Indigogenic substrates for detection and localization of enzymes

Indoxyl esters and glycosides are useful chromogenic substrates for detecting enzyme activities in histochemistry, biochemistry and bacteriology. The chemical reactions exploited in the laboratory are similar to those that generate indigoid dyes from indoxyl-beta-d-glucoside and isatans (in certain plants), indoxyl sulfate (in urine), and 6-bromo-2-S-methylindoxyl sulfate (in certain molluscs). Pairs of indoxyl molecules released from these precursors react rapidly with oxygen to yield insoluble blue indigo (or purple 6,6'-dibromoindigo) and smaller amounts of other indigoid dyes. Our understanding of indigogenic substrates was developed from studies of the hydrolysis of variously substituted indoxyl acetates for use in enzyme histochemistry. The smallest dye particles, with least diffusion from the sites of hydrolysis, are obtained from 5-bromo-, 5-bromo-6-chloro- and 5-bromo-4-chloroindoxyl acetates, especially the last of these three. Oxidation of the diffusible indoxyls to insoluble indigoid dyes must occur rapidly. This is achieved with atmospheric oxygen and an equimolar mixture of K(3)Fe(CN)(6) and K(4)Fe(CN)(6), which has a catalytic function. H(2)O(2) is a by-product of the oxidation of indoxyl by oxygen. In the absence of a catalyst, the indoxyl diffuses and is oxidized by H(2)O(2) (catalyzed by peroxidase-like proteins) in sites different from those of the esterase activity. The concentration of K(3)Fe(CN)(6)/K(4)Fe(CN)(6) in a histochemical medium should be as low as possible because this mixture inhibits some enzymes and also promotes parallel formation from the indoxyl of soluble yellow oxidation products. The identities and positions of halogen substituents in the indoxyl moiety of a substrate determine the color and the physical properties of the resulting indigoid dye. The principles of indigogenic histochemistry learned from the study of esterases are applicable to methods for localization of other enzymes, because all indoxyl substrates release the same type of chromogenic product. Substrates are commercially available for a wide range of carboxylic esterases, phosphatases, phosphodiesterases, aryl sulfatase and several glycosidases. Indigogenic methods for carboxylic esterases have low substrate specificity and are used in conjunction with specific inhibitors of different enzymes of the group. Indigogenic methods for acid and alkaline phosphatases, phosphodiesterases and aryl sulfatase generally have been unsatisfactory; other histochemical techniques are preferred for these enzymes. Indigogenic methods are widely used, however, for glycosidases. The technique for beta-galactosidase activity, using 5-bromo-4-chloroindoxyl-beta-galactoside (X-gal) is applied to microbial cultures, cell cultures and tissues that contain the reporter gene lac-z derived from E. coli. This bacterial enzyme has a higher pH optimum than the lysosomal beta-galactosidase of animal cells. In plants, the preferred reporter gene is gus, which encodes beta-glucuronidase activity and is also demonstrable by indigogenic histochemistry. Indoxyl substrates also are used to localize enzyme activities in non-indigogenic techniques. In indoxyl-azo methods, the released indoxyl couples with a diazonium salt to form an azo dye. In indoxyl-tetrazolium methods, the oxidizing agent is a tetrazolium salt, which is reduced by the indoxyl to an insoluble coloured formazan. Indoxyl-tetrazolium methods operate only at high pH; the method for alkaline phosphatase is used extensively to detect this enzyme as a label in immunohistochemistry and in Western blots. The insolubility of indigoid dyes in water limits the use of indigogenic substrates in biochemical assays for enzymes, but the intermediate indoxyl and leucoindigo compounds are strongly fluorescent, and this property is exploited in a variety of sensitive assays for hydrolases. The most commonly used substrates for this purpose are glycosides and carboxylic and phosphate esters of N-methylindoxyl. Indigogenic enzyme substrates are among many chromogenic reagents used to facilitate the identification of cultured bacteria. An indoxyl substrate must be transported into the organisms by a permease to detect intracellular enzymes, as in the blue/white test for recognizing E. coli colonies that do or do not express the lac-z gene. Secreted enzymes are detected by substrate-impregnated disks or strips applied to the surfaces of cultures. Such devices often include several reagents, including indigogenic substrates for esterases, glycosidases and DNAse.     

Suitability of the azocoupling reaction with 1-naphthyl-beta-D-glucoside for the histochemical demonstration of lactase (lactase-beta-glucosidase complex) in human enterobiopsies.

The suitability of the simultaneous azocoupling reaction with 1-naphthyl-beta-D-glucoside and hexazonium-p-rosanilin in the detection of the activity of lactase (or lactase-beta-glucosidase complex) in jejunal biopsies of patients with various forms of the malabsorption syndrome was tested. Results were compared with those obtained with the indigogenic method using 4-Cl-5-Br-3-indolyl-beta-D-fucoside which is the method of choice. Both methods gave identical results as far as the relative intensity of the brush border staining was concerned. The azocoupling method applied in unfixed cold microtome sections can be recommended for the routine diagnostics of the malabsorption syndrome when the indolyl substrate is not available.     

The presence and distribution of α and β glucosidase in root tip

A comparison was made of post and simultaneous azocoupling procedures for β glucosidase localization on unfixed and fixed root tips ofZea mays. Using this object, more detailed studies of β glucosidase distribution were undertaken, concerning the time course of enzyme reaction, its pH dependence, the effect of various buffers, the comparison of several diazonium salts and the use of different naphtholic substrates. The indigogenic reaction was also applied. Attempts were made by means of azocoupling procedures to localize a and β glucosidase in root tips ofCucurbita pepo, Lupinus albus, Piswm sativum andVicia faba in comparison withZea mays. In addition to certain technical problems, the questions of constitutive and adaptive enzymes, sugar distribution and histogenesis versus function are discussed in relation to the presence and distribution of ß glucosidase in the studied objects.     

Indoxyl alfa-D-galactoside as the temporarily last substrate for glycosidase histochemistry. The present state of the art in histochemical glycosidase research using indoxyl glycosidas

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.     

Detection of acyl esterase activity on electrophoresis membranes and separation from hyaluronidase.

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.     

Tetrazolium methods for the histochemical investigation of hydrolases (author's transl)]

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.     

The histochemical demonstration of aminopeptidase with bromoindolyl leucinamide.

The indigogenic method for aminopeptidase of Pearson et al. (1963) was critically evaluated. The localization obtained with it is not correct due to diffusion artifacts. Ferricyanide cannot be used as an oxidation agent. Based on experiments with other oxidation agents (phenazonium methosulfate, nitro BT, tetranitro BT) a new method was devised. The recommended incubation medium contains 0.9 mM L-N-(5-bromoindol-3-yl) leucinamide hydrobromide (chloride), 0.73 mM tetranitro BT, 0.27 mM phenazonium methosulfate and 0.67 M phosphate buffer pH 7.4. The enzyme activity is indicated by the deposition of tetranitro BT formazan. Results with this method in rat kidney, jejunum, liver, lung, and submaxillary gland, in monkey kidney and jejunum, and in human jejunal biosies are almost identical with those obtained with L-leucyl-4-methoxy-beta-naphthylamide applied in a simultaneous azocoupling procedure. The given principle of the demonstration of aminopeptidase activity with an indolylamine substrate deserves a further exploration in the demonstration of peptidases "in situ" both on optical as well as electronmicroscopical levels.     

Histochemical methods for acid β-galactosidase: Technics for semipermeable membranes

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.     

Carboxylic ester hydrolases in the thyroid gland of the guinea-pig. A light microscopic study

Synopsis The location of cholinesterase and non-specific esterase in the thyroid gland of the guinea-pig was studied with the light microscope. It was found that the inxodyl method for non-specific esterase activity under special conditions is superior to the cholinesterase method in a number of respects for the demonstration of the intra-, inter-and parafollicular cells. When using the indoxyl method the incubation period can be reduced from 2.5–3 hr to 40–50 min. Further, the reaction can be followed during the incubation. False localization of the reaction products is avoided, and nerves and erythrocytes are not stained.By varying the fixation time and the time of storage in gum arabic-sucrose, it was found that the miscellaneous activity of non-specific esterase in APUD cells (C-cells) and follicle cells may be due to both factors. In fresh tissue the activity of the enzyme was equal in follicle and C cells.Special cyst-like structures containing an esterase which is NaF-resistant whenn-naphthyl acetate is employed as a substrate and which gives a strong reaction at low pH values when 5-bromo indoxyl acetate is the substrate, are described, and their nature and possible origin are discussed.     

Azoindoxyl methods for the histochemical investigation of hydrolases. I. Lactase (lactase-beta-glucosidase complex) (author's transl)]

An azoindozyl method for the histochemical demonstration of lactase (lactase-beta-glucosidase complex) is described. The incubation medium consists of 5 mg 5-Br-4-Cl-3-indolyl-beta-D-fucoside (dissolved in 0.5 ml N,N-dimethylformamide) and 0.02 ml hexazotized prosaniline in 10 ml 0.1 M citric acid phosphate buffer, pH 6-6.5. By means of this method lactase can be exactly localized in the brush border of the enterozytes in the jejunum of suckling rats. Compared to the corresponding indigogenic method the azoindoxyl reaction proceeds faster and the reaction product is often precipitated more precisely.     

Simultaneous Demonstration of Nonspecific Esterase and Chloroacetate Esterase in Human Blood Cells

A new cytochemical method is described for the simultaneous demonstration of nonspecific esterase in monocytes and chloracetate esterase in granulocytes. The procedure uses both alpha-naphthyl butyrate and naphthol AS-D chloroacetate as substrates and hexazotized pararosaniline as the coupler. The enzyme reaction products are highly chromogenic and their localization is precise. This method is potentially useful for the accurate diagnosis of the acute monocytic leukemias. Its advantages and limitations are also discussed.     

Simultaneous demonstration of nonspecific esterase and chloroacetate esterase in human blood cells

A new cytochemical method is described for the simultaneous demonstration of nonspecific esterase in monocytes and chloracetate esterase in granulocytes. The procedure uses both alpha-naphthyl butyrate and naphthol AS-D chloroacetate as substrates and hexazotized pararosaniline as the coupler. The enzyme reaction products are highly chromogenic and their localization is precise. This method is potentially useful for the accurate diagnosis of the acute monocytic leukemias. Its advantages and limitations are also discussed.     

Histochemical detection of alpha-D-glucosidases and their molecular forms with 5-Br-4-Cl-3-indoxyl-alpha-D-glucoside

5-Br-4-Cl-3-Indoxyl-alpha-D-gluco(pyrano)side was found to be the most suitable synthetic substrate for the demonstration of alpha-D-glucosidases in situ. Using an azoindoxyl procedure with hexazotized pararosaniline or new fuchsine at pH 5 in freeze-dried celloidine-mounted cryostat sections acid alpha-D-glucosidase (EC 3.2.1.20) was shown for the first time in lysosomes of many cells of fetal and adult rat, mouse, guinea-pig, marmoset and human organs. At pH 6.5, in chloroform-acetone pretreated cryostat sections plasma membrane alpha-D-glucosidases were shown in the brush border of enterocytes of the small and large intestine, in the brush border of proximal renal tubule cells and in the stereocilia of the epididymal duct. In an indigogenic procedure with ferricyanide/ferrocyanide as redox catalysator plasma membrane alpha-D-glucosidases were depicted as well as with the azo-indoxyl method; the demonstration of the acid alpha-D-glucosidase was inferior to that achieved with the azo-indoxyl procedure. Using tetrazolium salts as capture reagent intracellular localization was unsatisfactory. In enterocytes, a localization in the Golgi apparatus was shown by the azo-indoxyl procedure only. Analytical isoelectric focusing revealed organ-dependent differences of plasma membrane and lysosomal alpha-D-glucosidases. Compared with the already existing methods the azo-indoxyl and indigogenic procedures are by far the most suitable techniques.     

Involvement of the carboxyl-terminal propeptide of beta-glucuronidase in its compartmentalization within the endoplasmic reticulum as determined by a synthetic peptide approach

The proenzyme form of beta-glucuronidase is compartmentalized in large quantities within the endoplasmic reticulum by binding to the esterase, egasyn. Also, the propeptide of the proenzyme form of beta-glucuronidase is likely located at the carboxyl terminus. We have, therefore, tested if this carboxyl-terminal peptide is important in binding to egasyn. A polyclonal antibody to a 30-mer synthetic peptide, corresponding to the carboxyl-terminal 30 amino acids of pro-beta-glucuronidase, provided evidence that egasyn binds to the carboxyl terminus of beta-glucuronidase. This antibody interacted with proenzyme beta-glucuronidase-egasyn complexes in which one, two, or three egasyn molecules were bound to the beta-glucuronidase tetramer, but not with those complexes (M4) which contained four egasyn molecules. We interpret these results as indicating that all available carboxyl termini of the beta-glucuronidase proenzyme tetramer are shielded by egasyn in the M4 complexes. The same antibody did not recognize the mature lysosomal form of beta-glucuronidase, indicating that only the proenzyme form of microsomal beta-glucuronidase contains the original carboxyl terminus. Also, the synthetic 30-mer was found to be a specific and potent inhibitor (50% inhibition at 1.3 microM) of the esterase activity of purified egasyn but exhibited little inhibitory activity toward other purified esterases including a rat trifluoroacetylated esterase or egasyn esterase from another species. Together, these data describe a potent interaction of the exposed carboxyl terminus of precursor glucuronidase with the esterase catalytic site of egasyn, which in turn results in the specific localization of glucuronidase within the lumen of the endoplasmic reticulum.     

An improved method for the simultaneous demonstration of mRNA and esterase activity at the human neuromuscular junction

The aim of this study was to develop a simple means of studying the distribution of mRNA coding for post-synaptic proteins at the human neuromuscular junction. A reliable method by which to identify the junctions in tissue sections after in situ hybridization was essential. A method is described for combining the histochemical demonstration of esterase activity at the neuromuscular junction with autoradiographic localization of mRNA by in situ hybridization in the same cryostat section of skeletal muscle. The indigogenic esterase method of Strum and Hall-Craggs (1982) was modified in such a way that it is able to survive the multiple steps involved in in situ hybridization and autoradiography. The protocol is simple and reproducible and has been used successfully on sections of both rat and human skeletal muscle. To demonstrate the method, sections were reacted to reveal esterase activity and were then processed for in situ hybridization using a 35S-labelled probe specific for the -s ubunit of the acetylcholine receptor. The reaction product was retained after the lengthy in situ hybridization and autoradiographic procedures. To our knowledge, this is the first demonstration of acetylcholine receptor mRNA by in situ hybridization at human neuromuscular junctions. © Chapman & Hall     

An important role for secreted esterase in disease establishment of the wheat powdery mildew fungus Blumeria graminis f. sp. tritici

The activity of esterase secreted by conidia of wheat powdery mildew fungus, Blumeria graminis f.?sp. tritici, was assayed using indoxyl acetate hydrolysis, which generates indigo blue crystals. Mature, ungerminated, and germinating conidia secrete esterase(s) on artificial media and on plant leaf surfaces. The activity of these esterases was inhibited by diisopropyl fluorophosphate, which is selective for serine esterases. When conidia were inoculated on wheat leaves pretreated with diisopropyl fluorophosphate, both appressorial germ tube differentiation and symptom development were significantly impaired, indicating an important role of secreted serine esterases in wheat powdery mildew disease establishment.     

The deacetylation of N-hydroxy-2-acetylaminofluorene by rat liver microsomes and carboxyl esterase

We have demonstrated that incubation of rat liver microsomes with N-hydroxy-2-acetylaminofluorene (N-OH-AAF) leads to formation of a 2-nitrosofluorene-membrane lipid adduct. This adduct exists as a nitroxyl free radical, termed N-O-LAF, in its oxidized state. When microsomes were incubated with the sulfhydryl binding agent, rho-hydroxymercuribenzoate, a larger amount of N-OL-LAF formed. We interpret this as a slowdown in the rate of endogenous chemical reduction of carcinogen-membrane lipid adduct. In this paper we present evidence that N-OH-AAF is deacetylated by a microsomal enzyme to form N-hydroxy-2-aminofluorene and this is then oxidized to 2-nitrosofluorene which adds covalently to membrane lipid double bonds to form N-O-LAF. Various antioxidants, peroxidase inhibitors, and P450 substrates and inhibitors were ineffective in altering the amount of N-O-LAF formed from N-OH-AAF; but two esterase inhibitors, dietyl-rho-nitrophenylphosphate and alpha-toluene-sulfonyl fluoride, prevented N-O-LAF formation. Of the following purified enzymes tested: porcine liver carboxyl esterase, pepsin, chymotrypsin, cathepsin D, ficin, papain, leucine aminopeptidase, Naja naja phospholipase, acetylcholinesterase (type I), trypsin (type I and V) and epoxide hydrase; only carboxyl esterase was effective in deacetylating N-OH-AAF.     

Partial purification and characterization of sialate O-acetylesterase from bovine brain

From bovine brain an esterase was purified 2,600-fold in an overall yield of 5.6%. For the isolation ion-exchange chromatographies, gel filtration, and preparative isoelectric focusing were used. The molecular mass is 56 kDa after gel chromatography on Sephacryl S-200 and 51 kDa after HPLC, the pH-optimum at 7.4, and the isoelectric point in the range of pH 5.8-6.1, as estimated from preparative isoelectric focusing. The substrate specificity of this enzyme was tested with various naturally occurring O-acylated sialic acids, synthetic carbohydrate acetates, and other esters. Besides aromatic acetyl esters such as e.g. alpha-naphthyl acetate, the highest preference was for N-acetyl-9-O-acetylneuraminic acid, followed by N-acetyl-4-O-acetylneuraminic acid. Other primary acetyl esters such as 6-O-acetylated D-glucose and 2-acetamido-2-deoxy-D-mannose were not hydrolyzed. The 9-O-acetyl derivative of the naturally occurring unsaturated sialic acid 2-deoxy-2,3-didehydro-N-acetylneuraminic acid, however, is a substrate for this esterase. Whereas N-acetyl-9-O-acetylneuraminic acid as a component of sialyllactose is nearly as well hydrolyzed as the corresponding free sialic acid, O-acetylated sialoglycoconjugates with high molecular weights (mucins, serum glycoproteins, gangliosides) are not hydrolyzed by this esterase. N-Acetylated sialic acids are better substrates than the analogous N-glycoloyl derivatives. Esterification of the carboxyl function of sialic acids prevents the action of the esterase on the O-acetyl groups. The enzyme has no carboxyl esterase or amidase activity, and does not act on acetylcholine. It hydrolyzes almost exclusively acetyl esters. Inhibition studies suggest that it has a catalytically active serine residue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of acid phosphatase, beta-glucuronidase, n-acetyl-beta-d-glucosaminidase and beta-galactosidase in cornea of albino rabbit.

Activities of acid phosphatase, beta-glucuronidase, N-acethyl-beta-D-glucosaminidase and acid beta-galactosidase were investigated histochemically in rabbit corneas. Frozen sections after block fixation in cold 4% formaldehyde with 1% CaCl2 followed by washing in cold physiological saline as well as cold microtome sections of corneas quenched in petroleter chilled with acetone-dry ice mixture, transferred to nonprecooled slides or semipermeable membranes were used. Standard aqueous media were employed in the case of free-floating frozen sections of fixed corneas as well as of cold mictrotome sections (postfixed in cold 4% formaldehyde). Agar media were used in connection with the technic of semipermeable membranes. Gomori method (in the case of acid phosphatase), simultaneous azocoupling methods (substrates derivated of naphthol-AS-BI with hexazonium-p-rosanilin) in the case of acid phosphatase, beta-glucuronidase and N-acetyl-beta-D-glucosaminidase and the indigogenic method in the case of acid beta-galactosidase were applied. Enzyme activities in sections of fixed corneas were minimal in comparison with those in cold microtome sections of unfixed material revealed particularly with the technic of semipermeable membranes which is to be preferred. This technic is recommended in studies concerned with lysosomal enzymes in the cornea, particularly in keratocytes. All enzymes investigated were present in corneal epithelium, keratocytes and endothelium. Acid phosphatase displayed the highest activity followed by beta-glucuronidase and acetyl-beta-D-glucosaminidase. The activity of beta-galactosidase was the lowest. For the demonstration of activities in keratocytes sections parallel to the surface are very suitable. In these sections enzyme activities were demonstrated in small granules (apparently lysosomes) present in the central part of their cytoplasm as well as in projections. Diffuse staining was also seen, being the highest in the case of acid phosphatase.