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PubMed1989

Synthesis of 2-acetamido-2-deoxy-3-O-beta-D-galactopyranosyl-D-galacto se by the sequential use of beta-D-galactosidases from bovine testes and Escherichia coli.

beta-D-Galp-(1----3)-D-GalNAc (1) was synthesised from lactose and GalNAc on a mmolar scale by transgalactosylation using beta-D-galactosidase from bovine testes. The large proportions of unwanted oligosaccharides in the product mixture were removed by treatment with beta-D-galactosidase from E. coli, which left 1, monosaccharides, and a small proportion of trisaccharides. Carbon-Celite chromatography then gave 1 in a yield of 21% based on the GalNAc added.

Acetylgalactosamine↗
PubMed1977

The effect of glycosaminoglycans on the in vitro activity of human skin fibroblast glycosphingolipid beta-galactosidases and neuraminidases.

The apparent low level of synthetic substrate beta-D-galactosidase activity in liver from patients with the Hurler-Hunter syndrome is caused by the inhibitory effect of accumulated glycosaminoglycans. We have demonstrated complete inhibition of GM1 ganglioside beta-galactosidase activity in vitro by both heparan sulfate and dermatan sulfate, but the effect on lactosylceramide and galactosylceramide hydrolysis was less marked. In contrast, lysosomal neuraminidase activity in vitro was enhanced by the addition of glycosaminoglycans. These observations are discussed in relationship to the observed storage pattern of glycosphingolipids in liver from patients with mucopolysaccharidoses.

Cells, Cultured↗
PubMed1978

Effects of macromolecular compounds on human leukocyte beta-galactosidase activity.

The enzyme properties of leukocyte beta-galactosidase and the effects of macromolecular compounds on its activity were studied in detail. It was demonstrated that various macromolecular compounds markedly influence the activity of beta-galactosidase. These phenomena should be taken into account when considering the biochemical abnormalities of tissues of patients with mucopolysaccharidoses and mucolipidoses.

Chromatography, Gel↗
PubMed1981

Highly sensitive sandwich enzyme immunoassay of human IgE with beta-D-galactosidase from Escherichia coli.

A highly sensitive sandwich enzyme immunoassay of human IgE was developed. Polystyrene balls were coated with goat anti-human IgE immunoglobulin (IgG) by physical adsorption. Goat anti-human IgE Fab' was purified by affinity chromatography and conjugated with beta-D-galactosidase from Escherichia coli. Using thus prepared anti-IgE-coated polystyrene balls and anti-IgE-beta-D-galactosidase conjugate, 0.2 mU (2 amol)--1 U of IgE per assay could be determined. When 0.1 microliter of serum per assay was used, the range of IgE levels in serum that could be determined was 2--10000 U/ml, and even 0.01 U/ml was measurable by using 20 microliters of serum. The regression equation and coefficient for correlation to radioimmunoassay were gamma (RIA) = 0.94 chi (EIA) + 18.2 and 0.96 (n = 81), respectively. The coefficients of within- and between-assay variations ranged from 5.4 to 8.5%. The mean levels of serum IgE determined by the present assay were 103 U/ml in 70 normal children and 1064 U/ml in 38 children with bronchial asthma.

Antibody Specificity↗
PubMed1984

Application of a GM1 ganglioside beta-galactosidase microassay method to diagnosis of GM1 gangliosidosis.

The enzymatic diagnosis of GM1 gangliosidosis, including the diagnosis of heterozygosity, requires a microassay of GM1 ganglioside beta-galactosidase activity in lymphocytes and cultured skin fibroblasts. We have adopted high-performance liquid chromatography (HPLC) to the assay of this enzyme and can measure the activity in crude samples fluorometrically. Reaction conditions were examined to determine those optimal for the assay of GM1 ganglioside beta-galactosidase activity in lymphocyte and skin fibroblast homogenates. Under these optimal conditions, reduced enzymatic activities could be detected in lymphocytes and cultured skin fibroblasts from three patients with GM1 gangliosidosis. Thus, this assay can be used for the diagnosis, rather than the usual assays employing radioactive or artificial substrates.

Adult↗
PubMed1985

Ecdysterone selectively stimulates the expression of a 23000-Da heat-shock protein-beta-galactosidase hybrid gene in cultured Drosophila cells.

To study the regulated expression of cloned heat-shock genes in homologous cells, hybrid Drosophila heat-shock-Escherichia coli beta-galactosidase genes were constructed. Segments of the ecdysterone-inducible 23,000-Da heat-shock protein (hsp23) gene and of two other hsp genes (hsp84 and 70), which are not hormone regulated, were functionally linked to the bacterial coding sequence, and the resulting hybrid genes were introduced into cultured, hormone-responsive Drosophila cells by transfection. All hybrid genes directed the synthesis of E. coli-specific beta-galactosidase in heat-treated cells. hsp23 hybrid gene expression was stimulated strongly by ecdysterone, while the activities of the other hybrid genes were not affected at all by the hormone. A hybrid gene with only 147 bp of hsp23 promoter sequence could not be activated by either heat or ecdysterone treatment. Thus, far upstream sequences contain signals required for the regulated expression of the hsp23 gene in Drosophila cells.

Animals↗
PubMed1983

ConA-mediated binding and uptake of purified alpha-galactosidase A in Fabry fibroblasts.

In most human tissues there are at least two different alpha-galactosidases, A and B. The former is deficient in patients hemizygous for Fabry disease. We have isolated it from human placenta and found that it was labile even at culture conditions, but was stabilized after binding to concanavalin A (conA). The alpha-galactosidase activity was markedly increased in Fabry fibroblasts when these were treated with conA and exposed to alpha-galA at 37 degrees C. The maximum activity was obtained after 1/2-2 h of incubation and was maintained for at least 4 h. The binding and uptake of conA into Fabry cells was followed by microscopical studies of fluorescein-labelled conA. We assume that alpha-galA is taken up by endocytosis of the enzyme-conA complex.

Biological Transport↗
PubMed1989

Expression of a mouse metallothionein-Escherichia coli beta-galactosidase fusion gene (MT-beta gal) in early mouse embryos.

We have microinjected DNA containing the inducible mouse metallothionein-I (MT-I) promoter, coupled to the structural gene for Escherichia coli beta-galactosidase (lacZ), into the pronuclei of one-cell mouse embryos. A qualitative histochemical assay, with 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-Gal) as a substrate, was used to detect expression of lacZ at several preimplantation stages. We observed staining indicative of exogenous beta-galactosidase activity in 5-17% of DNA-injected embryos assayed at preimplantation stages after 16-24 h treatment with ZnSO4. Thus, lacZ can be used as an indicator gene for promoter function during early mouse embryogenesis, and the incorporation of the MT-I promoter into fusion genes can be a useful means of controlling the expression of exogenous genes in preimplantation mouse embryos.

Animals↗
PubMed1985

Uptake and degradation of bovine testes beta-galactosidase by parenchymal and nonparenchymal rat liver cells.

The plasma half-life of beta-galactosidase in rat was about 1.5 min. Ten minutes after in vivo injection, 45% of the enzyme was recovered in liver, with hepatocytes and endothelial cells as the predominant cell types responsible for uptake. In vitro uptake of beta-galactosidase in hepatocytes and nonparenchymal liver cells was saturable, Ca2+-dependent and it could be partly inhibited by mannose or alpha-methyl-mannoside.

Animals↗
PubMed1982

Miniaturization of beta-galactosidase immunoassays using chromogenic and fluorogenic substrates.

Enzyme immunoassay techniques are widely use to quantify various antigens and antibodies. The final step of these techniques (i.e. enzyme reaction) may be carried out in several ways (e.g. chromogenic, fluorogenic, or radioactive substrate and thermometric measurement). This paper compares the effectiveness of the chromogenic and the fluorogenic substrates in the beta-galactosidase immunoassay. Using microtitration plates (150 microliter samples) coated with anti-human IgE, and anti-human IgE labeled with E. coli beta-galactosidase, the lowest concentrations of IgE that one could detect employing either the chromogenic (o-nitrophenyl-beta-D-galactopyranoside) or the fluorogenic (4-methyl-umbelliferyl-beta-D-galactopyranoside) substrate were determined. It was found that both substrates were almost equally effective in measuring the lowest concentration of IgE (0.075-0.13 IU/ml) under the optimal conditions. But, using fluorogenic substrate and suitable apparatuses the enzyme immunoassay can be miniaturized. Thus by using decreasing volumes of reagents, progressively smaller amounts of antigen were quantified: as the sample volumes were reduced from 150 to 10 microliter and finally to 0.3 microliter a progressive decrease from 7 x 107 molecules of IgE to 2.9 x 107 molecules and to 1.5 x 106 molecules was observed. The corresponding lowest detection limits were 0.075 IU/ml, 0.46 IU/ml and 0.8 IU/ml.

Animals↗
PubMed1983

An enzymatically active antigen-antibody probe to measure circulating immune complexes. II. E. coli beta-galactosidase in the probe and C1q as the recognition unit.

An enzymatically active probe (beta-galactosidase-anti-beta-galactosidase complex) is used to measure circulating immune complexes (CIC), in a competition assay where probe and CIC are confronted with a 'recognition unit'. The latter is bovine conglutinin in the original description of this method. Here we describe a version utilizing human or bovine C1q. The two techniques are compared for their sensitivity and specificity, on both in vitro formed tetanus toxoid-anti-toxoid complexes and on sera from patients with selected diseases. The results confirm that the two recognition units are sensitive to families of CIC that only partially overlap. The parallel use of conglutinin and C1q yields both quantitative and qualitative information on the nature of CIC in individual sera.

Animals↗
PubMed1984

Heterologous enzyme immunoassay for puromycin aminonucleoside using beta-D-galactosidase as a label.

A heterologous enzyme immunoassay (EIA) was developed to quantify puromycin aminonucleoside (PA). This double antibody assay was based on the use of anti-puromycin (PU) antibody and used beta-D-galactosidase-labeled PA conjugate prepared via N-(m-maleimidobenzoyloxy)succinimide. The standard curve of the assay ranged from 1 ng to 30 ng, and the lower limit of detection was 22.7 nM (1 ng/tube). The EIA was found to be approximately 20 times more sensitive than the homologous EIA for PA with anti-PA antibody and PA-beta-D-galactosidase conjugate. The heterologous EIA was free from interference by any purine or pyrimidine analogs and drug levels were easily determined in rat tissue following i.v. administration at a dose of 15 mg/kg. The sensitivity and specificity of the EIA should provide a valuable new tool for use in pharmacokinetic and toxicity studies of PA.

Animals↗
PubMed1987

A sensitive micro-immunoassay using beta-galactosidase/anti-beta-galactosidase complexes.

This paper describes a new sensitive microELISA based on enzyme/anti-enzyme complexes following an unlabelled antibody bridging step. beta-Galactosidase/anti-beta-galactosidase complexes were made using a monoclonal antibody raised against bacterial (E. coli) beta-galactosidase and enzyme activity was quantified with a fluorogenic substrate. Because of its high sensitivity the assay is particularly suitable for the detection of limited amounts of antigen. One application illustrated is the analysis of Class I and Class II histocompatibility antigens on peripheral blood lymphocytes using 5000 cells/well in 60-well Terasaki or 96-well microtitre plates.

Animals↗
PubMed1988

Sandwich enzyme immunoassay of tumor-associated antigen sialosylated Lewisx using beta-D-galactosidase coupled to a monoclonal antibody of IgM isotype.

Enzyme conjugates with antibody of IgG type have been used extensively in immunohistochemistry, but conjugates with antibody of IgM type have not been reported. This paper describes the beta-D-galactosidase (Gal) labeling of a monoclonal IgM antibody designated CSLEX1 (for cytotoxic sialosylated Lewisx), which is directed against a tumor-associated antigen sialosylated Lewisx (S-Lex). The antibody was first acylated with a heterobifunctional agent N-(gamma-maleimidobutyryloxy)succinimide (GMBS) to introduce the maleimide groups into the molecule; excess reagent was removed by gel filtration and then the activated antibodies were crosslinked to the thiol groups of Gal. The conjugates were partially purified of free Gal by DEAE-Toyopearl column chromatography with an increasing linear concentration of NaCl. The conjugates thus prepared retained almost full enzyme activity and were demonstrated to be free of CSLEX1 by affinity chromatography using anti-galactosidase antibody bound to Sepharose 4B. The conjugates were used as a label in a sandwich enzyme immunoassay (SEIA) to detect the antigen at concentrations as low as 0.2 U/well. The SEIA was used to measure serum S-Lex levels in both healthy subjects and lung cancer patients and mean concentrations of 70 U/ml and 198.6 U/ml were detected respectively.

Animals↗
PubMed1985

A frameshift mutation at the junction of an IS1 insertion within lacZ restores beta-galactosidase activity via formation of an active lacZ-IS1 fusion protein.

The insertion of IS1 elements into lacZ results in the loss of beta-galactosidase activity, and such insertions exert a severe polar effect on the expression of the distal genes of the operon. In addition to these properties, the mutation lacZ::IS1-MS319 has the unique property of reversion to Lac+ (ts) spontaneously or after treatment with the frameshift mutagen ICR-191; such revertants retain the IS1 element. We have determined that the site of integration of IS1 into lacZ is at position 4338, 18 nucleotides from the end of the sequence encoding the C-terminus of beta-galactosidase. Reversion to Lac+ promoted by ICR-191 results from the loss of a G residue from a GGG sequence located at the junction of lacZ and IS1. As a result an active, but temperature-sensitive, lacZ-IS1 fusion protein is formed containing six amino acids derived from IS1 which replace six amino acids encoded by lacZ. The IS1 element in MS319 is a new member of the iso-IS1 family, which we designate IS1T.

Base Sequence↗
PubMed1988

Insertion of d(pCpG)n.d(pCpG)n into the lacZ gene of Escherichia coli inhibits expression of beta-galactosidase in vivo.

Plasmids that contain a d(pCpG)13.d(pCpG)13 insert instead of a lac operator show a 34-fold decrease of beta-galactosidase synthesis. The same sequence causes a 24-fold decrease when inserted between codons 5 and 6 of the lacZ gene. In such constructs with d(pCpG)n.d(pCpG)n inserts, beta-galactosidase activity decreases approximately 1.6-fold per d(pCpG).d(pCpG) unit when n ranges from 5 to 16.

Base Sequence↗
PubMed1989

A comparison of alkaline phosphatase, beta-galactosidase, penicillinase and peroxidase used as labels for progesterone determination in milk by heterologous microtitre plate enzymeimmunoassay.

Assays for alkaline phospatase, beta-galactosidase, penicillinase and peroxidase were optimised for quantitation in microtitre plate wells. Their value as labels in microtitre plate enzymeimmunoassay (EIA) for progesterone was assessed following coupling with 11 alpha-hydroxyprogesterone 11-glucuronide using an active ester procedure. Bridge-heterologous antiserum (11 alpha-hydroxyprogesterone 11-hemisuccinate-bovine serum albumin as immunogen) was used to minimize bridge recognition. The limits of detection of the enzymes were in the order penicillinase greater than peroxidase greater than alkaline phosphatase greater than beta-galactosidase. Under appropriate conditions it was possible to achieve greater than 50% displacement of label with 50 pg of progesterone for all four labels.

Alkaline Phosphatase↗
PubMed1980

Adult mucolipidosis with beta-galactosidase and neuraminidase deficiencies.

An adult case of mucolipidosis with beta-galactosidase and neuraminidase deficiencies is reported. The patient was a 35-year-old Japanese female with coarse face, lumbar vertebral beaking, action myoclonus, cerebellar ataxia, clouding of the cornea, macular cherry-red spots, hearing loss and vacuolated lymphocytes, but without mucopolysacchariduria. Her clinical symptoms developed at a late age with a slow progression. The enzyme activities of beta-galactosidase were deficient in leukocytes and cultured skin fibroblasts but normal in serum. Sialic acid-rich glycopeptides and oligosaccharides were increased in the urine. Neuraminidase activities toward fetuin, alpha-N-acetylneuraminosyl-(2 leads to 3) lactose and alpha-N-acetylneuraminosyl-(2 leads to 6) lactose were deficient in cultured fibroblasts. It is suggested that the main disturbance in the present case might be the catabolic process of glycoproteins and oligosaccharides due to neuraminidase deficiency.

Adult↗