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PubMed1991

beta-Galactosidase expressing recombinant pseudorabies virus for light and electron microscopic study of transneuronally labeled CNS neurons.

A beta-galactosidase expression pseudorabies virus (Bartha strain) was constructed, injected into the adrenal gland of rats, and subsequently shown to transneuronally label the CNS autonomic neurons that project to the sympathoadrenal preganglionic neurons. Virally infected neurons were visualized with a one-step histochemical reaction using the Bluo-Gal substrate (halogenated indolyl-beta-D-galactoside) for the localization of beta-galactosidase activity. In some infected neurons, a Golgi-like staining of the primary and sometimes secondary dendrites could be obtained. For electron microscopic studies, the Bluo-Gal substrate produces an electron-dense reaction product that is easily identified at both low and high magnification. This virus may be useful for the study of the cell architecture and synaptic organization of transneuronally labeled neurons of functionally defined neural circuits. These results also demonstrate that it is possible to deliver foreign genes into specific chains of neurons in the mammalian CNS by means of the retrograde transneuronal vial labeling method.

Adrenal Glands↗
PubMed1991

Immobilisation of beta-D-galactosidase from Escherichia coli on cellulose beads and its use for the synthesis of disaccharide derivatives.

beta-D-Galactosidase, isolated from cloned E. coli, was immobilised on cellulose beads via oxidation with sodium periodate, activation by cyanuric chloride, or diazotisation. beta-D-Galactosidase immobilised via azo bonds showed the highest relative activity and thermostability, and was used for synthesis of disaccharide methyl glycosides.

Cellulose↗
PubMed1993

beta-Galactosidases of Escherichia coli with substitutions for Glu-461 can be activated by nucleophiles and can form beta-D-galactosyl adducts.

Nucleophiles activated the catalytic actions of beta-galactosidases with neutral or positively charged substitutions for Glu-461. Aliphatic carboxylic acids increased the rate of hydrolysis of o-nitrophenyl beta-D-galactopyranoside if the pKa values of the carboxyl groups were > approximately 3.5. Amino compounds activated if their pKa values were < approximately 8.5. Imidazole, azide, and 2-mercaptoethanol also activated. Nucleophiles with high pKa values were able to activate the catalysis if the pH was high, and this showed that the lack of activation at pH 7.0 was because of protonation. Kinetic analysis showed that most of the nucleophiles that activated were bound to the active site, since the activation followed Michaelis-Menten type saturation kinetics. The binding seemed to be dependent upon the hydrophobicity; the longer the aliphatic chain, the stronger the binding. Gas-liquid chromatographic analysis showed that adducts of some type were formed during the reactions in the presence of many of the nucleophiles. Three of these adducts were purified and the nucleophiles were found beta-linked to D-galactose. This indicates that if an intermediate covalent bond is formed in the mechanism of beta-galactosidase action and if the nucleophile reacts to displace it, the intermediate covalent bond must have the alpha configuration and involve a group other than Glu-461.

Carbohydrate Sequence↗
PubMed1993

Suicide-substrate inactivation of beta-galactosidase by diazomethyl beta-D-galactopyranosyl ketone.

Diazomethyl beta-D-galactopyranosyl ketone (1) has been proven to be a mechanism-based, irreversible (suicide-substrate) inactivator of Aspergillus oryzae beta-D-galactosidase, but not an inactivator of E. coli lacZ beta-D-galactosidase. Compound 1 is stable in buffers of normal physiological pH. It is decomposed by H+, but not by nucleophiles. Inactivation of A. oryzae beta-D-galactopyranosyl ketone (2) nor diazomethyl alpha-D-galactopyranosyl ketone inactivated the enzyme and therefore inactivation is stereospecific, excess inhibitor could be separated from inactive enzyme without regain of activity and therefore it is bound irreversibly, and a second pulse of enzyme is inactivated at the same rate as enzyme inactivated to 95% activity by the first pulse. Diazomethyl beta-D-glucopyranosyl ketone (2) inhibited sweet almond beta-D-glucosidase.

Aspergillus oryzae↗
PubMed1994

Regioselective transglycosylation in the synthesis of oligosaccharides: comparison of beta-galactosidases and sialidases of various origins.

N-Acetyl-lactosamine(beta-D-Gal p-(1-->4)-D-Glc pNAc) was synthesized regioselectively with the aid of the transglycosylation activity of beta-galactosidase isolated from Diplococcus pneumoniae using p-nitrophenyl beta-D-galactopyranoside as the donor. Also, transglycosylation of the sialyl group in an alpha-(2-->8)-linked sialic acid dimer or p-nitrophenyl glycoside of sialic acid to N-acetyl-lactosamine was performed using sialidases of various origins. When sialidase from Clostridium perfringens, Arthrobacter ureafaciens, or Vibrio cholerae was used, alpha-(2-->6)-linked sialyl N-acetyl-lactosamine was obtained regioselectively. In contrast, when sialidase from newcastle disease virus was used, the alpha-(2-->3)-linked isomer was obtained regioselectively. The regioselectivity of the transglycosylation reaction using beta-galactosidase and sialidase was compared with hydrolysis specificity toward the same linkages.

Amino Sugars↗
PubMed1982

Serum beta-N-acetylglucosaminidase, beta-D-glucosidase, alpha-D-glucosidase, beta-D-fucosidase, alpha-L-fucosidase and beta-D-galactosidase levels in acute viral hepatitis, pancreatitis, myocardial infarction and breast cancer.

The specific activities of several glycosidases (beta-N-acetylglucosaminidase, beta-D-glucosidase, alpha-D-glucosidase, beta-D-fucosidase, alpha-L-fucosidase and beta-D-galactosidase) were determined in human sera from a control group to 10 normal subjects and in four groups, each of 10 patients, with acute viral hepatitis, acute pancreatitis, acute myocardial infarction and breast cancer. The results show significantly higher activities in acute viral hepatitis for beta-N-acetylglucosaminidase, beta-D-glucosidase and alpha-D-glucosidase (p less than 0.001); in acute pancreatitis for the first two of these enzymes (p less than 0.001); and in breast cancer for beta-D-glucosidase (p less than 0.001). Further, lower differences were found in the patients with acute viral hepatitis for beta-D-fucosidase and alpha-L-fucosidase (p less than 0.01); in acute myocardial infarction for beta-N-acetylglucosaminidase, beta-D-glucosidase, alpha-D-glucosidase, beta-D-fucosidase and beta-D-galactosidase (p less than 0.01, p less than 0.05, p less than 0.05, p less than 0.01 and p less than 0.01, respectively); and in breast cancer for beta-N-acetylglucosaminidase (p less than 0.01). No significant differences were found for the other glycosidases.

Acetylglucosaminidase↗
PubMed1994

Enzyme-aided construction of medium-sized alditols of complete O-linked saccharides. The constructed hexasaccharide alditol Gal beta 1-4GlcNAc beta 1-6Gal beta 1-4GlcNAc beta 1-6(Gal beta 1-3)GalNAc-ol resists the action of endo-beta-galactosidase from Bacteroides fragilis.

We have constructed by enzyme-aided in vitro synthesis a hexasaccharide alditol Gal beta 1-4GlcNAc beta 1-6Gal beta 1-4GlcNAc beta 1-6(Gal beta 1-3) GalNAc-ol and shown that it resists the action of endo-beta-galactosidase from Bacteroides fragilis under conditions where a related pentasaccharide alditol, GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-6(Gal beta 1-3)GalNAc-ol, was completely cleaved. Together with earlier results from this laboratory, our present data imply that endo-beta-galactosidase from B. fragilis, apparently, can be used to distinguish between GlcNAc beta 1-6Gal and GlcNAc beta 1-3Gal units within linear backbone sequences of all known types of oligo-(N-acetyllactosamino)glycans.

Bacteroides fragilis↗
PubMed1995

Beta-glucosidase, beta-galactosidase, family A cellulases, family F xylanases and two barley glycanases form a superfamily of enzymes with 8-fold beta/alpha architecture and with two conserved glutamates near the carboxy-terminal ends of beta-strands four and seven.

Comparison of the recently determined crystal structures Pseudomonas fluorescens subsp. cellulosa family F xylanase, (1-3)-beta-glucanase and (1-3,1-4)-beta-glucanase and the catalytic domain of E. coli beta-galactosidase reveals that they belong to a superfamily of 8-fold beta/alpha-barrels with similar amino acid residues at their active sites. In the three families that these enzymes represent, the nucleophile is a glutamate, which is located close to the carboxy-terminus of beta-strand seven. In addition all three enzymes have the sequence asparagine-glutamate close to the carboxy-terminus of beta-strand four. This glutamate has been identified as the acid/base in the family F xylanases and is essential for catalysis in beta-galactosidase. We suggest that the equivalent residue in the barley glucanases is the acid/base. Analysis of the sequences of family 1 beta-glucosidases and family 5 cellulases shows that these enzymes also belong to this superfamily which we call the 4/7 superfamily.

Amino Acid Sequence↗
PubMed1983

A beta-galactosidase linked immunoassay for the analysis of antigens on individual cells.

Antibodies conjugated to enzymes such as horseradish peroxidase or alkaline phosphatase are widely used to detect antibody binding to individual cells or tissue sections through the deposit of insoluble colored reaction products. However, the presence of endogenous enzyme activity, especially in lymphomyeloid cell populations, necessitates the use of inhibitors which can be shown to decrease the sensitivity of the assay, a particular problem when monoclonal antibodies are used. As no endogenous beta-galactosidase activity can be demonstrated in lymphomyeloid cells, a cytoimmunoenzyme assay based on this enzyme should provide a preferable system for cytological investigations of antigens in these cells. Such as assay was developed using azo coupling of 2 alternative substrates with different diazonium salts to produce rapid and specific precipitation of reaction products of different colors. The sensitivity of the beta-galactosidase cytoimmunoenzyme assay was shown to be comparable with that of assays using FITC or peroxidase coupled antibodies.

Animals↗
PubMed1991

Synthetic peptide conjugates with horseradish peroxidase and beta-galactosidase for use in epitope-specific immunocytochemistry and ELISA.

Synthetic peptide-alkaline phosphatase conjugates can be used to detect the epitope specificity of (i) antibody-forming cells in vivo by immunocytochemistry; (ii) of antibody secreting cells in vitro by spot-ELISA; and (iii) antibodies in solution by capture ELISA. The availability of synthetic peptide-enzyme conjugates using detector enzymes other than alkaline phosphatase would offer several important advantages, for example in double staining approaches. This paper reports the production of synthetic peptide-horseradish peroxidase conjugates and synthetic peptide-beta-galactosidase conjugates. A peptide of 21 amino acids (SP 29) was coupled to peroxidase in seven differing molar ratios of peptide over peroxidase, ranging from 1:3.4 to 1:575, using periodate oxidation of the enzyme. SP 29 was coupled to beta-galactosidase in four molar ratios ranging from 1.25 to 10, using glutaraldehyde pre-activation of the enzyme. The enzyme activity of the different conjugates was determined, the conjugates were tested in direct capture-ELISA with peptide-specific monoclonal antibodies, and the conjugates were tested in immunocytochemistry to detect peptide-specific B cells. The results show that the conjugates perform best if the peptide is coupled to the enzyme at relatively low molar ratios (1-30). The availability of these new peptide-enzyme conjugates broadens the applicability of synthetic peptides for detection purposes in several assay systems.

Animals↗
PubMed1992

An in vivo study of a glycoprotein gIII-negative bovine herpesvirus 1 (BHV-1) mutant expressing beta-galactosidase: evaluation of the role of gIII in virus infectivity and its use as a vector for mucosal immunization.

We constructed a recombinant BHV-1 in which the glycoprotein gIII gene was replaced by the Escherichia coli lacZ gene. The resultant virus mimics the simple gIII deletion mutant in its growth characteristics in cell culture; however, it expresses beta-galactosidase in virus-infected cells. Further characterization of its virulence and the immune responses elicited by it was conducted in cattle. The mutant virus retained the ability to establish an infection when administered intranasally. Infected animals were also capable of transmitting virus to sentinel penmates. However, the mutant virus showed a reduced replication efficiency in the respiratory tract of cattle, as manifested by significantly lower virus shedding and a shorter duration of shedding when compared to wild-type (wt) BHV-1 infections. The mutant virus induced an efficient anti-BHV-1 antibody response and convalescent cattle were fully protected from subsequent wt virus challenge. In addition, cattle infected with the lacZ-expressing virus developed antibodies to beta-galactosidase. Our results demonstrate that the presence of gIII is not a prerequisite for BHV-1 infection; however, gIII does play an important role in maintaining virus replication efficacy in its natural host. With respect to developing BHV-1 as a vaccine vector, our results indicate that deletion of the gIII gene, which partially attenuates the virus and serves as a vaccine virus marker, does not compromise immunogenicity to BHV-1. Most importantly, this vector is effective in delivering foreign antigens to mucosal surfaces of the respiratory tract.

Animals↗
PubMed1976

A galactosidase immunosorbent test for human immunoglobulin E.

We report here the development of a galactosidase-immunosorbent test (GIST) for immunoglobulin E (IgE) antibodies in which the amount of galactosidase adsorbed to a cellulose disc is a single valued function of IgE concentration in human serum. Rabbit anti-IgE immunoglobulin insolubilized on cellulose discs is incubated sequentially with human serum, sheep anti-IgE serum, and a covalent conjugate of rabbit antisheep immunoglobulin with the enzyme beta-D-galactoside galactohydrolase (E.C.) 3.2.1.23). Colorimetric assay of enzyme conjugate adsorbed to discs permits quantitation of 1.0 to 25 ng of IgE per test. Concentrations of IgE in 48 sera as measured by the GIST gave a linear correlation coefficient of 0.97 with IgE concentrations as determined by radioimmunoassay. Preliminary studies indicate that the GIST makes possible nonisotopic measurement of ragweed-specific IgE antibiotics in human serum. The GIST for IgE is simple to perform and requires neither short-lived radioisotopes, expensive scintillation detection equipment, nor scarce, purified IgE.

Allergens↗
PubMed1983

The replication initiator protein of plasmid R6K tagged with beta-galactosidase shows sequence-specific DNA-binding.

We have tagged the replication initiator protein of the plasmid R6K near the C-terminal end by fusion, in the correct reading frame, with the 89 amino acid long N-terminal alpha-donor polypeptide of beta-galactosidase of E. coli. This fusion was carried out with recombinant DNA methods. The protein chimera thus generated retained the activities of both initiation of DNA replication in vivo at the replication origin gamma of R6K and hydrolysis of beta-galactopyranoside when complemented in vivo with the alpha-acceptor polypeptide coded by the lac Z gene containing the M15 deletion. Using the simple and convenient assay for detecting beta-galactosidase, we have partially purified the tagged replication initiator, and have demonstrated that the protein binds to specific DNA sequences of the R6K chromosome. The protein bound to DNA sequences located at two places in the 5' untranslated leader region of the initiator protein cistron.

Bacterial Proteins↗
PubMed1992

A straight correlation between mutagenic activity and beta-galactosidase activity induced by monofunctional alkylating agents.

Eight monofunctional alkylating agents were examined for their ability to induce mutation in Salmonella typhimurium. The assay was carried out in S. typhimurium TA100 with the preincubation method. The SN1-type agents were more mutagenic than the SN2-type ones; besides, methylating agents exerted more mutagenic activity than ethylating ones. Those responses in the reversion assay were quite similar to the results obtained previously with the beta-galactosidase assay in Escherichia coli CSH26/pMCP1000 (alkA'-lacZ') as to the induction of the adaptive response. A good correlation was found between mutagenic potency in the reverse mutation assay and inducing potency in the beta-galactosidase assay.

Alkylating Agents↗
PubMed1993

Rapid identification and quantitation of cells infected by recombinant herpesvirus (pseudorabies virus) using a fluorescence-based beta-galactosidase assay and flow cytometry.

We recently described construction and use of a beta-galactosidase expression cassette in isolating recombinant pseudorabies virus (PrV) mutants (Mettenleiter and Rauh, 1990). We report here the identification and exact quantitation of cells infected by these mutants using an assay based on the reaction of intracellular beta-galactosidase expressed during infection by the recombinant viruses with the fluorogenic substrate fluorescein di-beta-D-galactopyranoside (FDG) followed by detection of positive cells in flow cytometry (FACS-Gal assay; Nolan et al., 1988). The detection method is fast, sensitive, and reliable, and yields quantitative results on single cell basis.

Animals↗
PubMed1996

Inhibition of Escherichia coli beta-galactosidase by 2-nitro-1-(4,5-dimethoxy-2-nitrophenyl) ethyl, a photoreversible thiol label.

1-Nitro-2-phenylethene (beta-nitrostyrene, 1) which is a thiol-protecting reagent (Jung, G., Fouad, H. and Heusel, G. (1975) Angew. Chem. Int. Ed. Engl. 14, 817-818), was demonstrated in this work to be an irreversible inhibitor of beta-galactosidase (EC 3.2.1.23), an enzyme known to be inhibited by some thiol reagents or through modifying a methionine residue at the active site. No reversal of the inhibition was observed upon subsequent incubation with mercaptoethanol or irradiation (350 nm). 1-(4,5-dimethoxy-2-nitrophenyl)-2-Nitroethene 2) was also shown to be an irreversible inhibitor (94% inhibition, pH 8.3) of the enzyme. Kcat values of beta-galactosidase at pH 8.3 with o-nitrophenyl beta-D-galactopyranoside (ONPG) as the substrate and at the highest inhibitor concentrations employed for compound 1 (4.06 x 10(-4) M) ranged from 1.67 x 10(4) S-1 after 30 min of preincubation to <0.07 x 10(4) S-1 after 180 min preincubation. For compound 2 (9.5 x 10(-5) M) Kcat values ranged from 2.70 x 10(4) S-1 following 30 min preincubation to 1.15 x 10(4) S-1 after 180 min of preincubation; the changes in Km(app), however, were small. The activity was not recovered following incubation with mercaptoethanol. Since compound 2 and the inhibited enzyme are 2-nitrobenzyl derivatives, they are expected to be photosensitive and indeed, irradiation of the inhibited enzyme in the presence of mercaptoethanol resulted in recovery (89%, pH 8.3) of the enzyme activity.

Enzyme Inhibitors↗
PubMed1991

On-line determination of intracellular beta-galactosidase activity in recombinant Escherichia coli using flow injection analysis (FIA).

A flow injection analysis (FIA) system was developed for the determination of cytoplasmic beta-galactosidase activity in recombinant Escherichia coli. The FIA system and its application for on-line monitoring of beta-galactosidase production during cultivation of recombinant E. coli in a 60-l airlift tower loop reactor is described. The results demonstrate that an FIA assay in conjunction with a cell disintegration step can be applied successfully for on-line monitoring of intracellular protein formation.

Colorimetry↗
PubMed1983

Lysosomal sialidase deficiency in sialidosis with partial beta-galactosidase deficiency.

We analyzed the subcellular localization of sialidases in human lymphocytes from a patient with adult type sialidosis with partial beta-galactosidase deficiency and normal controls. Sialidase activities were measured with alpha,2 leads to 3 NeuAc-lactitol, 4-methylumbelliferyl-NeuAc and GM3 ganglioside as substrates. Sialidases in the lysosomes were sonication-labile and hydrolyzed mainly hydrophilic substrates such as NeuAc-lactitol and 4-methylumbelliferyl-NeuAc, but hydrolyzed subsidiarily GM3 ganglioside. On the other hand, sialidases in the plasma membrane were sonication-stable and hydrolyzed both hydrophilic substrates and GM3 ganglioside. In sialidosis with partial beta-galactosidase deficiency, the sialidases of the lysosomes showed 3-5% activity toward hydrophilic substrates and 25% activity toward GM3 ganglioside as compared with sialidase activities of the controls. However, there are no differences in the activities of the sialidases in the plasma membrane. These results demonstrate that the essential defect in this disease is the deficiency of a lysosomal sialidase.

Carbohydrate Metabolism, Inborn Errors↗