Multiple carbohydrate-cleaving specificities in human acidic and neutral glycosidases.

The common identity of human acidic beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) and beta-D-xylosidase (1,4-beta-D-xylan xylohydrolase, EC 3.2.1.37) as one enzyme and that of acidic beta-D-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23), beta-D-fucosidase (no allotted EC number) and alpha-L-arabinosidase (alpha-L-arabinofuranoside arabinohydrolase, EC 3.2.1.55) as another enzyme is indicated by similar binding patterns of glycosidase activities of each enzyme to various lectins. by similar ratios between their intra- and extracellular levels in normal and I-cell fibroblasts and by their deficiencies in liver tissues from patients with Gaucher disease and GM1 gangliosidosis, respectively. A third enzyme, neutral beta-D-galactosidase, purified to homogeneity from human liver has been shown to possess all these five glycosidase activities at neutral pH. These neutral enzymic activities were not bound by any of the lectins examined and found to be reduced in liver and spleen of a patient with neutral beta-D-galactosidase deficiency. An additional form of beta-D-xylosidase with optimal activity at pH 7.4 was bound by the fucose-binding lectin from Ulex eurpaeus while no binding was observed for the acidic (pH 4.8) and neutral (pH 7.0) beta-D-xylosidase activities of the multiple glycosidase enzymes.     

Characterization of beta-D-galactosidase isolated from I-cell disease liver.

The residual beta-D-galactosidase activity (10% of normal) present in an autopsy sample of liver derived from an I-cell patient has been characterized. The pH optima for both I-cell and normal acid 4-methylumbelliferyl beta-D-galactoside activities were 4.35. The adsorption and elution profiles of the I-cell enzyme from Con A-Sepharose were similar to those of normal liver beta-D-galactosidase. Although starch gel electrophoresis revealed the presence of beta-D-galactosidase A and B in I-cell disease liver, the A band was more diffuse and migrated less anodally than the A band from normal liver. The electrophoretic mobilities of both I-cell and normal beta-D-galactosidase A appeared to decrease after treatment with neuraminidase. Kinetic studies of the I-cell and normal level beta-D-galactosidase demonstrated similar apparent Km values with respect to the 4-methylumbelliferyl beta-D-galactoside and Gm1 ganglioside, whereas the Vmax values obtained for the I-cell enzyme were 10- to 12-fold lower than those of the normal enzyme for both substrates.     

Urinary lysosomal hydrolases in mucolipidosis II and mucolipidosis III.

Investigation of the binding characteristics of acid beta-D-galactosidase, N-acetyl-beta-D-glucosaminidase, alpha-D-galactosidase and alpha-L-fucosidase from patients with mucolipidosis II and mucolipidosis III to concanavalin A--Sepharose 4B revealed a 2--10-fold decrease in the proportion of enzyme activities from patients with mucolipidoses II and III that adsorbed on the lectin. Neuraminidase treatment of the unadsorbed enzyme fraction did not significantly increased the proportion of enzyme activities that bound to the concanavalin A--Sepharose 4B. Characterization of acid beta-D-galactosidase from the adsorbed and unadsorbed enzyme fractions of mucolipidosis II and mucolipidosis III patients demonstrated identical apparent Km values of 0.22 mM with respect to 4-methylumbelliferyl beta-D-galactopyranoside, altered pH--activity profiles and heterogeneous isoelectric-focusing patterns. The results of this study support the suggestion of an alteration of a post-translational modification (possibly glycosylation) occurring in mucolipidosis II and mucolipidosis III common to the lysosomal hydrolases that affects the mannoserelated properties of these enzymes.     

Plant and algal interference in bacterial beta-D-galactosidase and beta-D-glucuronidase assays.

Several commonly occurring freshwater and marine plants and algae were screened for beta-D-galactosidase and beta-D-glucuronidase activities by using a 60-min enzyme assay based on the hydrolysis by these enzymes of 4-methylumbelliferyl-beta-D-galactoside and 4-methylumbelliferyl- beta-glucuronide, respectively. All freshwater plant extracts tested showed beta-D-galactosidase activity several at relatively high levels, and a number also showed beta-D-glucuronidase activity. A number of the macroalgae showed no activity of either enzyme, but those showing beta-D-galactosidase activity also showed beta-D-glucuronidase activity. The majority of microalgae showed some beta-D-galactosidase activity, but few showed beta-D-glucuronidase activity. Further studies, using the commercial Colilert test and the marine water formulation of Colilert, revealed that 2 of 11 of the microalgal species and several of the plant extracts tested caused positive reactions. It was concluded that several plant extracts and algae could significantly interfere with the detection of coliform bacteria and Escherichia coli with the use of rapid assays, on the basis of their production of beta-D-galactosidase and beta-D-glucuronidase, respectively. The significance of the plant and algal interferences in tests such as Colilert is dependent on the levels of enzymes released under natural conditions, the dilution which they may undergo, and the numbers of algal cells present. This also applies to interferences in rapid enzyme assays.(ABSTRACT TRUNCATED AT 250 WORDS)     

I-Cell disease: isoelectric focusing, concanavalin A-Sepharose 4B binding and kinetic properties of human liver acid beta-D-galactosidases.

Isoelectric focusing of the acid beta-D-galactosidases (beta-D-galactoside galactohydrolase, EC 3.2.1.23) in normal crude liver supernatant fluids demonstrated multiple isoelectric forms in the pH range 4.58-5.15, while corresponding I-cell disease samples showed an absence of isoelectric forms in the pH range 4.99-5.15. Concanavalin A-Sepharose 4B chromatography of the I-cell disease mutant C.A. demonstrated a 31% and 37% decrease in the binding of 4-methyl-umbelliferyl-beta-D-galactosidase and GM1 beta-D-galactosidase activities, respectively, when compared to normal samples. Isoelectric focusing profiles of the concanavalin A-Sepharose 4B alpha-methyl-D-mannoside effluents containing normal and I-cell disease acid beta-D-galactosidase were generally similar, but the unadsorbed I-cell disease enzyme from concanavalin A-Sepharose 4B demonstrated more activity in the pH range 4.21-4.49 than normals. Normal and I-cell disease acid beta-D-galactosidase "A" and "B", separated by gel column chromatography were found to have similar properties with respect to apparent molecular weights pH vs. activity profiles and apparent Km values for the 4 methylumbelliferyl-beta-D-galactopyranoside, GM1-ganglioside and asialofetuin (ASF) substrates. However, the apparent V values for the ICD samples were consistently reduced when compared to the results obtained with the corresponding normal fractions. The greatest decreases in apparent V were obtained for acid beta-D-galactosidase activities in I-cell disease crude supernatant fluids, and for the separated I-cell disease "B" enzyme. The differences in the isoelectric focusing profiles, the altered binding to concanavalin A-Sepharose 4B, and the reduced V values with natural and synthetic substrates may be related to changes in carbohydrate composition of I-cell disease acid beta-D-galactosidase.     

beta-Fucosidase, beta-glucosidase and beta-galactosidase activities associated in bovine liver

1. beta-D-Fucosidase, beta-D-glucosidase and beta-D-galactosidase activities from bovine liver are associated in a single peak in isoelectric focusing. The isoelectric point is 4.35 for all these activities, suggesting that they are catalyzed by the same enzyme. 2. This enzyme shows the optimal pH in the range 4.5-6.5 for all the above mentioned activities. 3. The Km and Vmax are 0.26 mM and 31 mU mg-1, 0.10 mM and 24 mU mg-1, and 0.30 mM and 20 mM mg-1 for the p-nitrophenyl-fucoside, -glucoside and -galactoside, respectively. The glucoside derivative is the best substrate, with a Vmax/Km value of 0.24 ml . mg-1 . min-1. 4. The Lineweaver-Burk profiles are convex upward in most cases, suggesting a substrate-activation model, and the presence of more than one binding site in the enzyme. 5. The Ki for all the activities were determined with D-fucose, glucose and galactose as inhibitors. D-Fucose is the strongest inhibitor. The inhibition is competitive in all cases.     

Beta-D-glycosidase activities of Humicola grisea: biochemical and kinetic characterization of a multifunctional enzyme

A beta-D-glycosidase activity was purified from mycelium of Humicola grisea var. thermoidea grown on avicel as the main carbon source. The purified enzyme was a glycoprotein and migrated as a single polypeptide band on polyacrylamide gel electrophoresis under native or denaturing conditions. The apparent molecular weight of the enzyme was estimated to be 55 kDa by gel filtration and SDS-PAGE. The enzyme was active against o-nitrophenyl beta-D-galactoside; p-nitrophenyl beta-D-glucoside, p-nitrophenyl beta-D-fucoside, lactose and cellobiose, PNP fucoside (synthetic substrate) and cellobiose (natural substrate) being the best utilized. A comparison of the properties of beta-D-galactosidase, beta-D-glucosidase and beta-D-fucosidase showed that three activities exhibited similar pH and temperature optima and the same thermostability. The hydrolysis rate of substrate mixtures suggests that the enzyme possesses a common catalytic site for all the substrates assayed.     

Acid glycohydrolase in Chinese hamster with spontaneous diabetes. VII. The lack of short-term glucose-effect in cultured kidney cells

An epithelial cell line, designated CHK-ACE, was established from the kidney of a spontaneously diabetic Chinese hamster from the highly inbred AC line. CHK-ACE was separated into two sublines, CHK-ACE-100 and CHK-ACE-400, by successive passages in 100 and 400 mg/dl glucose respectively. Extra- and intracellular activities of N-acetyl-beta-D-glucosaminidase and beta-D-galactosidase were measured in these cultures after exposure to varying concentrations of glucose (100, 200, 300 and 400 mg/dl) for one passage and 10% heated fetal calf serum for 6.5 h before enzyme measurements were taken; no apparent dependence on medium-glucose concentration was found. In serum-free medium, the time-dependent release of both N-acetyl-beta-D-glucosaminidase and beta-D-galactosidase was sustained for up to 24 h; no significant difference in their activities was found between CHK-ACE-100 cultures grown in 100 and 400 mg/dl glucose for one passage.     

Detection of one milliattomole of ferritin by novel and ultrasensitive enzyme immunoassay

A novel and ultrasensitive enzyme immunoassay (immune complex transfer two-site enzyme immunoassay) for ferritin is described. Ferritin was reacted simultaneously with affinity-purified dinitrophenyl biotinyl anti-ferritin IgG and affinity-purified anti-ferritin Fab'-beta-D-galactosidase conjugate. The complex formed of the three components was trapped onto affinity-purified (anti-dinitrophenyl group) IgG-coated polystyrene balls. After eliminating excess conjugate by washing, the complex was eluted from the polystyrene balls with an excess of epsilon N-dinitrophenyl-L-lysine and transferred to streptavidin-coated polystyrene balls. The beta-D-galactosidase activity bound to streptavidin-coated polystyrene balls was assayed by fluorometry. Nonspecifically bound beta-D-galactosidase activity was remarkably lowered but there was much less decrease in specifically bound beta-D-galactosidase activity. As a result, the detection limit of ferritin was lowered to 1 milliattomole (1 x 10(-21) mol, 600 molecules as calculated from Avogadro's number). This technique will be useful for measuring, for example, antigens in single cells.     

Enzymic activities in the seminal plasma of normospermic, oligospermic and infertile azoospermic men

Activities of seminal plasma alpha-D-glucosidase, beta-D-galactosidase, alpha-amylase, chymotrypsin-like enzyme and RNAase were studied in normo spermic, oligospermic and non-obstructive azoospermic men. No significant change in the activities of the aforesaid enzymes were found amongst the three catagories, classified according to sperm density.     

Characterization and kinetics of beta-D-gluco/fuco/galactosidase from sheep liver

This enzyme shows beta-D-glucosidase, beta-D-fucosidase and beta-D-galactosidase activities, all associated in a single peak in Sephadex G-200, DEAE-cellulose, concanavalin A-Sepharose chromatographies, and in high resolution isoelectric focusing (pI 4.56), having the optimal pH in the range 4.5-5.5. The enzyme is very stable under different conditions: (i) at pH in the range 5.5-7.0; (ii) in successive freezing-thawing cycles; (iii) at 4 degrees C; (iv) after exhaustive ultrasonic treatment. It is not stable beyond 40 degrees C, and in the presence of urea, Triton X-100, SDS or mercaptoethanol. HgCl2, KCN, Tris, maltose and the lactones were inhibitors of the enzyme. With glucose, fucose and galactose the inhibition is competitive. In addition, a transglycosylation mechanism seems to occur. The kinetic studies suggest a substrate-activation model and the presence of two primary active sites: fuco-gluco and galacto.     

Changes in glycosidase activities during galactoglucomannan oligosaccharide inhibition of auxin induced growth

The inhibition of 2,4-D-induced elongation growth by galactoglucomannan oligosaccharides (GGMOs) in pea stem segments (Pisum sativum L. cv. Tyrkys) after 18 h of incubation results in changes of extracellular, intracellular and cell wall glycosidase activities (beta-D-glucosidase, beta-D-mannosidase, beta-D-galactosidase, beta-D-xylosidase, alpha-D-galactosidase, and alpha-L-arabinosidase). GGMOs lowered the glycosidase activities in the extracellular fraction, while in the cell wall fractions their activities were markedly increased. The intracellular enzyme alpha-d-galactosidase increased while the beta-d-galactosidase decreased in activity in response to the GGMO treatment. Extracellular enzymes showed low values of activities in comparison with intracellular and cell wall glycosidases. It is evident that GGMOs can alter auxin induced elongation and glycosidase activities in different compartments of the cell, however, the mode and site of their action remains unclear.     

Change in intracellular activity and secretion of various lysosomal glycosidases of human fibroblasts cultured in medium with sucrose

Intracellular activation of lysosomal glycosidases from human skin fibroblasts (alpha-L-fucosidase, beta-D-hexosaminidase, beta-D-galactosidase and beta-D-glucuronidase) was shown to occur on the 3rd-6th days of cultivation in media containing 0.04 M sucrose. The increase in the enzyme activity ranged from 40 to 300% depending on cell strain, nature of enzyme and cultivation time. Among pre- and postnatal fibroblast strains, those with a high and low response to sucrose load were identified. The maximal intracellular activation was observed in beta-D-galactosidase, the minimal one--in beta-D-glucuronidase. In pathological cells (Krabbe's disease) the highest activation by sucrose load was observed, as in normal cells, with beta-D-galactosidase, whereas the lowest one--with beta-D-glucuronidase. Secretion of lysosomal glycosidase is selective and noncoordinated. The maximal secretion of alpha-L-fucosidase and beta-D-hexosaminidase was observed within the first 24 hours (intensive sucrose endocytosis), but was considerably decreased at later times, i. e., by the 3rd and 6th days. The enzymes secreted during the 1st and 3rd days differed significantly in stability (37 degrees C, pH 7.0).     

Biosynthesis, processing, and subcellular localization of rat spermbeta-D-galactosidase

During spermatogenesis, spermatids synthesize constituent proteins present in mature spermatozoa; however, little information exists on the molecular processes involved. In previous studies, this laboratory reported the characterization of rat sperm beta-D-galactosidase. In this paper, we report the localization of this enzyme along with its biosynthesis and processing. An antibody against rat luminal fluid beta-D-galactosidase was used to immunolocalize the enzyme in the testis and in epididymal spermatozoa. We found that beta-D-galactosidase is localized within the acrosomal cap of spermatids and in the acrosome and cytoplasmic droplet of epididymal spermatozoa. A combination of germ cell radiolabeling, immunoprecipitation, SDS-PAGE, and autoradiography revealed that spermatids produce two forms of beta-D-galactosidase, 90 and 88 kDa. During pulse-chase analysis, a 56-kDa form appeared. Treatment of beta-D-galactosidase immunoprecipitates from testicular spermatozoa with N-glycanase or Endo H revealed that both the 90- and 88-kDa forms become a 70-kDa polypeptide on SDS-PAGE. Since Endo H or N-glycanase treatment provided similar results, the presence of extensive N-linked high mannose/hybrid-type glycans on these proteins is indicated. Treatment of the 56-kDa form of beta-D-galactosidase with Endo H or N-glycanase resulted in the appearance of 52- and 50-kDa forms, respectively. This result suggests that the 56-kDa form contains N-linked high mannose/hybrid as well as complex oligosaccharides. During epididymal maturation, the 90-kDa form of beta-D-galactosidase persists in caput epididymal spermatozoa and is gradually converted to a major 74-kDa form in cauda spermatozoa. In addition to the 90- to 74-kDa forms, cauda spermatozoa show a 56- to 52-kDa form on Western immunoblots. Since only the high-molecular weight forms of beta-D-galactosidase are present on immunoblots of isolated sperm heads, we suggest that they are acrosomal in origin and that the 56-kDa form, which is processed to 52 kDa in cauda spermatozoa, is associated with the cytoplasmic droplet.     

An enzyme immunoassay for secretin

A sensitive and specific enzyme immunoassay for secretin was developed with the use of enzyme-labeled antigens. Synthetic porcine secretin and its carboxy-terminal fragments (residues 11-27 and 18-27) were conjugated with beta-D-galactosidase for use in the immunoassay, and the assay method with the latter fragment (residues 18-27) linked to beta-D-galactosidase was found to be the most sensitive. The minimum amount of secretin detectable by this method was 1-2.5 pg/assay. Serum levels of secretin after intravenous injection of the peptide in rats were determined by both the enzyme immunoassay and a commercial radioimmunoassay kit. The correlation coefficient between the levels measured by the two methods was 0.984. The enzyme immunoassay could detect immunoreactive secretin levels in normal human sera, giving a value of 16.9 +/- 2.2 pg/ml (mean +/- SE of six human subjects).     

GM1 gangliosidosis (type 1) in a cat.

A kitten with clinical and morphological symptoms of a neurovisceral lysosomal-storage disease has been shown to have a marked deficiency of acidic beta-D-galactosidase in the brain, kidney and spleen. Chromatography on concanavalin A-Sepharose and inhibition studies with 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine, a selective inhibitor of the neutral broad-specificity beta-D-galactosidase, have shown that the residual beta-D-galactosidase at pH 4.0 in the tissues of the affected cat is due to the neutral beta-D-galactosidase and that there is a complete deficiency of the acidic (lysosomal) beta-D-galactosidase. There is marked accumulation in all tissues and excretion in the urine of neutral oligosaccharides. Analysis of these oligosaccharides by fast-atom-bombardment mass spectrometry and g.l.c. suggests that they arise from the incomplete catabolism of N-glycans of glycoproteins. The ganglioside content of all the tissues is elevated, and it has been shown by t.l.c. that the concentration of a ganglioside fraction with a mobility similar to that of GM1 ganglioside is particularly increased. There is also some evidence of accumulation of glycosaminoglycans in the brain. The clinical symptoms, the complete deficiency of acidic beta-D-galactosidase and the storage products in visceral organs all suggest that this is a case of feline GM1-type gangliosidosis comparable with the severe infantile (Type 1) form of the disease in humans.     

Genetic control of immunologic unresponsiveness to adjuvant-free solutions of beta-D-galactosidase. I. Inheritance of the Ir-Z1 and ir-Z2 loci in mice.

Two genetic loci, Ir-Z1 and ir-Z2, controlling the immune response to adjuvant-free bacterial beta-D-galactosidase (Z) are present in inbred mouse strains SJL/J and CE/J, respectively. Each locus segregates as a single, autosomal gene: Ir-Z1 as dominant and ir-Z2 as recessive. The response is characterized by production of activating and precipitating IgG. Maximal levels of circulating IgG occur between 16 and 20 days after immunization with a single, 50-microgram dose of enzyme. Failure of proteins other than Z to elicit an immune response indicates that the Ir-Z control is specific for determinant(s) of this enzyme. The immunogenicity of beta-D-galactosidase preparations cannot be attributed to either the catalytic activity of the enzyme or adjuvant contamination. Non-responder mice acquire immunologic memory without detectable increase in circulating specific IgG under the same conditions that elicit antibody production in responder strains.     

Purification of jack bean meal beta-D-galactosidase by a new affinity adsorbent.

A simple procedure has been developed for the purification of jack bean beta-D-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) by affinity chromatography employing a new affinity adsorbent. The ligand 6-N-beta-(4-aminophenyl)-ethylamino-3-O-beta-D-galactopyranosyl-6-deoxy-L-gulitol was prepared by the reaction between lactose and beta-(4-aminophenyl)-ethylamine and was coupled to cyanogen bromide activated Sepharose 4B via the amino groups of the 4-aminophenyl moiety. This affinity gel resulted in a 111-fold purification of beta-D-galactosidase with a 64% recovery of the enzyme. With p-nitrophenyl-beta-D-galactopyranoside as the substrate the apparent Km and V values were 0.59 mM and 1.87 mumol/min per mg, respectively. The method for purification of beta-D-galactosidase may be applicable to other glycosidases depending upon the choice of specific di- or oligosaccharides of known structures to be used in the preparation of ligands.     

Purification and characterization of a strictly specific beta-D-fucosidase from Aspergillus phoenicis.

Although beta-D-fucosidase (beta-D-fucohydrolase, EC 3.2.1.38) has been isolated from various sources, all those enzymes were associated with a high activity of beta-D-galactosidase and/or beta-D-glucosidase. We have purified a specific beta-D-fucosidase in electrophoretically homogeneous form from crude extracts of Aspergillus phoenicis by polyethyleneglycol 8000-phosphate buffer aqueous two-phase separation, and successive chromatography on DEAE-Sephadex A-50, hydroxyapatite, and Sephadex G-100 columns. The molecular weight of the enzyme was estimated to be 57,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 50,000 to 60,000 by gel filtration on Sephadex G-100. The enzyme showed optimum activity at pH 6.0 and 40 degrees C; it was stable in the pH range 5.5-6.5 and below 35 degrees C. The Km and the Vmax values for pNP-beta -D-fucoside were 2.4 mM, and 12.8 mumol.min-1.mg-1, respectively. The enzyme was strongly inhibited by sulfhydryl group reagents, p-chloromercuribenzoate, n-ethylmaleimide, and iodoacetate. It was also inhibited by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, diethyl pyrocarbonate, and N-bromosuccinimide. Thus, -SH and -COOH groups and histidyl and tryptophyl residues were essential for enzyme activity. The purified beta-D-fucosidase showed high specificity toward p-nitrophenyl-beta-D-fucoside. The enzyme was inhibited by D-fucose and D-fucono-gamma-lactone, but not by D-galactose, D-galactono-gamma-lactone, D-glucose, or D-glucono-gamma-lactone; the latter compounds are specific inhibitors of beta-D-galactosidase and beta-D-glucosidase, respectively. Thus, this enzyme is the most strictly specific beta-D-fucosidase when compared with those previously reported.     

Specific inhibition of the antibody-mediated activation of a defective beta-D-galactosidase by circulating activating epitope-binding molecules.

Activation of a defective Escherichia coli beta-D-galactosidase by specific activating antibody is inhibited competitively by a molecule with immunoglobulin properties but devoid of activating capacity. This molecule is found in the serum of nonimmunized rabbits and is no longer detectable after beta-D-galactosidase administration, but can be demonstrated in rabbits injected with antigens other than the enzyme. The data show that the inhibitory molecule recognizes and interacts specifically with the activating epitope of the activatable enzyme and that, although unable to activate the latter, it competes with the activating antibody and inhibits activation.