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At least 1,459 records · Page 81Linked to original sources
PubMed1992

A rapid and simple chemiluminescent assay for Escherichia coli beta-galactosidase.

We describe a chemiluminescent assay for E. coli beta-galactosidase using Lumi-Gal 530, a commercial formulation containing a stable phenylgalactose-substituted dioxetane as the substrate. Removal of the galactose moiety leads to the generation of an unstable dioxetane which decomposes to provide the observed chemiluminescence which is measured with a luminometer. Advantages of the assay are that it is simple, inexpensive and has 20-fold greater sensitivity than the standard spectrophotometric assay. Additional advantages are that the dioxetane is quite stable in the commercial formulation, and beta-galactosidase functions efficiently and is not degraded during the course of an assay. As luminometers are becoming commonplace in molecular biology laboratories, this assay provides a preferable alternative to the spectrophotometric assay.

Biotechnology↗
PubMed2005

[Preparation of transfusable human universal red blood cell with recombinant alpha-galactosidase].

In order to meet the demand for safe transfusion in special conditions and to utilize the donated blood supply efficiently, technology has been developed to convert erythrocytes from type A, B, or AB to "universal donor" blood. Conversion of blood type B to O was performed by means of recombinant alpha-galactosidase digestion. The results showed that blood type B to O was converted successfully, 1 transfusion unit of red cells of group B (100 ml totally) could converted to universal blood cells in the optimal conditions including pH 5.6, 26 degrees C, 2 hours, obturation and sterilization. It is concluded that the universal red blood cells converted from group B to group O are conformed to demand of identification rules of biological products, no harmful effects of alpha-galactosidase on cell structure and function are observed. The converted red cells can stored in 4 degrees C for 21 days.

ABO Blood-Group System↗
PubMedDate not supplied

[Regulation of beta-galactosidase synthesis in Escherichia coli by exogenous cyclic 3',5'-adenosine monophosphate].

The effect of cyclic 3',5'-adenosine monophosphate (cAMP) on the rate of beta-galactosidase biosynthesis was studied in the cells of Escherichia coli M-17 growing in MPB and mineral media with glucose and maltose, i.e. under the conditions of various catabolite repression, as well as upon lac-operon induction by isopropyl-beta-D-galactopyranoside (IPGP). The stimulating action of exogenous cAMP was found only in a medium with salts and glucose. The induction by IPGP was highest during the growth in a medium with glucose and maltose. When the medium contained IPGP, cAMP accelerated the enzyme synthesis in all media, but only at the early growth phases, while cAMP eliminated the effect of IPGP at the stationary phase of growth. The regulation of beta-galactosidase biosynthesis by cAMP demonstrated for the first time that this effect depended on the physiological state of E. coli: the expression of catabolite-sensitive E. coli genes was subject to both positive and negative regulation in one and the same inducible system. The effect exerted by cAMP depended on the nature of a carbon source in the growth medium.

Cyclic AMP↗
PubMed2006

Cloning and expression analysis of beta-galactosidase gene related to softening of banana (Musa sp.) fruit.

Beta-galactosidase is thought to be involved in fruit softening through cleaving beta(1-->4) galactan bonds in cell wall hemicellulose. To study the relationship between fruit softening and beta-Gal during banana (Musa sp.) fruit ripening, a beta-Gal cDNA fragment, named MA-Gal, has been cloned from banana fruit pulp using RT-PCR in this study. The results of sequence analysis showed that MA-Gal contained 927 bp, encoding a polypeptide of 309 amino acids, the deduced protein was highly homologous to plant beta-galactosidase expressed in fruit ripening. The MA-Gal putative amino acids have five homologous domains (typical eukaryotic beta-Gals have seven domains), contain the GGPIILSQIENEY(F) motif, which is a presumptive catalytic active site conserved in all beta-Gals. Phylogenetic analysis showed that MA-Gal was in plant beta-Gal group I, which was expressed in fruit tissue. The changes in beta-Gal activity and firmness in pulp during three different stages were also assayed. The Northern analysis showed that the MA-Gal transcripts in pulp were detected at low level at preclimacteric stage, while an increasing progression was observed during fruit ripening, and the highest transcript amount was found at the postclimacteric stage. These results suggest that MA-Gal may play a role during banana fruit ripening and softening.

Amino Acid Sequence↗
PubMed1990

[The effect of endo-beta-galactosidase on lactosamine sialoantigens Gd, Fl, Vo, Li].

Human red cells (RBC) were treated with endo-beta-galactosidase from Bacteroides fragilis removing type 2 polylactosamine chains [Galssl-4GlcNAc]n from the RBC surface by cleaving internal Galssl-4Glc(NAc) bonds of linear lactosamine sequences. I and i built up by unsubstituted branched and linear chains were inactivated. The sialo-antigen Vo resembling i antigen expression on adult, newborn and i adult RBC was also inactivated, demonstrating that the Vo determinant is created by sialylation of linear chains. Fl and Gd antigens known to be sialylated polylactosamine chains were not inactivated. Because Gd antigenicity is increased by increasing the length of sugar sequences and Vo determinants are obviously short chain sequences, the action of endo-beta-galactosidase could depend on the length of sialylated sequences on the RBC membrane.

Adult↗
PubMed1990

Screening of microorganisms for improvement of beta-galactosidase production.

The strain of Penicillium notatum 1 most effective for producing beta-galactosidase (see lactase 3.2.1.23), was selected out of 110 moulds belonging to 15 different species, by the test-tube microculture method. The dynamics of beta-galactosidase synthesis was investigated in P. notatum 1 during its culture by submerged method.

Fungi↗
PubMed1991

A sensitive method for the detection of beta-galactosidase in transfected mammalian cells.

A sensitive method has been developed for the detection of E. coli beta-galactosidase in transfected HeLa cells. The chromogenic substrate, CPRG (chlorophenol red-beta-D-galactopyranoside), was compared with ONPG (o-nitrophenyl-beta-D-galactopyranoside) by kinetic analysis with purified beta-galactosidase. The Km for CPRG was 1.35 mM and the Vmax was 21.4, whereas the Km for ONPG was 2.42 and the Vmax was 41.1. CPRG at 8.0 mM (6-fold Km) gave 86% of the Vmax and was used as the standard concentration for quantitation of enzyme levels. The Vmax for CPRG was half that for ONPG, and chlorophenol red has an extinction coefficient that is 21-fold higher than o-nitrophenol; these factors make CPRG about 10-fold greater in sensitivity for the quantitation of enzyme levels. The use of Nonidet P-40 to lyse the cells and the use of CPRG as substrate permitted the rapid detection of low levels of enzyme production from transfected human cells that could not be detected using ONPG.

Chlorophenols↗
PubMed1991

Human beta-galactosidase gene mutations in GM1-gangliosidosis: a common mutation among Japanese adult/chronic cases.

Molecular analysis of the human beta-galactosidase gene revealed six different mutations in 10 of 11 Japanese GM1-gangliosidosis patients. They were the only abnormalities in each allele examined in this study. A 165-nucleotide duplication (positions 1103-1267) was found in two infantile patients, producing an abnormally large mRNA; one patient was probably a homozygote, and the other was a heterozygote of this mutation. The other two infantile patients had different mutations; a 123 Gly(GGG)----Arg(AGG) mutation in one patient and a 316 Tyr(TAT)----Cys(TGT) mutation in the other. A 201 Arg(CGC)----Cys(TGC) mutation, eliminating a BspMI site, was detected in a late-infantile/juvenile patient; the restriction-site analysis of amplified genomic DNA confirmed his heterozygosity for this mutation. A 51 Ile(ATC)----Thr(ACC) mutation was found in all five adult/chronic patients examined in this study. It created a SauI site, and restriction-site analysis confirmed that four patients were homozygous mutants. The other was a compound heterozygote for this mutation and another 457 Arg(CGA)----Gln(CAA) mutation. These mutant genes expressed markedly decreased or completely deficient enzyme activities in beta-galactosidase-deficient human fibroblasts transformed by adenovirus-SV40 recombinants. We conclude that gene mutations are heterogeneous in GM1-gangliosidosis but that the 51 Ile(ATC)----Thr(ACC) mutation is common among the Japanese adult/chronic cases. Genotype-phenotype correlations in GM1-gangliosidosis are briefly discussed.

Adolescent↗
PubMed1991

Sequence homology between a beta-galactosidase and some beta-glucosidases.

A significant sequence homology was found between a thermostable beta-galactosidase from Sulfolobus solfataricus and two beta-glucosidases, respectively, from Caldocellum saccharolyticum and from Agrobacterium sp. These glycosidases appear to form a new protein family, since no homology could be detected with established beta-galactosidase or beta-glucosidase families.

Amino Acid Sequence↗
PubMed1991

Purification and characterization of a sea squirt beta-galactosidase.

A beta-galactosidase was extracted from the internal organs of a sea squirt, Styela plicata, and purified 959-fold, with an 18% yield, by successive gel chromatography, anion-exchange chromatography, chromatofocusing, and affinity chromatography on a Con A-Sepharose column. The purified enzyme was fairly homogeneous, as judged on disc PAGE, SDS-PAGE, and gel chromatography on a Sephadex G-200 column. The molecular weight of the enzyme was estimated to be 77,000 and 75,000 by gel chromatography and SDS-PAGE, respectively, and its isoelectric point was determined to be 4.9 by the isoelectric focusing method. The enzyme was substantially stable in the pH range of 3.5 to 7.5, the optimum pH being 4.0. The enzyme was significantly inhibited by 9 mM HgCl2 and 9 mM DFP, while the inhibition by 0.9% PCMB was only 60% at 0 degrees C for 30 min. The purified beta-galactosidase apparently liberated galactose from a sea squirt antigen (H-antigen), two allergenically active glycopeptides (Gp-1 and Gp-2) derived from another sea squirt antigen (Gi-rep), asialo-ovomucoid glycopeptide, asialo-fetuin glycopeptide, GA1, CDH, and an ABEE-derivative (Gal beta 1----3ThrNAc-ABEE) of Gal beta 1----3GalNAc-ol isolated from bovine submaxillary gland mucin.

Animals↗
PubMed1977

[Isolation and characterization of beta-D-galactosidase from gastric juice].

A method for beta-D-galactosidase isolation from cattle gastric juice has been developed. Gastric juice mucus was removed by and addition of equimolar amounts of Na2HPO4 and CaCl2. The removal of proteases and other proteins was achieved by the treatment with resins KB-51X2 and AN-22. The resulting preparation had specific activity of 0.14 units per mg of protein. Gel filtration on Sepharose 4B led to an increase of specific activity of the preparation up to 0,4 units per mg of protein. Some properties of the beta-D-galactosidase obtained were compared to relation of lactose and o-nitrophenyl-beta-D-galactoside (pH optima, temperature optimym and thermostability).

Animals↗
PubMed1990

Expression of acid phosphatase-beta-galactosidase hybrid proteins prevents translocation by depleting a soluble factor.

We have shown that hybrid proteins composed of the yeast repressible acid phosphatase (PHO5) and bacterial beta-galactosidase (lacZ) interfere with secretion of native acid phosphatase (Wolfe, P. B. (1988) J. Biol. Chem. 263, 6908-6915). We now report that PHO5-LacZ hybrid proteins have a more general effect on secretion and prevent translocation of several secreted proteins. Translocation of both the mating pheromone alpha-factor and the vacuolar protease carboxypeptidase Y is partially blocked when PHO5-LacZ hybrids are expressed. Cell fractionation and protease sensitivity indicate that alpha-factor and carboxypeptidase Y accumulate in precursor form on the cytoplasmic surface of the endoplasmic reticulum. Indirect immunofluorescence with antibody directed against beta-galactosidase supports the localization of hybrid proteins to the endoplasmic reticulum. Analysis of the hybrid protein phenotype in vivo and in vitro suggests that the hybrid proteins deplete a soluble factor required for efficient translocation across the endoplasmic reticulum. First, a decrease in the expression of a hybrid protein in vivo decreases its effect on translocation. Second, an in vitro translation/translocation reaction, prepared from a hybrid-bearing strain, is deficient in its ability to translocate prepro-alpha-factor across yeast microsomal membranes. This deficiency is complemented by addition of cytosol prepared from wild type cells. Finally, the hybrid protein phenotype is shown to be independent of the requirement for SSA gene products.

Acid Phosphatase↗
PubMedDate not supplied

[Factors influencing the biosynthesis of beta-galactosidase in Alternaria tenuis].

The effect of pH, carbon sources, and some organic substances on the biosynthesis of beta-galactosidase was studied in Alternaria tenuis on a defined medium that had been optimized by the method of mathematical planning of experiments. Optimal conditions for the production of the enzyme and its liberation into the cultural broth were maintained by adding 2 per cent soya flour to the medium and 0.1 M phosphate-citrate buffer pH 3.0. The production of the enzyme was increased by 3 to 4 times. The biosynthesis of beta-galactosidase by Alternaria tenuis is of an induced nature.

Alternaria↗
PubMedDate not supplied

[Production and properties of a beta-galactosidase preparation from Alternaria tenuis].

Different methods of the preparation of fungal beta-galactosidase from the 72-hour culture of Alternaria tenuis were tested: lyophilization of the culture liquid, precipitation with ethanol, acetone, ammonium sulphate. Optimal results were obtained with precipitation by 1.5 acetone volume. Studies of the properties of fungal beta-galactosidase demonstrated that the preparation retained its activity during 22 month storage at 5 degrees C. The fungal preparation had pH optimum at a more acidic zone (4.2 versus 6.9), was active in a wider pH range 2.8-5.7 and 6.2-7.5), had a much higher temperature optimum (65 degrees and 30 degrees) and better thermostability as compared with the yeast preparation. Data on other properties of the preparation are presented.

Alternaria↗
PubMed1987

A new endo-beta-galactosidase releasing Gal alpha 1----3Gal from carbohydrate moieties of glycoproteins and from a glycolipid.

Culture fluid of Clostridium perfringens contained an endoglycosidase releasing a galactosyl disaccharide from glycans derived from glycoproteins of teratocarcinoma OTT6050. The endoglycosidase was purified by ammonium sulfate precipitation, Sephadex G-200 column chromatography, and DEAE-Sephadex A-25 column chromatography. The structure of the disaccharide product was determined to be Gal alpha 1----3Gal. The enzyme also released the disaccharide from bovine thyroglobulin glycopeptide and from a pentaglycosyl ceramide (Gal alpha 1----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc-Cer). Therefore, we concluded that the enzyme was an endo-beta-galactosidase cleaving Gal beta 1----4GlcNAc linkage. Keratan sulfate and the antigenic determinant of blood group type B were resistant to the enzyme: the specificity of the enzyme is different from other endo-beta-galactosidases so far described. The enzyme could act on intact thyroglobulin and defucosylated ovarian cyst mucin.

Animals↗
PubMed1986

Enzyme immunoassay for the quantification of mithramycin using beta-D-galactosidase as a label.

A sensitive enzyme immunoassay for mithramycin (MTM) has been developed by using antibody induced in rabbits, beta-D-galactosidase-labeled MTM, and a double-antibody separation technique, which allowed us to measure accurately as little as 100 pg of MTM per assay tube. MTM-antibody was produced against MTM-bovine serum albumin conjugate prepared by the use of diazotized p-aminobenzoic acid as a cross-linker. The beta-D-galactosidase-labeled MTM conjugate was similarly prepared by a geometric m-isomer of diazotized aminobenzoic acid. This enzyme immunoassay was specific to MTM and showed a very slight cross-reactivity with MTM analogues, chromomycin A3 (5.6%) and olivomycin (2.4%), but no cross-reactivity with drugs commonly used with MTM in combination chemotherapy for cancer treatment. The values of MTM concentrations detected by this assay were comparable to those detected by the high-pressure liquid chromatography method. However, the enzyme immunoassay method was 100 times more sensitive in detecting MTM in lower concentrations. Using this assay, drug levels were easily determined in the blood and urine of rats during 6 h after i.v. administration of MTM in a single dose of 2.0 mg/kg. Since MTM has long been used against a variety of human cancers, the enzyme immunoassay of the drug will be a valuable new tool in clinical pharmacological studies.

Animals↗
PubMed1987

Morquio's disease type B (beta-galactosidase deficiency) in three siblings.

The clinical and biochemical findings in 3 siblings with Morquio's disease type B (mucopolysaccharidosis (MPS) IV B) are presented. Their phenotype is characterised by short trunk dwarfism with kyphoscoliosis and thoracic deformity. Radiographic findings include general platyspondyly, dysplasia of the pelvis and epiphyseal abnormalities. The patients are of normal intelligence. In the urine of all 3 affected children abnormal oligosaccharide excretion was found by thin-layer chromatography and in 1 of them keratosulphaturia was detected. The clinical diagnosis was confirmed biochemically by demonstration of a profound deficiency of beta-galactosidase activity in cultured fibroblasts. The clinical picture is compared with that of other cases in the literature and the possible molecular basis of the different phenotypes of beta-galactosidase deficiency (variants of monosialo-ganglioside-1 (GM1)-gangliosidosis, Morquio's disease type B) is discussed.

Adolescent↗