Comparative studies on blood serum alpha-L-fucosidases from several mammalian species.

1. Peripheral blood serum alpha-L-fucosidases have been studied from various mammalian species: Sus scropha var domestica L. (pig), Capra hircus L. (goat), Bos taurus L. (bull, races Morucha and Charolais), Equus caballus L. (horse) and Equus asinus L. (donkey). 2. Fluorimetric and spectrophotometric procedures were used for determination of alpha-L-fucosidases. 3. alpha-L-Fucosidases were more active towards fluorescent substrates than towards chromogenic substrates. 4. pH optima values of the enzymes are: (A) 5.5 for sera from all above-mentioned species when fluorescent substrates were employed; (B) 4.0 for goat, 4.5 for bull, 5.0 for pig and 4.5-5.0 for horse and donkey sera when chromogenic substrates were used. 5. pH activity profiles are very similar for two races (Morucha and Charolais) of the same species (Bos taurus L.) and also for two species of the same genus (Equus caballus and Equus asinus L.). 6. These serum alpha-L-fucosidases are very labile under heat treatment, even at 37 degrees C.     

Comparative study on the activity, pH optimum and thermal stability of some glycosidases and acid phosphatase from pig and mule leukocytes.

1. alpha-D-mannosidase, beta-D-galactosidase, alpha-L-fucosidase, beta-N-acetylgalactosaminidase, alpha-D-glucosidase and acid phosphatase activities were studied in circulating blood leukocytes from Sus scropha var. domestica L. (pig) and Equus asinus x caballus (mule) by spectrophotometric procedures using p-nitrophenyl derivatives as substrates and three different buffer solutions. 2. The highest specific activity corresponds to acid phosphatase. The specific activities of the glycosidases, all relatively close together in all cases, were low in comparison with that of phosphatase. 3. Generally, each of the above-mentioned enzymes shows one common peak for the pH optimum between 3.5 and 6.0, except alpha-D-glucosidase, which shows two peaks. 4. The pH optima values are generally similar in three buffer solutions employed. 5. Specific activities of the studied enzymes show a parallelism in leukocytes from both pig and mule. Also, this parallelism is observed in their pH optima values. 6. Thermal stability of alpha-D-mannosidase is high whereas that of acid phosphatase is low, in both materials. For other enzymes, differences in the thermal stability was observed according to their source.     

Effect of pH on protease activities in the large intestine

The influence of pH on proteolysis in different fractions of human faeces was studied with a variety of chromogenic substrates. The pH optima of proteases in the washed cell fraction and washed particulate fraction were neutral to alkaline, whereas extra-cellular proteolysis in the cell-free supernatant fraction was relatively insensitive to pH over the range 6·4–8·0. Measurements with p -nitroanilide substrates suggested the presence of more than one elastase-like, trypsin-like and chymotrypsin-like protease in the gut.     

Stability of the fluorogenic enzyme substrates and pH optima of enzyme activities in different Finnish soils

Fluorogenic artificial substrates facilitate sensitive enzyme activity measurements for a variety of processes in soil and other environmental samples. It is possible to use in situ pH for measurements on condition that the substrates are chemically stable. We studied the stability of 12 different methyl umbellipherone (MUF) and amino methyl coumarine (AMC) derivatives used as substrates for arylsulphatase, alpha-glucosidase, beta-glucosidase, beta-xylosidase, cellobiosidase, chitinase, phosphomonoesterase (PME), phoshodiesterase (PDE), esterase, lipase and alanine- and leucine aminopeptidases (AP) over the pH range from 4.0 to 8.0 in modified universal buffer (MUB). Stability of the substrates for lipase (4-MUF-heptanoate) and esterase (4-MUF-acetate) measurements was poor, especially at the higher pH values. Chitinase substrate, 4-MUF-N-acetyl-beta-D-glucosamide, was unstable at high pH values whereas the substrate for PME activity measurement (4-MUF-phosphate) disintegrated at low pH. The other substrates and MUF and AMC standard solutions were stable over the pH range studied. The optima between pH 4 and 8 of the 11 different enzyme activities were measured in three forest and two agricultural soil samples and in one activated sludge sample. In soil, for alanine and leucine AP the pH optima were usually 7.5 or higher, for arylsulphatase, beta-glucosidase, beta-xylosidase, esterase and PDE between 4 and 5.5, and for cellobiosidase between 4 and 5. alpha-Glucosidase had an optimum below 5.5 but also exhibited high activity at pH 7. Soil-dependent variation in pH optima were observed for chitinase, esterase, PDE and PME. Enzyme activities were also measured in 0.5 M acetate buffer at pH 5.5. This buffer yielded the highest activities in all soil samples for arylsulphatase, PDE and PME.     

Properties of extracellular plasmin-like proteases of <Emphasis Type="Italic">Aspergillus ochraceus</Emphasis> micromycete

The properties of two extracellular proteases of Aspergillus ochraceus VKM F-4104D micromycete with plasmin-like activity have been studied. It has been shown that the enzymes differ in pI (5.05 and 6.83) and have similar molecular weights (about 32 and 35 kDa), pH optima (pH 9.0–10.00 at 45°C), and specificities of action on a limited set of chromogenic peptide substrates of trypsin-like proteases. According to inhibitory analysis, both enzymes belong to the serine proteases. Their properties appeared to be similar to those of the protease, protein C activator, which is the main proteolytic enzyme of A. ochraceus VKM F-4104D. Most likely, proteases of this micromyсetes are isoenzymes.     

Effects of mitogenic stimulation on lymphocyte alpha-D-mannosidases

Three types of alpha-D-mannosidase are present in human and murine lymphocytes. Their levels increased substantially when the cells were activated by T-cell mitogens, concanavalin A (Con A) and phytohaemagglutinin (PHA), and in the murine cells also by lipopolysaccharide (LPS), a B-cell mitogen. The intracellular localization of the alpha-D-mannosidases in the non-stimulated and activated murine cells was investigated by fractionation of lymphocyte lysates on colloidal silica (Percoll) and discontinuous sucrose gradients. In both types of cell, an enzyme having optimal activity at neutral pH was obtained in the cytosolic fraction and another alpha-D-mannosidase most active at an intermediate pH was obtained partly in membrane-bound form. In contrast, an acidic alpha-D-mannosidase, which was particularly elevated in the activated murine spleen cells, had a distribution in these lymphoblasts which was markedly different from that in non-stimulated lymphocytes. In the latter, the major proportion of the activity was obtained in a cytosolic fraction and the remainder in a particulate fraction of light density, whereas the enzyme in activated lymphocytes was distributed between vesicles of light and heavy density comparable with lysosomal organelles. Moreover, the acidic alpha-D-mannosidase still remained membrane bound even when cell lysates were prepared under hypotonic conditions which disrupt lysosome integrity. These results suggest that lymphocyte activation involves either stabilization of fragile lysosomes present in resting cells or de novo synthesis of lysosome-like structures. The acidic alpha-D-mannosidase present within isolated, intact lysosomes was found to be in a form, A, whereas a different form, B, was most prominent in whole-cell extracts of both types of lymphocyte.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of pH on chemiluminescence of different probes exposed to superoxide and singlet oxygen generators

The compromised optima for high intensity chemiluminescence (CL), using superoxide generators, were all above pH 9.0 for the CL probes luminol and lucigenin. With luminol the optima were at pH 9.0 and 9.4 for the generators KO₂ and hypoxanthine/xanthine oxidase (HX/XO), respectively. Lucigenin, with the same generators, produced optima at pH 9.5 and 10.0, respectively. The probe methyl-Cypridina-luciferin analogue (MCLA) produced optima closer to neutral pH, which is preferred for physiological assessments. MCLA had optima at pH 6.0, 8.7 and 9.5 with KO₂ and with HX/XO optima at pH 4.8, 6.0, 7.0 and 8.7. When CL was assessed at physiological pH, MCLA observed superoxide radicals with a sensitivity of 100- and 330-fold more than luminol or luicigenin respectively. For singlet oxygen, the sensitivity of MCLA at this pH was 45- and 5465-fold more than for the said probes respectively. H₂O₂ did not elicit CL between pH 4 and 9.5 with any of the probes and did not influence the production of superoxide or singlet oxygen when co-assessed. Therefore CL could only be obtained when enzymes were used as converters. The optima for the enzyme-conversion system horseradish peroxidase (HRP)/H₂O₂, and luminol, were at pH 8.0 and 9.2. Lucigenin and HRP/H₂O₂ also had a biphasic CL profile with optima at pH 7.4 and 9.6. MCLA and HRP/H₂O₂ had five optima, with the major ones at pH 6.1 and beyond 10. The optima for the myeloperoxidase/H₂O system were at 8.6 and beyond 10.0 when luminol and 0.15 mol/L NaBr were used. © 1997 John Wiley & Sons, Ltd.     

Rapid procedures for determination of endo-N-acetyl-alpha-D-galactosaminidase in Clostridium perfringens, and of the substrate specificity of exo-beta-D-galactosidases

Culture fluid of Clostridium perfringens hydrolyzed the synthetic, chromogenic substrates beta-Gal-(1 leads to 3)-alpha-GalNAc-1 leads to OPh and beta-Gal-(1 leads to 3)-alpha-GalNAc-1 leads to OC6H4-NO2-o or -p to beta-Gal-(1 leads to 3)-GalNAc and the aglycon. Such assays facilitated the characterization and purification of this endo-N-acetyl-alpha-D-galactosaminidase activity. This activity was purified 1200-fold by fractionation with ammonium sulfate and chromatography on columns of Sephadex-G200, DEAE-Sephadex, and hydroxylapatite. The final preparation showed activity over a broad range of pH, with an optimum at 9.0, but less-pure material had two pH optima, 4.0 and 9.0. Another assay method, which employed the synthetic, chromogenic substrates beta-Gal-(1 leads to 3)-beta-GlcNAc-1 leads to OC6H4NO2-p, beta-Gal-(1 leads to 4)-beta GlcNAc-1 leads to OC6H4NO2-p, and beta-Gal-(1 leads to 6)-beta-GlcNAc-1 leads to OC6H4NO2-p, was developed for the rapid identification of the linkage specificity of exo-beta-D-galactosidases from any source via a coupled reaction with N-acetyl-beta-D-hexosaminidase.     

Visualization of protein digestion in the midgut of the acarid mite Lepidoglyphus destructor

The ingestion of chromogenic or fluorescent substrates for protease detection enables the visualization of digestive processes in mites in vivo due to their transparent bodies. The substrates for protease detection were offered to Lepidoglyphus destructor, and the resulting signals were observed in specimens under a compound microscope. The protease activity was successfully localized using chromogenic substrates (azoalbumin, AAPpNA, SAAPFpNA, elastin-orcein, SA(3) pNA, ZRRpNA, ArgpNA, and MAAPMpNA) and fluorescent substrates (casein-fluorescein, albumin-fluorescein, AAPAMC, BAAMC, ZRRAMC, ArgAMC, and AGPPPAMC). No activity was detected using the keratin azure and BApNA substrates. In the mesodeum, trypsin-like activity generated by hydrolysis of the BApNA substrate was not observed, but the BAAMC substrate allowed the visualization of trypsin-like activity in food boli in the posterior mesodeum. The results indicate that cathepsins B, D, and G and cathepsin H or aminopeptidase-like activities are present in the midgut of L. destructor. Among these activities, cathepsin D-like activity was identified for the first time in the gut of L. destructor. All proteases mentioned are produced in the mesodeal lumen and form the food bolus together with ingested food, afterward passing through the gut to be defecated. The method used enables the visualization of protease activities in the gut of transparent animals.     

Evaluation of the use of chromogenic and fluorogenic substrates in solid-phase enzyme linked immunosorbent assays (ELISA)

Fluorogenic and chromogenic substrates were used in direct and trapping enzyme-linked immunosorbent assays (ELISA) for the detection of mouse IgG and foot-and-mouth disease virus (FMDV). The detection limits for both antigens were compared using different combinations of enzymes and substrates. Various times and concentrations of chemicals were used to obtain maximum sensitivity for both systems. Similar sensitivities were found using fluorogenic and chromogenic substrates. Tetramethyl benzidine substrate for horse-radish peroxidase enzyme conjugates was found to attain the highest sensitivity levels for chromogenic assays (0.12 ng IgG/ml and 1.0 ng/ml FMDV respectively), after 10 min incubation. Of the two fluorogenic enzyme/substrates studied, B-galactosidase was the most sensitive but required extended incubation times (2-3 h) as compared with chromogenic systems. Special microplates for fluoro-immunoassay (FIA) were compared with conventional microplates and no advantage was found to justify their use. An alkaline phosphatase anti-guinea-pig conjugate was used to confirm the equivalence of fluorogenic and chromogenic substrates in terms of sensitivity. A comparison of the amount of signal generated using various concentrations of enzyme in the absence of antigen was made for two different alkaline phosphatase conjugates to obtain theoretical sensitivity limits. One possible advantage of fluorogenic substrates is that high binding ratio can improve the confidence in discrimination of positive results.     

Effect of swainsonine on rat epididymal glycosidases

Each epididymis of control and swainsonine-fed rats (5 micrograms/ml drinking water) was divided into 5 segments, and tissue, spermatozoa and sperm-free supernatants were prepared from each segment. When levels of 3 lysosomal glycosidases and total protein were determined, the proximal cauda contained the greatest concentration of glycosidase. The specific-activity profile for beta-glucuronidase and beta-galactosidase was similar in swainsonine-fed and control rats. However, the concentration of alpha-D-mannosidase in tissue of all segments was significantly greater in swainsonine-fed rats than in age-matched controls. Enzyme activity for alpha-D-mannosidase after swainsonine treatment was significantly greater in spermatozoa from the caput, than in spermatozoa from the corpus and the cauda epididymidis. Since the alpha-D-mannosidase activity was optimal at pH 4.5 and studies with highly specific antibody to lysosomal alpha-D-mannosidase immunoprecipitated all of the alpha-D-mannosidase present in detergent extracts of epididymal tissue, spermatozoa, and sperm-free supernatant, the enzyme studied is of lysosomal origin.     

Fluorogenic substrates for assay of chymosin

The use of fluorogenic substrates with intramolecular fluorescence quenching as substrates for chymosin was studied. It was shown that chymosin hydrolyzes the Phe-Phe peptide bond. The effect of pH on the hydrolysis of substrates by chymosin was investigated. The catalytic characteristics of the hydrolysis of the fluorogenic substrates were obtained at the pH optima. The influence of dimethylformamide on chymosin activity was studied.     

alpha-D-mannosidase forms in chicken liver

Two forms (I and II) of alpha-D-mannosidase have been separated by ion-exchange chromatography on DEAE-cellulose from embryonic chicken liver. A third form (III), which is absent in embryos, was also separated from 4-day-old chickens. The optimum pH of form I is at pH 5.0. Form II is named "neutral" because it shows maximal activity at pH 6.5. The optimum pH of form III is 4.5. Forms I and III are heat-stable at 50 degrees C for 1 hr, whereas form II is very unstable under these conditions. Zn2+ and Mg2+ have been found to increase the alpha-D-mannosidase activity of forms I and II. In contrast, Co2+ increases mannosidase I activity and inhibits form II from 18-day-old embryos. alpha-Methyl-D-mannoside, N-acetyl-D-mannosamine and D-mannosamine were found to be inhibitors of both forms I and II. "Neutral" mannosidase was also inhibited by chloride. Competitive inhibition by D-mannose was also studied and Ki values are given.     

Glycosphingolipid glycosyl hydrolases and glycosidases of synchronized human KB cells.

KB cells were synchronized by a double thymidine block procedure. An investigation was made of the activities of alpha-L-fucosidase (EC 3.2.1.51), alpha-D-galactosidase (EC 3.2.1.22), beta-D-galactosidase (ec 3.2.1.23), alpha-D-glucosidase (EC 3.2.1.20), beta-D-glucosidase (EC 3.2.1.21), alpha-D-mannosidase (EC 3.2.1.24), beta-D-N-acetylgalactosaminidase (EC 3.2.1.53), and beta-D-N-acetylglucosaminidase (EC 3.2.1.52) from synchronized cultures, using appropriate artificial substrates. Ceramide glucosidase (EC 3.2.1.45) and ceramide trihexosidase levels (EC 3.2.1.47) were also investigated at various stages in the cell cycle, using appropriate glycosphingolipid substrates. Whereas each of these enzymes exhibited some activity throughout the cell cycle, peak activity (2- to 6-fold increase) occurred late in the S phase. Two molecular forms of ceramide glucosidase (optimal activity at pH 4.0 and pH 6.0) and two forms of ceramide trihexosidase (pH 4.0 and pH 7.5) were identified. Peak levels of the forms that preferred the relatively acid pH occurred earlier in the S phase of the cell cycle than those of the forms that were more active at the higher pH. The possibility that the forms with optimal activity at pH 4 are precursors of those with optimal activity at pH 6 to 7.5 is discussed. Precipitation of beta-galactosidase of synchronized KB cells with specific antibody revealed that changes in the activity of this enzyme during the cell cycle were the result of fluctuations in the amount of the enzyme.     

Nicotinamide adenine dinucleotide-dependent and nicotinamide adenine dinucleotide-independent lactate dehydrogenases in homofermentative and heterofermentative lactic acid bacteria

Three homofermentative (Lactobacillus plantarum B38, L. plantarum B33, Pediococcus pentosaceus B30) and three heterofermentative (Leuconostoc mesenteroides 39, L. oenos B70, Lactobacillus brevis) lactic acid bacteria were examined for the presence or absence of nicotinamide adenine dinucleotide (NAD)-dependent and NAD-independent d- and l-lactate dehydrogenases. Two of the six strains investigated, P. pentosaceus and L. oenos, did not exhibit an NAD-independent enzyme activity capable of reducing dichlorophenol indophenol. The pH optima of the lactic dehydrogenases were determined. The NAD-dependent enzymes from homofermentative strains exhibited optima at pH 7.8 to 8.8, whereas values from 9.0 to 10.0 were noted for these enzymes from heterofermentative organisms. The optima for the NAD-independent enzymes were between 5.8 and 6.6. The apparent Michaelis-Menten constants determined for both NAD and the substrates demonstrated the existence of a greater affinity for d- than l-lactic acid. A comparison of the specific NAD-dependent and NAD-independent lactate dehydrogenase activities revealed a direct correlation of the d/l ratios of these activities with the type of lactic acid produced during the growth of the organism.     

Some kinetic properties of polyphenol oxidase from Thymbra spicata L. var. spicata

Polyphenol oxidase (PPO) of Thymbra (Thymbra spicata L. var. spicata) was isolated by (NH₄)₂SO₄ precipitation and dialysis. A diphenolase from Thymbra plant, active against 4-methylcatechol, catechol and pyrogallol was characterized in detail in terms of pH and temperature optima, stability, kinetic parameters and inhibition behaviour towards some general PPO inhibitors. 4-Methylcatechol was the most suitable substrate, due to the lowest K_m and the biggest V_max/K_m values, followed by catechol and pyrogallol. The Thymbra PPO had maximum activity at pH 5.0, 7.0 and 8.0 with 4-methylcatechol, catechol and pyrogallol substrates, respectively. The optimum temperature of activity for Thymbra PPO was 30, 40 and 50 °C for 4-methylcatechol, catechol and pyrogallol substrates, respectively. It was found that optimum temperature and pH were substrate-dependent studied. The enzyme activity decreased due to heat denaturation of the enzyme with increasing temperature and inactivation time. Inhibition of Thymbra PPO was investigated with inhibitors such as l-cysteine and glutathione using 4-methylcatechol, catechol and pyrogallol as substrates. It was found that l-cysteine was a more effective inhibitor than glutathione owing to lower K_i. The type of inhibition depended on the origin of the PPO studied and also on the substrate used. Furthermore, the IC₅₀ values of inhibitors sudied on PPO were determined by means of activity percentage (I) diagrams.     

Purification and properties of major alpha-D-mannosidase in the luminal fluid of porcine epididymis.

A lysosomal type alpha-D-mannosidase was successfully purified by DEAE-Sephacel, Red-Amicon and Superdex 200 column chromatographies from porcine cauda epididymal fluid. The purified enzyme consisted of 63 and 51 kDa subunits at equimolar amounts. It cleaved alpha1-2 linked mannosyl residues and less but significantly cleaved alpha1-3 and alpha1-6 linked mannosyl residues in the high-mannose oligosaccharides. The optimal pH to hydrolyze oligosaccharide was in the acidic pH range (pH 3.5 approximately 4.0).Total alpha-D-mannosidase activities in the porcine epididymal fluid increased from proximal to distal caput epididymis, which maintained to cauda epididymis. At least two kinds of alpha-D-mannosidase (lysosomal type enzyme and 135 kDa alpha-D-mannosidase (MAN2B2)) were contained in the porcine epididymal fluid. The activity of the lysosomal type enzyme is much higher than MAN2B2 at the physiological pH.These results suggest that the lysosomal type alpha-D-mannosidase is the predominantly active enzyme in the luminal fluid of porcine epididymis and that it participates in the glycoprotein modification on the sperm surface during epididymal transit.     

Fluorogenic surrogate substrates for toluene-degrading bacteria--are they useful for activity analysis?

Cultivated bacterial toluene degraders use one or several of four described pathways for the aerobic degradation of this priority groundwater contaminant. To be able to identify un-cultivated toluene-degrading bacteria within enriched or natural consortia, we attempted to develop a set of staining techniques that invariably label toluene-degrading bacteria while differentiating between the different degradation pathways. In the literature, we found suggestions for pathway-specific labels of individual cells that rely on the conversion of toluene surrogates into specific colored and fluorescent products. These surrogate substrates were phenylacetylene (PA), cinnamonitrile, 3-hydroxyphenylacetylene (3-HPA), and indole. We were able to confirm that the chromogenic reactions reliably verified the pathway-specific reactions of well-characterized toluene-degrading bacterial species. However, it was most surprising to find out that three (PA, 3-HPA and cinnamonitrile) of the four supplied surrogate substrates did not lead to any product fluorescence above the cultures' autofluorescence, neither inside of cells nor in supernatants. More disturbingly, the original surrogate compound 3-HPA was inherently fluorescent and found to stain cells at intensities that depended on their states in the cell cycle. Indoxyl originating from the surrogate substrate indole was the only fluorescent product that was formed. It was detected intracellularly when the cells were sealed with para-formaldehyde, but its appearance was unrelated to the presence of expressed toluene degradation pathways. These findings were scrutinized by fluorescence spectroscopy, fluorescence microscopy, and flow cytometry. Activity and growth of the test bacteria were determined by analyzing chromosome numbers and membrane integrity. Our results contradict literature reports that propose the surrogate fluorogenic substrates for the identification of toluene degraders and the identification of specific pathways used by them.     

Intracellular serine protease-4, a new intracellular serine protease activity from Bacillus subtilis

A previously undiscovered intracellular serine protease activity, which we have called intracellular serine protease-4, was identified in extracts of stationary Bacillus subtilis cells, purified 260 fold from the cytoplasmic fraction, and characterized. The new protease was stable and active in the absence of Ca²⁺ ions and hydrolyzed azocasein and the chromogenic substrate carbobenzoxy-carbonyl-alanyl-alanyl-leucyl-p-nitroanilide, but not azocollagen or a variety of other chromogenic substrates. The protease was strongly inhibited by phenylmethylsulfonylfluoride, chymostatin and antipain, but not by chelators, sulfhydryl-reactive agents or trypsin inhibitors. Its activity was stimulated by Ca²⁺ ions and gramicidin S; its pH and temperature optima were 9.0 and 37°C, respectively. Although intracellular serine protease-4 was immunochemically distinct from intracellular serine protease-1, it was absent from a mutant in which the gene encoding the latter was disrupted.     

Extracellular amylase production by cassava-fermenting bacteria

Summary Fermentation of cassava tubers was accompanied by a gradual decrease in pH, increased amylase activity in the steep liquor, and increased microbial load and lactic acid concentration. Amylase-producing bacterial strains associated with cassava fermentation were isolated and identified asBacillus subtilis, Bacillus licheniformis andBacillus cereus. The pH optima for the partially purified enzymes of these organisms were 7.0, 5.5 and 7.5, whilst their temperature optima were 30, 37 and 80°C. There was no significant difference in amylase activities when starch, dextrin, amylopectin, glucose and maltose were used as growth substrates.