC 1.1.3.4) and peroxidase (EC 1.11.1.7) as ancillary enzymes have been developed for the measurement of trehalase (α,α′-trehalose-1-d-glucohydrolase, EC 3.2.1.28) activity from the cellular slime mold, Dictyostelium discoideum. With these methods, product formation was linear with time and the coupled reaction rate was directly proportional to the level of enzyme assayed. The validity of both the discontinuous and continuous fluorometric assays was confirmed by comparative studies with discontinuous spectrophotometric assays for glucose. Levels of glucose as low as 0.02 nmol were measurable with the discontinuous fluorometric procedures, thereby making the latter about 500-fold more sensitive than routine spectrophotometric assays. With the continuous fluorometric trehalase assays, the lower limits of sensitivity correspond to enzyme levels of the order of 5 to 25 μunits. The high level of sensitivity achieved with these assays makes them ideally well suited for: (i) elucidation of the regulatory mechanisms underlying the dramatic changes in trehalase activity that occur during spore germination and cellular aggregation in Dictyostelium and (ii) characterization studies involving electrophoresis or isoelectric focusing of trehalase in solid matrices in which enzymatic activity is measured either quantitatively in gel eluates or qualitatively by the in situ localization of the enzyme histochemically. 相似文献
2.
Rabbit intestinal trehalase (alpha,alpha-trehalose glucohydrolase, EC 3.2.1.28) was solubilized with Triton X-100 and purified in the presence of EDTA. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis in the presence of Triton X-100 or SDS. It showed amphiphilic properties on gel filtration. polyacrylamide gel electrophoresis, charge-shift electrophoresis and phenyl-Sepharose chromatography. Its molecular weight was estimated to be about 330 000 by gel filtration under nondenaturing conditions and in the presence of Triton X-100, the value being in satisfactory agreement with the sum of the weight of one Triton X-100 micelle and twice the molecular weight (105 000) of purified hydrophilic trehalase which had been deprived of the anchor segment. The two purified trehalases gave almost the same molecular weights (about 75 000) on SDS-polyacrylamide gel electrophoresis. These results suggest that intestinal trehalase consists of two subunits with a molecular weight of 75 000 and that its anchor segment is small (less than 5000). Triton X-100 extracts freshly prepared from intestinal microvilli essentially showed one form of trehalase, which behaved on phenyl-Sepharose and Con A-Sepharose chromatography in the same manner as purified amphiphilic trehalase. 相似文献
3.
Solubilization of trehalase from rabbit renal and intestinal brush-border membranes by a phosphatidylinositol-specific phospholipase C 总被引:4,自引:0,他引:4
Trehalase (EC 3.2.1.28) associated with renal and intestinal brush-border membranes was solubilized by highly purified phosphatidylinositol-specific phospholipase C (EC 3.1.4.10) from Bacillus thuringiensis, but not by phosphatidylcholine-hydrolyzing phospholipase C (EC 3.1.4.3) from Clostridium welchii or phospholipase D (EC 3.1.4.4) from cabbage. The solubilized trehalase was not adsorbed on phenyl-Sepharose, indicating that it was hydrophilic. Phosphatidylinositol-specific phospholipase C also converted Triton X-100-solubilized amphipathic trehalase into a hydrophilic form. These results suggest that trehalase is bound to the membrane through a direct and specific interaction with phosphatidylinositol. 相似文献
4.
Peroxidase Activity in Relation to Suberization and Respiration in White Spruce (Picea glauca [Moench] Voss) Seedling Roots 总被引:2,自引:1,他引:1
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Peroxidase (EC 1.11.1.7) activity is associated with suberization during endodermal development and metacutization in roots of white spruce (Picea glauca [Moench] Voss) seedlings. Histochemical analysis indicates a relationship between suberization and peroxidase activity, but peroxidase is ubiquitous. Increased peroxidase activity results from the induction of four anodic peroxidase isozymes in addition to quantitative increases in two anodic peroxidase isozymes. Four of these polymerized eugenol. Cold temperatures induce formation of two anodic isozymes and result in suberization. The increased peroxidase activity associated with suberization is correlated to residual respiration. In an attempt to elucidate this relationship, the effect of respiratory inhibitors on respiration and peroxidase activity are compared. 相似文献
5.
Kathleen A. Killick 《Preparative biochemistry & biotechnology》2013,43(5):547-557
A rapid and reliable method for the preparation of homogeneous trehalase from the cellular slime mold, Dictyostelium discoideum for usage in enzyme characterization studies and trehalose assays was developed. This procedure takes advantage of the fact that trehalase activity is secreted by Dictyostelium during the course of development, the major fraction being released late in fruiting body formation. Purification of trehalase to electrophoretic homogeneity was accomplished utilizing the techniques of ultrafiltration, streptomycin sulfate precipitation, ammonium sulfate fractionation, DEAE-Sephacel chromatography and preparative disc gel electrophoresis. Analysis of the purified enzyme by analytical polyacrylamide disc gel electrophoresis demonstrated the presence of a single protein band which was stainable with Coomassie blue. Assay of trehalase activity in eluates from segments of a companion gel indicated that all of the recovered trehalase activity was associated with this band of protein. Examination of the substrate specificity of the purified enzyme indicated absolute specificity for trehalose. 相似文献
6.
Abstract Candida utilis cells contain a regulatory trehalase enzyme (280 kDa) which can be activated by cAMP-dependent phosphorylation. A 100-fold purification of this enzyme activity results in the enrichment of a protein band of apparent M r 70 000 as identified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). This component is phosphorylated in vivo under conditions in which trehalase activation occurs in whole cells. It is concluded that the trehalase enzyme might be a tetramer, composed of 4 identical 70-kDa subunits. 相似文献
7.
Genetic and biochemical evidence that trehalase is a substrate of cAMP-dependent protein kinase in yeast 总被引:20,自引:0,他引:20
In Saccharomyces cerevisiae, trehalase activity in crude extracts obtained from wild type cells was activated about 3-fold by preincubation with cAMP and ATP. The inactive trehalase fractionated by DEAE-Sephacel chromatography was activated by the addition of the cAMP-dependent protein kinase fraction from wild type cells in the presence of cAMP and ATP. Using the crude extract obtained from bcy1 mutant cells which were deficient in the regulatory subunit of cAMP-dependent protein kinase, the stimulation of trehalase activity was observed in the absence of cAMP. The cAMP-dependent protein kinase of CYR3 mutant cells which had a high Ka value for cAMP in the phosphorylation reaction required a high cAMP concentration for activation of trehalase. Increased activation of partially purified inactive trehalase (Mr = 320,000) was observed to correlate with increased phosphorylation of a protein (Mr = 80,000) identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The assay results using various mutants altered in cAMP metabolism indicated that the activation and phosphorylation of inactive trehalase fractions depended on the cAMP concentration accumulated in mutant cells. Inactivation and dephosphorylation of active trehalase fractions were observed by treatment with alkaline phosphatase or crude cell extracts. The results indicated that the conversion of inactive form of trehalase to the active form is regulated by cAMP through cAMP-dependent protein kinase. 相似文献
8.
Changes in antioxidant and lignifying enzyme activities in sunflower roots (Helianthus annuus L.) stressed with copper excess 总被引:1,自引:0,他引:1
Treatment with 50 microM CuSO4 for five days caused significant decrease in dry-matter production and protein level of ten-day-old sunflower seedling roots. An increase of lipoperoxidation product rate was also observed. The involvement of some enzyme activities in the sunflower root defence against Cu-induced oxidative stress was studied. Copper treatment induced several changes in antioxidant enzymes. SOD (superoxide dismutase, EC 1.15.1.1) activity was reduced but CAT (catalase, EC 1.11.1.6) and GPX (guaiacol peroxidase, EC 1.11.1.7) activities were significantly enhanced. The lignifying peroxidase activities, assayed using coniferyl alcohol and syringaldazine, were also stimulated. Analysis by native gel electrophoresis of syringaldazine peroxidase activity showed the stimulation of an isoform (A2) and the induction of another one (A1) under cupric stress conditions. On the other hand, the activity of PAL (phenylalanine ammonia lyase, EC 4.3.1.5), which plays an important role in plant defence, was also activated. The possible mechanisms by which Cu-induced growth delay and changes in enzymatic activities involved in plant defence processes are discussed. 相似文献
9.
Disaccharidases from the gut of Ascaris suum were investigated to determine whether they were synthesized by the worm or whether they were host enzymes adsorbed to the worms' intestinal cells. Alpha-d-glucoside glucohydrolase (maltase) (EC 3.2.1.20), Beta-d-fructofuranoside fructohydrolase (invertase) (EC 3.2.1.26) and 1-glucohydrolase (trehalase) (EC 3.2.1.28) from Ascaris were studied in both a membrane (brush border)-bound and solubilized form with regard to temperature stability and pH optima. Data collected were compared to similar data on hog intestinal enzymes. Worm maltase and trehalase were relatively heat labile, whereas the hog enzymes were more stable to heat inactivation. Worm invertase was heat stable in comparison to the hog enzyme. The pH optima for Ascaris maltase and invertase were different from those of hog disaccharidases, whereas the pH optimum for trehalase from both parasite and host were similar. Tissue homogenates of second-stage larvae contained measurable maltase, but not sucrase, or trehalase activity. Results suggested that Ascaris intestinal disaccharidases represent three distinct enzymes of parasite rather than host origin. 相似文献
10.
Induction of trehalase in Arabidopsis plants infected with the trehalose-producing pathogen Plasmodiophora brassicae 总被引:1,自引:0,他引:1
Brodmann A Schuller A Ludwig-Müller J Aeschbacher RA Wiemken A Boller T Wingler A 《Molecular plant-microbe interactions : MPMI》2002,15(7):693-700
Various microorganisms produce the disaccharide trehalose during their symbiotic and pathogenic interactions with plants. Trehalose has strong effects on plant metabolism and growth; therefore, we became interested to study its possible role in the interaction of Arabidopsis thaliana with Plasmodiophora brassicae, the causal agent of clubroot disease. We found that trehalose accumulated strongly in the infected organs (i.e., the roots and hypocotyls) and, to a lesser extent, in the leaves and stems of infected plants. This accumulation pattern of trehalose correlated with the expression of a putative trehalose-6-phosphate synthase (EC 2.4.1.15) gene from P. brassicae, PbTPS1. Clubroot formation also resulted in an induction of the Arabidopsis trehalase gene, ATTRE1, and in a concomitant increase in trehalase (EC 3.2.1.28) activity in the roots and hypocotyls, but not in the leaves and stems of infected plants. Thus, induction of ATTRE1 expression was probably responsible for the increased trehalase activity. Trehalase activity increased before trehalose accumulated; therefore, it is unlikely that trehalase was induced by its substrate. The induction of trehalase may be part of the plant's defense response and may prevent excess accumulation of trehalose in the plant cells, where it could interfere with the regulation of carbon metabolism. 相似文献
11.
Neutral trehalase was purified from stationary yeast ABYS1 mutant cells deficient in the vacuolar proteinases A and B and the carboxypeptidases Y and S. The purified electrophoretically homogeneous preparation of phosphorylated neutral trehalase exhibited a molecular mass of 160,000 Da on nondenaturing gel electrophoresis and of 80,000 Da on sodium dodecyl sulfate-gel electrophoresis. Maximal activity (114 mumol of trehalose min-1 x mg-1 at 37 degrees C) was observed at pH 6.8-7.0. The apparent Km for trehalose was 34.5 mM. Among seven oligosaccharides studied, the enzyme formed glucose only from trehalose. Neutral trehalase is located in the cytosol. A polyclonal rabbit antiserum raised against neutral trehalase precipitates the enzyme in the presence of protein A. The antiserum does not react with acid trehalase. Dephosphorylation by alkaline phosphatase from Escherichia coli of the active phosphorylated enzyme is accompanied by greater than or equal to 90% inactivation. Rephosphorylation by incubation with the catalytic subunit of beef heart protein kinase is accompanied by reactivation and incorporation of 0.85 mol of phosphate/mol subunit (80,000 Da). The phosphorylated amino acid residue was identified as phosphoserine. 相似文献
12.
To utilize specific fetal markers in amniotic fluid for prenatal detection of fetal anomalies, it is necessary to determine the precise tissue origin of these markers. In rabbit fetuses, we distinguished between intestinal and renal forms of trehalase (alpha,alpha'-trehalose-1-D-glucohydrolase, EC 3.2.1.28) in amniotic fluid on the basis of differences in net electric charges. Trehalase was solubilized from purified brush-border membranes of fetal rabbit kidney and intestine by Triton X-100 treatment, whereas the trehalase activity in amniotic fluid was soluble. The kinetic properties of trehalase from intestine, kidney and amniotic fluid were very similar. The Mr of the soluble amniotic fluid trehalase was between 72,600 and 66,300 from hydrodynamic parameters, depending on the amount of sugar bound to the enzyme, and 48,500 by radiation inactivation, a method which detects only the protein part of the enzyme. For membrane-bound trehalase from kidney and intestine in situ the radiation inactivation method also gave a molecular size of around 49,000. Isoelectric focusing of freshly solubilized membranes allowed us to distinguish between renal and intestinal forms of trehalase in rabbit fetuses on the basis of different isoelectric points. Each trehalase form was also present in the amniotic fluid but in varying proportions depending on the gestational age at which the amniotic fluid was collected. The results suggest that early in gestation amniotic fluid trehalase activity originates exclusively from the fetal kidney but that more and more intestinal enzyme is released into the amniotic cavity as the fetus develops. Similar results were also obtained when ion-exchange chromatography was used to separate the various trehalase forms. The development of trehalase activity in rabbit fetal kidney and intestine correlates well with its occurrence in the amniotic fluid; trehalase activity in the kidney develops early in gestation whereas the intestinal trehalase activity develops just before term. 相似文献
13.
D Thompson K Norbeck L I Olsson D Constantin-Teodosiu J Van der Zee P Moldéus 《The Journal of biological chemistry》1989,264(2):1016-1021
The oxidation of eugenol (4-allyl-2-methoxyphenol) by horseradish peroxidase was studied. Following the initiation of the reaction with hydrogen peroxide, eugenol was oxidized via a one-electron pathway to a phenoxyl radical which subsequently formed a transient, yellow-colored intermediate which was identified as a quinone methide. The eugenol phenoxyl radical was detected using fast-flow electron spin resonance. The radicals and/or quinone methide further reacted to form an insoluble complex polymeric material. The stoichiometry of the disappearance of eugenol versus hydrogen peroxide was approximately 2:1. The addition of glutathione or ascorbate prevented the appearance of the quinone methide and also prevented the disappearance of the parent compound. In the presence of glutathione, a thiyl radical was detected, and increases in oxygen consumption and in the formation of oxidized glutathione were also observed. These results suggested that glutathione reacted with the eugenol phenoxyl radical and reduced it back to the parent compound. Glutathione also reacted directly with the quinone methide resulting in the formation of a eugenol-glutathione conjugate(s). Using 3H-labeled eugenol, extensive covalent binding to protein was observed. Finally, the oxidation products of eugenol/peroxidase were observed to be highly cytotoxic using isolated rat hepatocytes as target cells. 相似文献
14.
Localization of renal and intestinal trehalase with immunofluorescence- and enzyme-labeled antibody techniques 总被引:1,自引:0,他引:1
M Nakano 《The journal of histochemistry and cytochemistry》1982,30(12):1243-1248
The localization of trehalase with fluorescein isothiocyanate-conjugated and peroxidase-conjugated antibody techniques was examined. Antiserum against purified rabbit renal trehalase was produced against guinea pigs. Anti-renal trehalase immunoglobulin (Ig)G was isolated from the serum and used for the immunohistochemical localization of intestinal and renal trehalases. Specific fluorescence and peroxidase staining were observed in the brush borders of proximal tubules and of intestinal epithelial cells. These results are in good agreement with the biochemical results. Thus, it is concluded that trehalase is specifically localized in the renal and intestinal brush borders. Sections of rabbit intestine and of rabbit kidney treated with anti-rabbit renal trehalase IgG were observed to have a specific fluorescence at the brush borders. Sections of rat intestine treated with the same antibody, however, showed no specific fluorescence at the brush borders. From these results, it is strongly suggested that renal trehalase and intestinal trehalase from the rabbit have common antigenic determinants and that these differ from those in rat intestinal trehalase. 相似文献
15.
Zhao H Charnley AK Wang Z Yin Y Li Z Li Y Cao Y Peng G Xia Y 《Journal of biochemistry》2006,140(3):319-327
Trehalose is the main sugar in the haemolymph of insects and is a key nutrient source for an insect pathogenic fungus. Secretion of trehalose-hydrolysing enzymes may be a prerequisite for successful exploitation of this resource by the pathogen. An acid trehalase [EC 3.2.1.28] was purified to homogeneity from a culture of a locust-specific pathogen, Metarhizium anisopliae, and its properties were characterized. The gene (ATM1) of this acid trehalase was also isolated. The pure enzyme can efficiently hydrolyze haemolymph trehalose into glucose in vitro. The new acid trehalase appearing in the haemolymph of Locusta migratoria infected with M. anisopliae had the same pI and substrate specificity as the purified fungal acid trehalase, and the concentration of trehalose in the haemolymph decreased sharply after infection. RT-PCR also revealed the ATM1 gene's expression in the haemolymph of the infected insects. Our results indicated that the acid trehalase may serve as an "energy scavenger" and deplete blood trehalose during fungal pathogenesis. 相似文献
16.
Physical properties and Tris inhibition of an insect trehalase and a thermodynamic approach to the nature of its active site 总被引:1,自引:0,他引:1
The midgut from Rhynchosciara americana larvae display a trehalase (alpha,alpha'-trehalose glucohydrolase, EC 3.2.1.28) which is soluble with a molecular weight of 122 000 and pI 4.6. The optimum pH of the enzyme is 6.0, its apparent Km for trehalose is 0.67 mM and its energy of activation is 16.7 kcal/mol. Sulfhydryl reagents do not inhibit the trehalase. The results suggest the existence of two carboxyl groups in the active site, one of which has a very high (8.3) pK. The increase of the pK values of the essential groups of the free enzyme in the presence of increasing concentrations of dioxane supports the hypothesis that these groups are carboxyls. The purified enzyme hydrolyzes only alpha,alpha'-trehalose and it is competitively inhibited by several compounds. 相似文献
17.
The effect of eugenol on the antioxidant status of the rat intestine after short and long term (15 days and 90 days respectively) oral administration of 1000 mg/kg.b.wt (a dosage which has been reported to be highly hepatoprotective) was studied. The level of lipid peroxidation products (TBARS) and the activities of glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD) and catalase (CAT) were found to be near normal on eugenol treatment. The level of glutathione (GSH) did not show any change on 15 days of eugenol treatment, but it was increased significantly on 90 day eugenol treatment. The activity of glutathione-S-transferases (GSTs) was increased significantly in both 15 day eugenol treated and 90-day eugenol treated groups. The results suggest that eugenol is nontoxic, protective and induces glutathione-S-transferases (GSTs) and thereby it may facilitate the removal of toxic substances from the intestine. 相似文献
18.
Kathleen A. Killick 《Archives of biochemistry and biophysics》1983,222(2):561-573
Trehalase (α-α′-trehalose 1-d-glucohydrolase, EC 3.2.1.28) was solubilized from myxamoebae of the cellular slime mold Dictyostelium discoideum by a freeze-thaw cycle and was subsequently purified to homogeneity using the techniques of ethanol fractionation, molecular sieve chromatography, DEAE-cellulose ion-exchange chromatography, chromatofocusing, and preparative polyacrylamide disc gel electrophoresis. The 1000-fold purified enzyme had a specific activity of about 104 units/mg, which was accompanied by a net recovery of 5 to 7% of the original activity. The purified enzyme was maximally active at pH 5.5, showed high specificity for trehalose, and exhibited a typical hyperbolic response as a function of trehalose concentration with a Km of 1.2 mm. The enzyme was maximally active at 50 °C and had an energy of activation of 12–13 kcal/mol. Thermal stability studies demonstrated that full enzymatic activity was recovered following a 5-min incubation of trehalase at temperatures up to 45–50 °C. Analysis of various compounds for inhibitory effects indicated that Tris and urea were slightly effective, reducing enzymatic activity by 28 and 6% at concentrations of 100 and 10 mm, respectively. Of five heavy metals tested, HgCl2 was the most inhibitory, reducing activity by 58% when present at a final concentration of 1.0 mm. Enzymatic activity was not affected by any adenine derivative examined (e.g., ATP, ADP, AMP, cAMP, adenosine, and adenine). The molecular weight of the native enzyme was determined by molecular sieve chromatography, pore gradient electrophoresis, and electrophoresis as a function of acrylamide concentration. All three methods yielded a value of about 105 ± 5 × 103. Estimation of the subunit or monomer molecular weight by sodium dodecyl sulfate-gel electrophoresis indicated a value of 95–100 × 103. The isoelectric point as determined in 7.5% polyacrylamide gels with pH 3–10 ampholytes was 7.2–7.3. The purified enzyme adsorbed to concanavalin A-Sepharose in the presence of KCl (0.1 m) and was eluted with α-methylmannoside, thereby suggesting an association between trehalase and carbohydrate. In agreement with this conclusion was the observation that trehalase could be specifically stained for carbohydrate with the Alcian blue and periodic acid-Schiff's reagents following polyacrylamide disc gel electrophoresis. 相似文献
19.
K A Killick 《Journal of general microbiology》1985,131(2):273-278
Previous studies have indicated that during development in the slime mould Dictyostelium discoideum, compartmentation of the isoenzymes of trehalase (alpha, alpha'-trehalose 1-D-glucohydrolase, (EC 3.2.1.28) occurs between the extracellular and intracellular environments. The compartmentation of trehalase between soluble and particulate cell fractions was examined in this work. The trehalase present in crude homogenates prepared during the first 12 h of development was completely soluble. Starting at about the pseudoplasmodial stage (i.e. the 14th hour of development), trehalase activity became associated with insoluble cellular material and this increased to a maximal value in homogenates from mature sorocarps, where 50% of the activity was insoluble. Spore cells accounted for only 2 to 3% of the trehalase associated with mature sorocarps, with the remaining 97% being localized in stalk cell material. Although trehalase recovered from spores was completely soluble, more than half of that from the stalk was recovered in the buffer-insoluble pellet fraction. 相似文献
20.
Only one molecular form of trehalase (E.C. 3.2.1.28) was detectable in adult Drosophila melanogaster by polyacrylamide gel electrophoresis and isoelectric focusing. An examination of duplication- and deletion-bearing aneuploids exhibiting dosage sensitivity indicated that the enzyme is encoded by a gene, Treh+, located between 55B and 55E of the second chromosome. The tissue-specific soluble and particulate forms of trehalase appear to be manifestations of a single protein encoded by a single gene. 相似文献