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1.
Initiated by the recently published histochemical method for the investigation of alfa-D-galactosidas with an indoxyl substrate, the current state of this group of synthetic compounds in light and electron microscopic histochemical glycosidase research is evaluated whereby historical, functional, methodological and applied aspects are considered. Beginning with the introduction of indoxyl acetate for non-specific esterase in 1951 and 1952 numerous other indoxyl substrates and mostly substituted in the 5- and 4-position of the indol ring by Br and Cl were developed to study histochemically non-specific phosphatases and glycosidases and frequently used in indigogenic, azoidoxyl, tetrazolium salts and metal salt techniques for catalytic (activity) histochemical and less often for immunohistochemical, affinity histochemical and hybridohistochemical purposes. The last substrate which became available and was validated for activity histochemistry was 5-Br-4-Cl-3-indoxyl alfa-1-galactoside for alfa-1-galactosidase. At present, the indoxyl glycosides are more widely used than 5-Br-4-Cl-3-indoxyl acetates and phosphates when compared with the alternative synthetic (artificial) naphthol, 6-Br-2-naphthol or ternative synthetic (artificial) naphthol, 6-Br-2-naphthol AS substrates, and among the indoxyl glycosides those for the oxoglycosidases lactase, maltase-glucoamylase, glucoamylase, acid beta-D-galactosidase, neuroaminidase and alfa-D-galactosidase are superior to other artificial compounds. When one considers in addition, electron microscopic catalytic glicosidase histochemistry (ultracytochemistry, 5-Br-4-Cl-3-indoxyl is the only suitable moiety for this purpose. These glycosidase can mostly be localized in plasma membranes or lysosomes and also measured there in tissue sections but are also found in secretion granules, endoplasmic reticulum and organ lumina.  相似文献   

2.
Summary The possibility of the use of indoxyl substrates for enzyme localization in plant material was tested and proved. Studying the localization of the carboxyl esterase in broad bean root tip a) the time course of the indigogenic reaction was investigated, b) the concentration of the ferro/ferri redox buffer was examined, c) various substances were used to control the indigogenic visualizing reaction, d) the azocoupling procedure was applied with indoxyl substrates, e) unhalogenated and halogenated substrates were tried, and, finally, f) acetate and butyrate were employed in the indigogenic procedure. Using the last mentioned (f) trial the existence and localization was verified in the materal tested of carboxyl esterase fractions recognized there previously on the basis of azocoupling reactions with naphtholic substrates.  相似文献   

3.
Chemiluminescent assays of various enzymes have been developed using indoxyl derivatives as substrates. The principle of the method is as follows: an enzyme causes hydrolysis of an indoxyl derivative to an intermediate indoxyl that is readily oxidized to indigo dye and simultaneously produces hydrogen peroxide (H2O2). Hydrogen peroxide is detected chemiluminescently using isoluminol-microperoxidase. Alkaline phosphatase (ALP), beta-D-galactosidase (beta-gal), and beta-glucosidase were assayed by this method using 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal), and 5-bromo-4-chloro-3-indolyl-beta-D-glucoside, respectively, as substrates. Using BCIP and X-Gal substrates, we have been able to detect 10(-19) mol of ALP and beta-gal, respectively. This assay system can be applied to enzyme immunoassay and DNA probe assay.  相似文献   

4.
Summary In contrast to human and animal tissues, little information is available on the activity, distribution and functional role of acid and neutral hydrolases in plant cells and tissues. Because it is known that these enzymes are relatively active during germination, they were analysed histochemically during this process using light microscope azo, azoindoxyl, indigogenic and tetrazolium methods. Proteases, glucosidases and glucuronidases could not be detected. Non-specific acid phosphatases were species-independent and showed considerable activities in aleuron and nutritional cells, in other cell types of cotyledon or endosperm tissue and in different types of embryonic cells. Acid glycosidases and non-specific esterases, in contrast displayed a species-dependent activity and differences in localization. Of the glycosidases, -d-galactosidase was the most active. Non-specific esterases, acid phosphatase and glucosaminidase were also present in the extracellular matrix. During germination, acid hydrolase activity either decreased or increased, depending on the seedling species and enzyme.  相似文献   

5.
R Gossrau 《Histochemistry》1978,58(3):203-218
Using freeze-dried or sections from fresh-frozen or aldehyde-fixed material nitro BT (NBT), tetranito BT (TNBT), distyryl nitro BT (DS-NBT), thiocarbamyl nitro BT (TC-NBT) or benzothiazolylstyrylphthalhydrazidyl tetrazolium chloride (BSPT) were tested as auxiliary reagents for the localization of glycosidases, phosphatases and non-specific esterases with indoxyl substrates in rat tissues. By means of NBT or TNBT as a tetrazolium salt acid beta-D-galactosidase, beta-D-N-acetylglucosaminidase, acid phosphatase, neuraminidase and non-specific esterase can only be localized at the cellular level; a more precise localization is possible for lactase-beta-D-glucosidase in the intestinal brush border, and the best results are obtained in the demonstration of alkaline phosphatase; among all methods described previously the tetrazolium procedure with TNBT is the method of choice for the light microscopic localization of this enzyme. Reverse data are observed with BSPT as a tetrazolium salt; then, all acid and neutral hydrolases can be exactly localized in lysosomes, secretion granules, cytoplasm and/or microvilli of many cells and tissues provided BSPT-formazan is stabilized by osmification. Furthermore, this procedure enables the reliable ultracytochemical demonstration of these enzymes. However, in the case of alkaline phosphatase only sites with high enzyme activity reveal a positive reaction. -DS- and TC-NBT are inferior to NBT, TNBT or BSPT.  相似文献   

6.
Zymogram patterns of nine strains of Toxoplasma gondii were studied using the API enzyme research kit. This system uses chromogenic substrates to detect the presence or absence of 84 enzymes. Enzyme classes assayed for included amino-peptidases, glycosidases, esterases, lipases, phosphoamidase and phosphatases.
All strains were positive for 24 enzymes: 15 arylamidases, seven esterases, alkaline phosphatase and acid phosphatase. In contrast, 27 enzyme tests were negative in all strains. Thirty-three enzymatic reactions were different in one or more strains.  相似文献   

7.
Indoxyl glycosides proved to be valuable and versatile tools for monitoring glycosidase activities. Indoxyls are released by enzymatic hydrolysis and are rapidly oxidized, for example by atmospheric oxygen, to indigo type dyes. This reaction enables fast and easy screening in vivo without isolation or purification of enzymes, as well as rapid tests on agar plates or in solution (e.g., blue-white screening, micro-wells) and is used in biochemistry, histochemistry, bacteriology and molecular biology. Unfortunately the synthesis of such substrates proved to be difficult, due to various side reactions and the low reactivity of the indoxyl hydroxyl function. Especially for glucose type structures low yields were observed. Our novel approach employs indoxylic acid ester as key intermediates. Indoxylic acid esters with varied substitution patterns were prepared on scalable pathways. Phase transfer glycosylations with those acceptors and peracetylated glycosyl halides can be performed under common conditions in high yields. Ester cleavage and subsequent mild silver mediated glycosylation yields the peracetylated indoxyl glycosides in high yields. Finally deprotection is performed according to Zemplén.  相似文献   

8.
The esterases are studied histochemically in the pharyngeal bulb of local earthworms using tweens, naphthols and indoxyl substrates. Lipase and esterases are located mainly in the pharyngeal epithelial cells and chromophill cells. No activity is seen in the nonchromophill cells. The connective tissue and musculo-vascular tissue contain some esterases activity. Possible role of the esterases in the cellular elements of pharynx has been discussed.  相似文献   

9.
Nanogram amounts of soluble aminopeptidases, dehydrogenases, and esterases were detected by nondenaturing ultralow gelling point agarose-polyacrylamide gel electrophoresis (ULGA-PAGE). Cytosolic fractions from Frankia sp. were electrophoresed at 4 degrees C in the presence of Co2+, Zn2+, or Mg2+ ions. Then, aminopeptidases and esterases were revealed by simultaneous capture staining by using fast garnet GBC diazonium salt as the chromogenic coupling compound. Dehydrogenases were revealed by using nitro blue tetrazolium salt as electron acceptor. A variety of aminopeptidases, dehydrogenases, and esterases could be identified by their migration in ULGA-PAGE and by their sensitivities to NaCl, CoSO4, ZnSO4, and MgCl2 when assayed "ingel." The presence of agarose was essential for the detection of the complex enzyme patterns. The patterns were remarkably similar for the five Frankia strains isolated from Allocasuarina and Casuarina host plants and differed from those of Frankia strains isolated from Comptonia and Hippopha? host plants. A nomenclature is proposed for aminopeptidases and other Frankia enzymes. The richness of the Frankia amino-peptidases and esterases zymograms makes them adequate marker enzymes for taxonomical, genetic, or biochemical studies. Dehydrogenases might also be useful, although a more restricted number of bands were found with L-lactic and L-malic acid as substrates.  相似文献   

10.
Summary Indigogenic and azocoupling reactions for the detection of acid -galctosidase in unfixed cold microtome sections adherent to semipermeable membranes are described. The indigogenic method is the method of choice. The described procedure prevents the leakage of the enzyme activity of sections (the diffusion is limited to the closest surroundings of the actual localization of enzyme activity) and is recommended as a routine method in studies concerning acid -galactosidase.  相似文献   

11.
In this work we describe the conditional toxic effect of the expression of enzymes that cleave 5-bromo-4-chloro-3-indolyl (BCI) substrates and its use as a new counterselection principle useful for the generation of clean and unmarked mutations in the genomes of bacteria. The application of this principle was demonstrated in the thermophile Thermus thermophilus HB27 and in a mesophile for which currently no counterselection markers are available, Micrococcus luteus ATCC 27141. For T. thermophilus, the indigogenic substrate BCI-β-glucoside was used in combination with the T. thermophilus β-glucosidase gene (bgl). For M. luteus, a combination of BCI-β-galactoside and the E. coli lacZ gene was implemented. We observed a strong growth-inhibiting effect when the strains were grown on agar plates containing the appropriate BCI substrates, the inhibition being proportional to the substrate concentration and the level of bgl/lacZ expression. The growth inhibition apparently depends on intracellular BCI substrate cleavage and accumulation of toxic indoxyl precipitates. The bgl and lacZ genes were used as counterselection markers for the rapid generation of scar-less chromosomal deletions in T. thermophilus HB27 (both in a Δbgl and in a wild type background) and in M. luteus ATCC 27141.  相似文献   

12.
Glucuronoyl esterases (GEs) are microbial enzymes with potential to cleave the ester bonds between lignin alcohols and xylan-bound 4-O-methyl-d-glucuronic acid in plant cell walls. This activity renders GEs attractive research targets for biotechnological applications. One of the factors impeding the progress in GE research is the lack of suitable substrates. In this work, we report a facile preparation of methyl esters of chromogenic 4-nitrophenyl and 5-bromo-4-chloro-3-indolyl β-D-glucuronides for qualitative and quantitative GE assay coupled with β-glucuronidase as the auxiliary enzyme. The indolyl derivative affording a blue indigo-type product is suitable for rapid and sensitive assay of GE in commercial preparations as well as for high throughput screening of microorganisms and genomic and metagenomic libraries.  相似文献   

13.
Summary A mitochondrial pellet, prepared from rat skeletal muscle, contained a number of carboxylic ester hydrolase isoenzymes. The esterases which split -naphthyl acetate were organophosphate sensitive, whereas two out of three indoxyl acetate hydrolysing enzymes were resistant to both organophosphate and organomercury. The activity of the indoxyl acetate esterases was enhanced by the non-ionic detergents Tween-40 and Lubrol. After freezing, thawing and high speed centrifugation most of the -naphthyl acetate splitting enzymes were found in the supernatant, indicating that the enzymes are loosely bound to mitochondrial membranes.  相似文献   

14.
A method is described for the detection of acyl esterase activity on cellulose acetate membranes following electrophoresis of the enzyme. It uses the indigogenic substrate, indoxyl acetate, which directly forms the colored product visualized in the test. This substrate also detects activity of acetyl cholinesterase and pseudocholinesterase. With this method, bovine testicular hyaluronidase is shown to contain acyl esterase activity. By electrophoresis of hyaluronidase preparations at pH 6.8, esterase and hyaluronidase activities are separated, further assuring the specificity of the method for hyaluronidase.  相似文献   

15.
Broad-substrate-range monooygenase enzymes, including toluene-4-monooxygenase (T4MO), can catalyze the oxidation of indole. The indole oxidation products can then condense to form the industrially important dye indigo. Site-directed mutagenesis of T4MO resulted in the creation of T4MO isoforms with altered pigment production phenotypes. High-pressure liquid chromatography, thin-layer chromatography, and nuclear magnetic resonance analysis of the indole oxidation products generated by the mutant T4MO isoforms revealed that the phenotypic differences were primarily due to changes in the regiospecificity of indole oxidation. Most of the mutations described in this study changed the ratio of the primary indole oxidation products formed (indoxyl, 2-oxindole, and isatin), but some mutations, particularly those involving amino acid G103 of tmoA, allowed for the formation of additional products, including 7-hydroxyindole and novel indigoid pigments. For example, mutant G103L converted 17% of added indole to 7-hydroxyindole and 29% to indigoid pigments including indigo and indirubin and two other structurally related pigments. The double mutant G103L:A107G converted 47% of indole to 7-hydroxyindole, but no detectable indigoid pigments were formed, similar to the product distribution observed with the toluene-2-monooxygenase (T2MO) of Burkholderia cepacia G4. These results demonstrate that modification of the tmoA active site can change the products produced by the enzyme and lead to the production of novel pigments and other indole oxidation products with potential commercial and medicinal utility.  相似文献   

16.
Three different monoacetates of 4-nitrophenyl beta-D-xylopyranoside were tested as substrates for beta-xylosidase and for microbial carbohydrate esterases and a series of non-hemicellulolytic esterases. The acetyl group in 2-O-acetyl, 3-O-acetyl, and 4-O-acetyl 4-nitrophenyl beta-D-xylopyranoside makes the glycoside resistant to the action of beta-xylosidase (EC 3.2.1.37). This fact was explored to introduce a new enzyme-coupled assay of acetylxylan esterases (EC 3.1.1.72) and other carbohydrate-deacetylating enzymes. The deacetylation converts the monoacetates into the substrate of beta-xylosidase, the auxiliary enzyme. The effect of the acetyl group migration along the xylopyranoid ring in aqueous media can be avoided by shortening the assay duration. The assay enables an easy examination of the positional specificity of the enzymes, which is important for classification of acetylxylan esterases and for elucidation of the structure-function relationship among carbohydrate esterases in general. Non-hemicellulolytic esterases showed different positional specificity of deacetylation than did acetylxylan esterases.  相似文献   

17.
Broad-substrate-range monooygenase enzymes, including toluene-4-monooxygenase (T4MO), can catalyze the oxidation of indole. The indole oxidation products can then condense to form the industrially important dye indigo. Site-directed mutagenesis of T4MO resulted in the creation of T4MO isoforms with altered pigment production phenotypes. High-pressure liquid chromatography, thin-layer chromatography, and nuclear magnetic resonance analysis of the indole oxidation products generated by the mutant T4MO isoforms revealed that the phenotypic differences were primarily due to changes in the regiospecificity of indole oxidation. Most of the mutations described in this study changed the ratio of the primary indole oxidation products formed (indoxyl, 2-oxindole, and isatin), but some mutations, particularly those involving amino acid G103 of tmoA, allowed for the formation of additional products, including 7-hydroxyindole and novel indigoid pigments. For example, mutant G103L converted 17% of added indole to 7-hydroxyindole and 29% to indigoid pigments including indigo and indirubin and two other structurally related pigments. The double mutant G103L:A107G converted 47% of indole to 7-hydroxyindole, but no detectable indigoid pigments were formed, similar to the product distribution observed with the toluene-2-monooxygenase (T2MO) of Burkholderia cepacia G4. These results demonstrate that modification of the tmoA active site can change the products produced by the enzyme and lead to the production of novel pigments and other indole oxidation products with potential commercial and medicinal utility.  相似文献   

18.
Fluorescence and dye histochemical methods are compared for the investigation of hydrolases in sections and culture cells. At present, only some of the synthetic substrates with fluorescent leaving groups may be used for the fluorescence localization of these enzymes in sections. This limitation is due to a reduced fluorescence intensity and/or diffusion of the fluorescent tags. Satisfactory results are obtained for alkaline phosphatase, non-specific esterases and proteases with naphthol AS and 4-methoxy-2-naphthylamine coupled to nitrosalicylaldehyde. If, however, cultured monolayer cells are investigated, all synthetic substrates with fluorescent tags are suitable, including those that have so far only been used for biochemical hydrolase measurements. The fluorescent leaving groups are naphthol AS and its derivates, 4-methoxy-2-naphthylamine, aminomethylcoumarin, aminomethyltrifluoromethylcoumarin, methylumbelliferon, fluorescein and, with some limitations, also 1- and 2-naphthol. These fluorescence methods are more sensitive than the corresponding dye procedures. In addition, the fluorescence techniques allow the use of more synthetic substrates and therefore more information become available than with dye histochemistry about the enzymic properties of culture cells.  相似文献   

19.
Glycosyl fluorides in enzymatic reactions   总被引:4,自引:0,他引:4  
Glycosyl fluorides have considerable importance as substrates and inhibitors in enzymatic reactions. Their good combination of stability and reactivity has enabled their use as glycosyl donors with a variety of carbohydrate processing enzymes. Moreover, the installation of fluorine elsewhere on the carbohydrate scaffold commonly modifies the properties of the glycosyl fluoride such that the resultant compounds act as slow substrates or even inhibitors of enzyme action. This review covers the use of glycosyl fluorides as substrates for wild-type and mutant glycosidases and other enzymes that catalyze glycosyl transfer. The use of substituted glycosyl fluorides as inhibitors of enzymes that catalyze glycosyl transfer and as tools for investigation of their mechanism is discussed, including the labeling of active site residues. Synthetic applications in which glycosyl fluorides are used as glycosyl donors in enzymatic transglycosylation reactions for the synthesis of oligo- and polysaccharides are then covered, including the use of mutant glycosidases, the so-called glycosynthases, which are able to catalyze the formation of glycosides without competing hydrolysis. Finally, a short overview of the use of glycosyl fluorides as substrates and inhibitors of phosphorylases and phosphoglucomutase is given.  相似文献   

20.
Indole is presumably a product of indole-3-glycerol phosphate catabolism in Isatis tinctoria. It is oxidized into indoxyl and stored in young leaves as indigo precursor. Further oxidation and dimerization of indoxyl produces indigoid pigments. In this work, we describe an HPLC method dedicated to the identification and quantification of indigoid pigments (indigo, indirubin, isoindigo and isoindirubin) and indigo precursors produced in I. tinctoria (Woad). This work, carried out with two cultivars of I. tinctoria, has confirmed that the quantity of indigo precursors is dependent on the species and the harvest period. In addition we have shown for the first time that young leaves of I. tinctoria, harvested in June contained a new indigo precursor in addition to isatan B (indoxyl-5-ketogluconate) and indican (indoxyl-beta-D-glucoside). We suggest the name "isatan C" for this new indigo precursor in I. tinctoria. Its chemical characteristics point to an dioxindole ester with PM of 395. We have shown that isatan C reacts with isatan B increasing the red pigment production.  相似文献   

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