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PubMed1991

Purification and characterization of different "acid" beta-galactosidases from sheep kidney.

1. Two "acid" forms, Am and Al, of beta-galactosidase from sheep kidney have been isolated and purified 349- and 154-fold, respectively, with a recovery of about 8%. 2. Their mol. wts were about 450,000 and 230,000, respectively. Am seems to be a dimer of Al. The aggregation is stimulated by NaCl. 3. The "acid" beta-galactosidase has a pH optimum between 4.0 and 5.0 for both forms. They are located in the lysosomes. The optimal temperature is 37 degrees C and 40 degrees C for Al and Am forms, respectively. 4. Three peaks were detected by isoelectric focusing. After sialidase treatment, these peaks were obtained at higher pH values. 5. The activation energy values were 10.75 and 11.72 kcal/mol for Am and Al, respectively. 6. A variety of chemicals were tested as possible activators or inhibitors. The enzyme is strongly inhibited by gamma-D-galactonolactone, and the kinetic evidence suggests a competitive inhibition in all cases.

Animals↗
PubMed1993

Purification and immunological characterization of acid beta-galactosidase from dog liver.

1. Dog liver acid beta-galactosidase was isolated in high yield and purified to homogeneity using a series of chromatographies on Con A-Sepharose, decyl-agarose, anion-exchange HPLC and gel-filtration HPLC. 2. Non-denaturing gel filtration by HPLC gave a single homogeneous peak corresponding to molecular mass of 180-190 kDa. During SDS-PAGE analysis, the single peak dissociated into a major band corresponding to molecular mass of 32 kDa with minor bands at 18 and 13 kDa. 3. Polyclonal antibodies raised against the purified enzyme immunoprecipitated beta-galactosidase activity specifically from dog liver extracts and recognized a single 32 kDa band in Western blot analysis of dog tissue homogenates. This antibody did not crossreact with any protein band in tissue homogenates from other species examined except cat. 4. Western blot analysis of tissue extracts from dogs affected with GM1-gangliosidosis showed the presence of a 32 kDa band similar to that of controls.

Animals↗
PubMed1991

Low copy number vectors for expression of fused genes to beta-galactosidase in Escherichia coli.

A series of six expression vectors, pXM184Lac.A, B, C, pXM184Z.A, B, C, based on the low copy plasmid pACYC184 that allow for expression of proteins fused to beta-galactosidase in Escherichia coli is described. A level of 50,000 units of beta-galactosidase is routinely observed and is easily identifiable on protein gels. This paper also reports the tight regulation of expression of the Trc promoter in these vectors using the LacIq repressor.

Amino Acid Sequence↗
PubMed1991

Improved methods for the detection of beta-galactosidase activity in colonies of Escherichia coli using a new chromogenic substrate: VBzTM-gal (2-(2-(4-(beta-D-galactopyranosyloxy)-3-methoxyphenyl)-vinyl)-3- methylbenzothiazolium toluene-4-sulphonate).

The use of a new substrate 2-(2-(4-(beta-D-galactopyranosyloxy)-3- methoxyphenyl)-vinyl)-3-methylbenzothiazolium toluene-4-sulphonate (VB-zTM-gal) is described for the detection of beta-galactosidase activity in colonies of wild type and mutant strains of Escherichia coli. On enzymic hydrolysis this substrate, which is soluble in water, released a chromophore which is red at pH 7 and bound to cellulose and nitrocellulose. The best procedure for the detection of activity was to grow colonies on standard nitrocellulose membranes (pore size 0.45 microns) laid onto an agar plate and to float the membranes over a solution of the substrate. Coloured colonies developed within 3 min, which were stable at 4 degrees C for several days, and this identified the expression of beta-galactosidase activity. This was found to be more specific than methods using triphenyltetrazolium or Eosin Methylene Blue media, and more economical than methods using X-gal (5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside). VBzTM-gal should have applications in gene cloning technology and in the detection of coliform organisms in polluted water.

Chromogenic Compounds↗
PubMed1984

Expression of canine parvovirus-beta-galactosidase fusion proteins in Escherichia coli.

Cloned DNA fragments encoding portions of canine parvovirus (CPV) structural proteins were inserted into plasmid expression vectors. These plasmids expressed CPV-beta-galactosidase fusion proteins under the transcriptional control of the Escherichia coli lac promoter-operator. The fusion proteins were purified and used to immunize rabbits. Rabbit antibodies raised against these fusion proteins were shown to immunoprecipitate authentic CPV structural proteins from infected cell extracts. This demonstrated that the CPV-beta-galactosidase fusion proteins expressed in bacteria elicit antibodies which can recognize determinants of authentic CPV proteins. However, none of the antibodies neutralizes CPV virus particles.

Animals↗
PubMed1995

The functional role of glutamine-280 and threonine-282 in human alpha-galactosidase.

Our previous study on chimeric mutants of alpha-galactosidase suggested that two peptide regions encoded by exons 1-2 and 6 of the enzyme gene contribute to substrate recognition (Ishii, S. et al. (1994) Biochim. Biophys. Acta 1204, 265-270). In this study, we constructed five single amino acid substitutions for functional analysis of the amino acid residues around glutamine-279, the mutation site detected in an atypical Fabry disease patient. Two mutants, Q280S (Gln280-->Ser; CAA-->TCA) and T282A (Thr282-->Ala; ACT-->GCT), showed increased Km and decreased thermostability as compared with normal enzyme. Circular dichroism spectrum was not modified. An additional chimeric mutation in the exon 1-2 region by substitution with the homologous sequence of alpha-N-acetylgalactosaminidase cDNA restored catalytic activity and thermostability in both mutants. These data indicated the functional significance of glutamine-280 and threonine-282 for expressing the activity and stability of alpha-galactosidase molecule, and also the presence of an intramolecular interaction between the two peptide regions encoded by exons 1-2 and 6.

Amino Acid Sequence↗
PubMed1996

The expression of a reporter protein, beta-galactosidase, is preserved during maturation and aging in some cells of the adult Drosophila melanogaster.

The effects of maturation and aging on cell stability and maintenance of protein expression have been examined in adult Drosophila melanogaster. Counting the number of cells present in the antenna of the adult fly revealed little loss in cell number with aging. Enhancer map-marked genes expressing beta-galactosidase (beta-gal) in the antenna and an Rh1 opsin reporter gene construct expressing beta-gal in the R1-6 photoreceptor cells of the compound eye revealed no alteration in spatial distribution or amount of beta-gal with aging. A heat shock-inducible promoter coupled to the expression of beta-gal, hsp70-lacZ, revealed that the rate and amount of induction of beta-gal after heat shock is preserved during aging but the rate of decay of beta-gal may be slightly delayed in older animals. These studies suggest that the ability to express a reporter protein, beta-galactosidase, is preserved in at least a subset of cells in the aging fly.

Animals↗
PubMed1992

An import-competent precursor of small subunit of ribulose-1,5-bisphosphate carboxylase generated by factor Xa cleavage from a beta-galactosidase fusion expressed in Escherichia coli.

A plasmid-encoding fusion protein interlinked by factor Xa recognition sequence between beta-galactosidase and a precursor of the small subunit of wheat ribulose-1,5-bisphosphate carboxylase has been constructed. The plasmid directed abundant synthesis of the fusion protein in Escherichia coli. The recombinant protein was accumulated in an aggregated form that was associated with the bacterial membranes. A procedure was developed to isolate the fusion protein in a relatively pure and soluble form. Bovine factor Xa cleaved the isolated chimera to generate the complete chloroplast precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase from the fused beta-galactosidase. The cleaved precursor protein was imported into the isolated chloroplasts and processed to yield its mature counterpart.

Amino Acid Sequence↗
PubMedDate not supplied

Prospective study of the enzymatic activities in urine of N-acetyl-beta-D-glucosaminidase, alpha-D-mannosidase, alpha- and beta-D-glucosidases, alpha-L- and beta-D-fucosidases, and beta-D-galactosidase in type I diabetes mellitus with early nephropathy.

Different surveys have been carried out on the plasma activities of different glycosidases in patients with insulin-dependent diabetes mellitus, but research on urinary glycosidases in this disease is scanty and incomplete. To elucidate the behavior of these lysosomal enzymes in the metabolic alterations occurring in the glomerular basal membrane during the initial stages of diabetic nephropathy, we conducted a prospective study to examine the urinary activities of N-acetyl-beta-D-glucosaminidase (NAG), alpha-D-mannosidase, alpha- and beta-D-glucosidase, alpha-L- and beta-D-fucosidase, and beta-D-galactosidase in patients with type I insulin-dependent diabetes mellitus, surveyed over 18 months, whose early diabetic nephropathy was detected by the presence of microalbuminuria. The simultaneous determination of beta 2-microglobulin in urine confirmed the glomerular origin of the albuminuria. No statistically significant correlation was found between the levels of albuminuria and the activities of any of the glycosidases analyzed. In the diabetic patients, a significant decrease was observed in the activities of all the enzymes (p < 0.05), except NAG and alpha-D-mannosidase, although the decrease in the latter was very close to statistical significance (p = 0.028, unilateral; p = 0.057 bilateral). Similarly, in the patients, there was a significant negative correlation (p < 0.05) with the serum levels of fructosamine, except with beta-D-galactosidase, which showed a positive correlation (p < 0.05) with fructosamine and blood HbA1c.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylglucosaminidase↗
PubMed2004

Sesquiterpene lactones inhibit luciferase but not beta-galactosidase activity in vitro and ex vivo.

Reporter enzymes such as firefly luciferase or beta-galactosidase of Escherichia coli are frequently used to study transcriptional activity of genes and to investigate the effects of novel compounds on gene or transcription factor activity. It is generally assumed that the activity of these enzymes is unaffected by the treatment conditions. Therefore, this factor is not considered when interpreting the data obtained. Biologically active compounds such as sesquiterpene lactones (SLs) have also been tested in reporter gene assays for their influence on gene expression. Here we show in in vitro and ex vivo experiments that SLs inhibit firefly luciferase activity probably by direct targeting of the enzyme while beta-galactosidase remains almost completely unaffected. The loss of luciferase activity after SL treatment could be an effect of their sulfhydryl-modifying potency and the subsequent alteration of the enzyme's tertiary structure. These results demonstrate that the effect of the test substance on the reporter enzyme used should be taken into consideration when the transcriptional effect of novel compounds is investigated.

Animals↗
PubMed2005

Co-production of alpha-amylase and beta-galactosidase by Bacillus subtilis in complex organic substrates.

Various nutrients belonging to three categories, carbon, organic nitrogen and complex organic sources, were investigated for the first time in terms of their effect on the co-production of extracellular thermostable alpha-amylase and beta-galactosidase by Bacillus subtilis, a bacterium isolated from fresh sheep's milk. Among the organic nitrogen sources tested, tryptone and corn steep liquor favored their production. Substitution of soluble starch by various starchy substrates, such as corn flour, had a positive effect on both enzyme yields. Furthermore, a two-fold higher production of both enzymes was achieved when corn steep liquor or tryptone was used in combination with the different flours. Among the divalent cations examined, calcium ions appeared to be vital for alpha-amylase production. The crude alpha-amylase and beta-galactosidase produced by this B. subtilis strain exhibited maximal activities at 135 degrees C and 65 degrees C, respectively, and were also found to be significantly stable at elevated temperatures.

Bacillus subtilis↗
PubMed2006

Purification and characterisation of an intracellular enzyme with beta-glucosidase and beta-galactosidase activity from the thermophilic fungus Talaromyces thermophilus CBS 236.58.

An intracellular beta-glycoside hydrolase with beta-glucosidase and beta-galactosidase activity, designated beta-glucosidase BGL1, was isolated to apparent homogeneity from the thermophilic ascomycete Talaromyces thermophilus CBS 236.58. The monomeric enzyme has a molecular mass of 50 kDa (SDS-PAGE) and an isoelectric point of 4.5-4.6. The enzyme is active with both glucosides such as cellobiose and galactosides including lactose; based on the catalytic efficiencies determined glucosides are the preferred substrates. beta-Galactosidase activity of BGL1 is activated by various mono and divalent cations including Na+, K+ and Mg2+, and it is moderately inhibited by its reaction products glucose and galactose. Its pH optimum for the hydrolysis of galactosides is in the range of 5.5-6.0, and its optimum temperature was found to be 50 degrees C (15 min assay). In addition to its hydrolytic activity, BGL1 shows a significant transferase activity which results in the formation of galacto-oligosaccharides. These have recently attracted interest because of possible applications in food industry. The highest yields of oligosaccharides was approximately 20% when using 38 gl(-1) lactose as the starting material.

Cellulases↗
PubMed2004

Msx1 expression in the adult mouse brain: characterization of populations of beta-galactosidase-positive cells in the hippocampus and fimbria.

We have analyzed Msx1 expression in the mature mouse brain using in situ hybridization and beta-galactosidase activity in Msx1(nLacZ) mice. The study revealed that Msx1 is strongly expressed in the circumventricular organs, such as the subcommissural organ and choroid plexus, and in some epithelia, such as that of the dorsal, but not the ventral part of the third ventricle. Immunohistochemical analysis revealed that the Msx1-expressing cells of the hippocampus and fimbria are astrocytes, oligodendrocytes or immature oligodendrocytes. In contrast, no co-expression was detected in these structures using several neuronal markers. These results were confirmed, using transmission electron microscopy, by the presence of 5-bromo-3-indolyl-beta-D-galactopyranosideprecipitates in astrocytes and oligodendrocytes in both sites. Moreover, using an anti-glial fibrillary acidic protein antibody (GFAP), our study reveals two populations of astrocytes in the adult hippocampus and other areas, such as the fimbria, namely Msx1+/GFAP+ and Msx1-/GFAP+. Beta-galactosidase activity was also observed in endothelial cells of hippocampal fissure blood vessels. We also observed co-localization of polysialic acid neural cell adhesion molecule, a marker of the polysialylated form of the neural cell adhesion molecule, in Msx1-expressing cells in the fimbria. These cells may be precursors of glial cells and originate from the epithelium of the fimbria. The present study indicates, in the mature mouse brain, that Msx1 may be linked to secretory activity in circumventricular organs, and to glial proliferation and differentiation in the hippocampus and fimbria, and presumably also in other cerebral areas. We suggest that Msx1 could be associated with brain homeostasis and blood-brain barrier function.

Aging↗
PubMed2004

Papaya beta-galactosidase/galactanase isoforms in differential cell wall hydrolysis and fruit softening during ripening.

The potential significance of the previously reported papaya (Carica papaya L.) beta-galactosidase/galactanase (beta-d-galactoside galactohydrolase; EC 3.2.1.23) isoforms, beta-gal I, II and III, as softening enzymes during ripening was evaluated for hydrolysis of pectins while still structurally attached to unripe fruit cell wall, and hemicelluloses that were already solubilized in 4 M alkali. The enzymes were capable of differentially hydrolyzing the cell wall as evidenced by increased pectin solubility, pectin depolymerization, and degradation of the alkali-soluble hemicelluloses (ASH). This enzyme catalyzed in vitro changes to the cell walls reflecting in part the changes that occur in situ during ripening. beta-Galactosidase II was most effective in hydrolyzing pectin, followed by beta-gal III and I. The reverse appeared to be true with respect to the hemicelluloses. Hemicellulose, which was already released from any architectural constraints, seemed to be hydrolyzed more extensively than the pectins. The ability of the beta-galactanases to markedly hydrolyze pectin and hemicellulose suggests that galactans provide a structural cross-linkage between the cell wall components. Collectively, the results support the case for a functional relevance of the papaya enzymes in softening related changes during ripening.

Carica↗
PubMed2005

Biochemical and molecular characterization of alpha-D-galactosidase from coffee beans.

Alpha-D-Galactosidase (alpha-Gal; EC 3.2.1.22) is one of three principal enzymes involved in the modification or degradation of plant cell wall galactomannans. In the present paper it is shown that alpha-galactosidase activities in field-grown coffee beans are variable amongst cultivars of the two species investigated (Coffea arabica and C. canephora var. Robusta). Higher activities were found in Arabica cultivars. Using beans from greenhouse-cultivated C. arabica as a model, we showed that alpha-Gal activity was undetectable in the bean perispem tissue, but increased gradually during the endosperm development, to reach a peak at approximately 30 weeks after flowering (WAF) which coincided with the hardening of the endosperm. Alpha-Gal-specific transcripts detected at 22 and 27 WAF accompanied the peak of alpha-Gal activity, but were reduced to be undetectable in mature beans at 30 WAF, while alpha-Gal activity still persisted. Two isoforms were distinguished in 2-DE profiles of crude protein extracts by N-terminal sequencing analysis. Analysis of two-dimensional gel electrophoresis profiles demonstrated that both isoforms accumulated in a linear fashion throughout grain maturation. Alpha-Gal activity was also observed to increase to high levels during in vitro germination of coffee beans suggesting an important function of this enzyme in this process. Alpha-Gal cDNA sequences from Arabica and Robusta were sequenced and their deduced proteins appeared to be very similar, differing by only eight amino acids. Southern-blot analysis suggests that the enzyme was encoded by at least two genes in C. arabica that could explain the existence of the two isoforms identified in 2-DE profiles.

Amino Acid Sequence↗
PubMed1996

Transgene expression in the rhesus cervix mediated by an adenovirus expressing beta-galactosidase.

OBJECTIVES: More than 90% of cervical cancers are positive for human papillomavirus, which functionally represses p53 and pRb. The remainder have been found to contain p53 mutations. Gene therapy involves insertion of a functioning gene into a patient to correct a genetic abnormality. STUDY DESIGN: The ability of a beta-galactosidase adenovirus to mediate transgene expression in the rhesus cervix was evaluated. Three different doses and two different entry techniques of virus were investigated. RESULTS: The ideal dose determined by X-galactosidase staining was 2 x 10(10) plaque-forming units, and the injection method yielded better staining than did abrasion with topical application. Increased adenoviral-specific immunoglobulin G antibody response in the injected monkeys confirmed the results. CONCLUSION: High transduction efficiency by use of adenoviral vectors can be achieved in the cervix. Reversing the effects of human papillomavirus and p53 mutations with gene therapy may become a novel therapy for invasive and preinvasive cervical cancer.

Adenoviridae↗
PubMed1990

Molecular cloning of mouse acid beta-galactosidase cDNA: sequence, expression of catalytic activity and comparison with the human enzyme.

A full-length cDNA coding for mouse lysosomal acid beta-galactosidase has been isolated on the basis of homology with the human gene. Catalytic activity toward 4-methylumbelliferyl beta-D-galactoside in the COS-1 cell expression system provided positive proof for its authenticity. The sequence analysis showed that the degree of similarity between the human and mouse enzymes was approximately 70% in the nucleotide sequence and nearly 80% in the amino acid sequence. The deduced primary amino acid sequences of the enzymes from the two species indicated that, of the seven possible N-glycosylation sites in the human enzyme, five are conserved in the mouse enzyme. Three additional possible N-glycosylation sites, not present in the human enzyme, are found in the primary amino acid sequence of the mouse enzyme. All seven cysteine residues in the mouse enzyme are conserved in the human enzyme. Although the nucleotide sequence could be aligned to 60% identity with the E. coli beta-galactosidase, similarity in the amino acid sequence was minimal.

Amino Acid Sequence↗
PubMed2000

Purification and characterisation of a beta-galactosidase from Aspergillus aculeatus with activity towards (modified) exopolysaccharides from Lactococcus lactis subsp. cremoris B39 and B891.

Beta-galactosidase from Aspergillus aculeatus was purified from a commercial source for its hydrolytic activity towards (modified) exopolysaccharides (EPSs) produced by Lactococcus lactis subsp. cremoris B39 and B891. The enzyme had a molecular mass of approximately 120 kDa, a pI between 5.3 and 5.7 and was optimally active at pH 5.4 and 55-60 degrees C. Based on the N-terminal amino acid sequence, the enzyme probably belongs to family 35 of the glycosyl hydrolases. The catalytic mechanism was shown to be retaining and transglycosylation products were demonstrated using lactose as a substrate. The beta-galactosidase was also characterised using its activity towards two EPSs having lactosyl side chains attached to different backbone structures. The enzyme degraded O-deacetylated EPS B891 faster than EPS B39. Furthermore, the presence of acetyl groups in EPS B891 slowed down the hydrolysing rate, but the enzyme was still able to release all terminally linked galactose.

Acetylation↗