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PubMed1988

Beta-galactosidase as a marker in the peripheral and neural tissues of the herpes simplex virus-infected mouse.

We have inserted a modified Escherichia coli lacZ gene, placed under the control of herpes simplex virus alpha 4 or beta 8 regulatory signals, into the HSV-1 genome disrupting the viral thymidine kinase gene. Using beta-galactosidase as an in situ indicator of viral gene expression, we detected expression from these recombinant HSV in dermal and neural tissues of the BALB/c mouse. Our detection of beta-galactosidase expression in neuronal cells indicates that TK-deficient viruses are capable of invading mouse neuronal cells and expressing up to the beta class of gene product.

Animals↗
PubMed1983

New heat shock puffs and beta-galactosidase activity resulting from transformation of Drosophila with an hsp70-lacZ hybrid gene.

A hybrid gene that consists of the Drosophila heat shock gene, hsp70, fused to the E. coli beta-galactosidase gene has been introduced into the Drosophila germline by the P element microinjection method. This hybrid includes 194 bp of sequence upstream of the start of the hsp70 transcript. Three strains of transformed flies were isolated and characterized by DNA blotting experiments and by in situ hybridization to polytene chromosomes. Strain Bg61 has a single insert of the hybrid gene at the tip of chromosome 3L, site 61A, and the insert consists of a structure that is consistent with P-element-mediated transposition. Strain Bg9,61 has inserts at both 61A and 9E, while Bg64 has a single insert at 64D. Heat shock induces the formation of a large chromosomal puff at all three sites. These puffs appear and regress with kinetics indistinguishable from the puffing of the heat shock locus, 87C, from which the hsp70 gene, used in these studies, was isolated. The beta-galactosidase activity in the transformants is inducible by heat shock and shows a widespread distribution throughout the tissues of larvae and adults.

Animals↗
PubMed1983

A solid-phase beta-galactosidase ELISA for detecting and quantifying monoclonal antibody binding to dissociated cell cultures of postnatal rodent cerebellum.

A solid-phase, indirect beta-galactosidase-linked immunoassay (ELISA) is described for screening large numbers of monoclonal antibodies that recognize cell surface antigens of primary monolayer cerebellar cultures. Target cultures were prepared from perikaryal suspensions of postnatal rodent cerebellum seeded into poly-L-lysine pre-coated, flat-bottom microtiter wells and fixed with glutaraldehyde after growth in vitro. Hybridoma supernatants were then incubated on these cultures. After the addition of beta-galactosidase-linked anti-mouse IgG F(ab')2 fragments, antigen-positive supernatants were detected with the enzyme substrate o-nitrophenyl-beta-D-galactopyranoside. Using a monoclonal antibody specific for rat brain Thy-1 glycoprotein, this solid-phase ELISA was found to be useful in quantifying changes in the developmental expression of cerebellar surface antigens in these cultures.

Animals↗
PubMed1983

Effect of dextran and dextran modifications on the thermal and proteolytic stability of conjugated bovine testis beta-galactosidase and human serum albumin.

In order to study carbohydrate-induced protein stabilization bovine testis beta-galactosidase and human serum albumin were conjugated with dextran, partially acetylated dextran and partially methylated dextran. The conjugates and the free proteins were compared with respect to thermal stability at 50 degrees C and resistance to proteolytic digestion by subtilopeptidase A. Both beta-galactosidase and serum albumin were stabilized by conjugation with polysaccharide. However, higher stability was achieved by conjugating the proteins with the hydrophilic polysaccharides, dextran and acetylated dextran, than by conjugation with the hydrophobic polysaccharide, methylated dextran. The results are discussed in relation to possible explanations of carbohydrate-induced protein stabilization.

Animals↗
PubMed1990

Expression and characterization of a tripartite fusion protein consisting of chimeric IgG-binding receptors and beta-galactosidase.

Using protein engineering, a tripartite fusion protein was constructed consisting of five IgG-binding regions of protein A from Staphylococcus aureus, two IgG-binding regions of protein G from Streptococcus strain G148 and beta-galactosidase from Escherichia coli. The resulting protein lacks the serum albumin binding regions of native protein G. The fusion protein, which is a tetramer of approximately 660 kDa, was designed as a tool for immunological assays taking advantage of its broad spectrum of antibody affinity. The gene was placed under control of two promoters, the PR promoter and the lac UV5 promoter and the expression from the two promoters was studied in a bioreactor. Induction of the PR promoter gave an intracellular product concentration corresponding to 20% of the cell dry weight. By utilizing the properties of beta-galactosidase, the protein was purified by extraction in an aqueous two-phase system. The fusion protein was not proteolytically degraded during the cultivation and purification steps. The biological activity of all three parts of the protein was demonstrated with a competitive ELISA.

Antigens, Differentiation↗
PubMed1994

Appearance and fate of a beta-galactanase, alpha, beta-galactosidases, heparan sulfate and chondroitin sulfate degrading enzymes during embryonic development of the mollusc Pomacea sp.

The characterization and properties of a beta-galactanase and alpha- and beta-galactosidases as well as heparan sulfate and chondroitin sulfate degrading enzymes which appear during the 15 days of the embryonic development of the mollusc Pomacea sp. is reported. The beta-galactanase, which appears around day 7 of development, was separated from alpha- and beta-galactosidase which emerge at day 1 and 4 after oviposition, respectively. The galactanase seems to be responsible for the degradation of an acidic beta-galactan (which is also synthesized by the eggs around day 5) to galactose and di- and tri-galactosides. Heparan sulfate appears around day 10 of development together with a heparan sulfate endoglucuronidase responsible for the degradation of its N-acetylated region. An alpha-N-acetylglucosaminidase and a beta-glucuronidase which act upon the N-acetylated fragments formed from heparan sulfate emerge around day 4 of development. Chondroitin sulfate and a chondroitin sulfate sulfatase emerge around day 9 of development whereas a beta-N-acetylgalactosaminidase and the beta beta-galactan, heparan and chondroitin sulfate, respectively. The possible role of these elements in the migration of mesenchymal cells, in the processes of cell-cell recognition and control of cell growth is discussed.

Acetylglucosaminidase↗
PubMed1983

A beta-galactosidase isoenzyme from Turbo cornutus with substrate specificity toward GM1-ganglioside and glycoproteins.

beta-Galactosidase from T. cornutus was resolved into two activity peaks by gel filtration column chromatography. The pH optima of the two peaks designated P1 and P2, were 5.5 and 3.0, respectively, when p-nitrophenyl-beta-D-galactopyranoside was used as the substrate. The molecular weights of P1 and P2 were 700,000 +/- 70,000 and 78,000 +/- 7800, respectively, when estimated by gel filtration chromatography. The activities of both forms of the enzymes are stimulated by anions such as Cl-, Br- and NO-3. While the activity of P1 was stimulated by low anion concentrations, P2 requires 700 times higher anion concentration for similar enhancement of activity. P1, the high molecular weight form hydrolyzes mainly galactose from small molecular weight beta-galactosides, such as p-nitrophenyl-beta-D-galactopyranoside, 4-methylumbelliferyl-beta-D-galactopyranoside, lactose, lactosylceramide and 3-O-beta-D-galactopyranosyl-D-arabinose, whereas P2, the low molecular weight form cleaves, in addition, all the beta-galactosides tested, including 2-hexadecanoylamino-4-nitrophenyl-beta-D-galactopyranoside, GM1-ganglioside, asialo-GM1-ganglioside, asialo fetuin, alpha 1-acid glycoproteins and the tryptic peptides of the glycoproteins. The optimal conditions for the hydrolysis of the terminal galactose from GM1-ganglioside which does not occur in gastropods, such as T. cornutus, was found to require 40 mM NaCl and 1 mM sodium taurodeoxycholate at pH 3.0 in 50 mM sodium citrate buffer, conditions similar to those by mammalian beta-galactosidase.

Animals↗
PubMed1983

New versatile plasmid vectors for expression of hybrid proteins coded by a cloned gene fused to lacZ gene sequences encoding an enzymatically active carboxy-terminal portion of beta-galactosidase.

A new class of plasmid cloning vectors has been constructed with cleavage sites in a variety of translational reading phases of the promotorless lacZ gene. Fused hybrid proteins can be produced by these vectors by cloning DNA fragments containing the promoter, translation initiation site, and the amino terminal portion of a gene, all with proper orientation, into the correct translational reading frame of the lacZ gene. Enzymatically active hybrid-beta-galactosidase proteins are formed, which have amino-terminal amino acids encoded by the cloned gene segment. Another class of these vectors retains an active lac promoter and lacZ translation-initiation region, which can direct hybrid protein synthesis from DNA fragments that do not have gene initiation regions. These vectors allow transcription from the lacZ initiation region to proceed across, or to stop and restart within, an inserted fragment into the essential part of the beta-galactosidase gene. Also described is a small lacZ gene fragment (cartridge), without a plasmid replicon and without any other lac genes, which can be inserted directly into other genes to form hybrid protein fusions. Polyrestriction site sequences were easily moved into some of these vectors by incorporating drug-resistance genes that serve as markers for the selection and detection of these sequences; those markers can be easily removed afterwards.

Bacterial Proteins↗
PubMed1989

Beta-galactosidase overexpression in SV40-transformed Chinese hamster fibroblasts exposed to mutagens as a result of amplification of transfected bacterial lacZ DNA sequences.

Genetic constructions in which the bacterial lacZ gene, encoding the enzyme beta-galactosidase, is fused to a viral (SV40) origin of replication have been introduced in an SV40-transformed hamster cell line (C1102). We have studied in detail 3 clones in which beta-galactosidase-specific activity increases after treatment with genotoxic agents. We show that this increase is dependent on the activity of the viral T protein and correlates with an amplification of lac sequences. This system provides a basis for the study of the induction of gene amplification by genotoxic agents in mammalian cells.

Animals↗
PubMed1988

Cloning, sequencing, and expression of cDNA for human beta-galactosidase.

We cloned and sequenced the full-length cDNA for human placental beta-galactosidase. The 2379-nucleotide sequence contains 2031 nucleotides which encode a protein of 677 amino acids. The amino acid sequence includes a putative signal sequence of 23 amino acids and 7 potential asparagine-linked glycosylation sites. The cDNA in the expression vector pSVL was used to transfect COS cells. Expression of the cDNA in transfected COS cells produced immunoprecipitable proteins and led to an increase in beta-galactosidase activity.

Amino Acid Sequence↗
PubMed1988

GM1-gangliosidosis: abnormalities in biosynthesis and early processing of beta-galactosidase in fibroblasts.

Biosynthesis and early processing of beta-galactosidase were analyzed by pulse-chase technique in human fibroblasts. In normal cells, an 84 kDa precursor was processed first to an intermediate form of higher molecular weight (88 kDa), and then to a 64 kDa mature enzyme. This intermediate form was detected also in the culture medium. Biosynthesis of the precursor was apparently normal in four cases of GM1-gangliosidosis, and a precursor of abnormally high molecular weight (86 kDa) was observed in one case. No further processing occurred to the 88 kDa form. It was concluded that the enzyme deficiency was caused by heterogeneous molecular mutations of beta-galactosidase with a defect in early processing in this disease.

Adult↗
PubMed1988

Site-directed mutagenesis of beta-galactosidase (E. coli) reveals that tyr-503 is essential for activity.

By using the technique of site-directed mutagenesis we have succeeded in replacing tyr-503 of beta-galactosidase (E. coli) with a phe. A study of the kinetic and stability properties of this mutant enzyme (F-503 beta-galactosidase) showed that the loss in activity upon this change is due to the loss of a catalytic group (rather than a detrimental change in the enzyme's overall structure or a change in the enzyme's binding capacity). This confirms previous suggestions that this tyr residue is involved in catalysis.

Cloning, Molecular↗
PubMed1982

Attempted affinity-labelling of beta-D-galactosidase from Escherichia coli with 2,6:3,4-dianhydro-1-deoxy-D-talo-hept-1-enitol.

The epoxides methyl 2,3-anhydro-beta-D-talopyranoside (1) and 2,6:3,4-dianhydro-1-deoxy-D-talo-hept-1-enitol (2), both prepared by improved methods, and 2,6:3,4-dianhydro-1-deoxy-D-gulo-hept-1-enitol (3) were applied as potential reagents for the affinity labelling of E. coli beta-D-galactosidase. Compounds 1 and 3 are ineffective as labelling reagents, whereas compound 2 irreversibly inhibits the enzyme activity. Deactivation is complete only when high concentrations (0.5M) of the inhibitor are applied over a relatively long period of time (24 h). Saturation kinetics cannot be observed. Nevertheless, the competitive inhibitor isopropyl 1-thio-beta-D-galactopyranoside protects the enzyme from irreversible deactivation by 2, indicating that the latter also reacts with the active site. Treatment of beta-D-galactosidase with 2,6:3,4-dianhydro-1-deoxy-D-talo-[4-3H]hept-1-enitol under conditions that effect deactivation of the enzyme to only a minor extent causes labelling of the protein at the molar ratio of 48:1. Specific, radioaffinity labelling of the active site of the enzyme cannot be thus achieved.

Affinity Labels↗
PubMed1988

Isolation of recombinant mycobacterial antigens by an automatic and generally applicable purification method for beta-galactosidase fusion proteins.

An automated two-dimensional chromatographic method has been developed for the isolation and concentration of recombinant fusion proteins with beta-galactosidase. The system consists of an immunoaffinity column with anti-beta-galactosidase antibodies as ligand, followed by an anion-exchange column. It was used for the purification and concentration of recombinant fusion proteins from Mycobacterium tuberculosis and M. leprae. Small amounts of crude lysates of Escherichia coli were loaded stepwise onto the immunoaffinity column with intermittent washing, elution and re-equilibration. After several cycles the eluate was passed through the anion-exchanger. Using an immunoaffinity gel of 5-ml volume and the anion-exchanger Mono Q HR 5/5, from 10 ml of crude E. coli lysate (containing up to 50 mg of protein) up to 100 micrograms of recombinant protein in a 2-ml volume could be isolated overnight.

Antibodies, Bacterial↗
PubMed1985

Headspace gas chromatographic determination of beta-galactosidase activity using electron-capture detection.

A headspace gas chromatographic method for the determination of beta-galactosidase (E.C. 3.2.1.23) activity is described. The method, in which 2,2,2-trichloroethyl beta-D-galactopyranoside (beta-TCG) is used as substrate, involves determination of the liberated 2,2,2-trichloroethanol by gas chromatography with electron capture detection. The preparation of beta-TCG and of 2,2,2-trichloroethyl alpha-D-galactopyranoside is described. A Km = 0.80 mM was found for the enzymatic hydrolysis of beta-TCG employing beta-galactosidase from Escherichia coli. The assay has been applied to the quantitative determination of E. coli bacteria.

Chromatography, Gas↗
PubMed1988

Expression of cDNA encoding the human "protective protein" associated with lysosomal beta-galactosidase and neuraminidase: homology to yeast proteases.

The "protective protein" is a glycoprotein that associates with lysosomal beta-galactosidase and neuraminidase and is deficient in the autosomal recessive disorder galactosialidosis. We have isolated the cDNA encoding human "protective protein". The clone recognizes a 2 kb mRNA in normal cells that is not evident in fibroblasts of an early infantile galactosialidosis patient. The cDNA directs the synthesis of a 452 amino acid precursor molecule that is processed in vivo to yield mature "protective protein," a heterodimer of 32 kd and 20 kd polypeptides held together by disulfide bridges. This mature form is also biologically functional since it restores beta-galactosidase and neuraminidase activities in galactosialidosis cells. The predicted amino acid sequence of the "protective protein" bears homology to yeast carboxypeptidase Y and the KEX1 gene product. This suggests a protease activity for the "protective protein."

Amino Acid Sequence↗
PubMed1986

Properties and kinetics of a neutral beta-galactosidase from rabbit kidney.

A neutral beta-galactosidase has been purified by concanavalin A-Sepharose affinity chromatography, DEAE-cellulose chromatography, Sephadex G-200 gel filtration and hydroxylapatite chromatography. The enzyme was purified 126-fold with a yield of about 21%. This form has a neutral optimal pH (7.5) and it is located in the cytosolic fraction. It shows a wide pH stability from pH 4.5 to 8.0, but it is very unstable at low pH values. Its isoelectric point is 4.9 and this value does not change on neuraminidase treatment. The estimated molecular weight was 47 000. The neutral form shows beta-D-galactosidase, beta-D-fucosidase and beta-D-glucosidase activities, all of them associated in a single peak in all the purification steps. p-Nitrophenyl beta-D-galactosides, p-nitrophenyl beta-D-fucosides and p-nitrophenyl beta-D-glucosides competed fully for a common active site in mixed-substrate experiments. Using gamma-D-galactonolactone as competitive inhibitor the Ki values were always coincident for the three activities. The effect of NaCl, methyl mannoside and some sugars (fucose, galactose and glucose) was studied.

Animals↗
PubMed1988

Microassay for GM1 ganglioside beta-galactosidase activity using high-performance liquid chromatography.

A simple and sensitive assay for GM1 ganglioside (GM1) beta-galactosidase activity was devised by direct measurement of released D-galactose using high-performance liquid chromatography (HPLC). GM1 beta-galactosidase activity in crude samples such as brain homogenates could be measured by this method. After incubation of brain homogenate for 1 h with GM1 at 37 degrees C and pH 4.4 in the presence of sodium taurodeoxycholate, the reaction was terminated by heating at 100 degrees C for 2 min and the supernatant from the centrifuged sample was analysed directly by HPLC. D-Galactose isolated by HPLC was converted into a fluorescent compound by a post-column reaction with arginine at 150 degrees C and the fluorescence intensity at 430 nm was measured with excitation at 320 nm. By this method 10 pmol of D-galactose could be measured and the fluorescence intensity was linear up to 1 mmol of D-galactose. Using this method, the optimal conditions for the activity of this enzyme were re-examined. As an application, the enzyme activity in the brain of a patient with GM1 gangliosidosis was examined. This method can be applied to any natural substrates, glycolipids or glycoproteins, the terminal galactose of which is hydrolysed by this enzyme.

Brain↗