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PubMed1978

Distribution of alpha-Galactosidase in Cucurbita pepo.

The distribution of alpha-galactosidase (alpha-d-galactoside galactohydrolase [EC 3.2.1.22]) in Cucurbita pepo has been determined in an attempt to assess its involvement in hydrolysis of transport sugars of the raffinose oligosaccharide series ([alpha-1-6-0-galactopyranosyl](n) sucrose). Extracts prepared from leaves and petioles at different stages of development, roots, flowers, dry and germinating seeds, all contained appreciable levels of alpha-galactosidase activity. Chromatography of these extracts on DEAE-Sephadex resolved the enzyme into three active isozymic forms. These isozymes were present in all regions of the plant analyzed but their relative proportions varied between tissues and changed within leaf and petiole tissues during development and in seeds during their germination. The level of total alpha-galactosidase activity in the leaf blade measured on a fresh weight or total protein basis remained constant at all developmental stages analyzed. The occurrence of these isozymes in mature exporting leaves indicates an effective intracellular compartmentation between their location and the sites of galactosyl oligosaccharide biosynthesis, accumulation and movement in the tissue. We have used these results to comment on the transport pathway of galactosyl oligosaccharides between the phloem and surrounding tissues in this plant.

Journal Article↗
PubMed1980

Characterization of alpha-Galactosidase from Cucumber Leaves.

Two forms of alpha-galactosidase (alpha-d-galactoside galactohydrolase, E.C. 3.2.1.22) which differed in molecular weight were resolved from Cucumis sativus L. leaves. The enzymes were partially purified using ammonium sulfate fractionation, Sephadex gel filtration, and diethylaminoethyl-Sephadex chromatography. The molecular weights of the two forms, by gel filtration, were 50,000 and 25,000. The 50,000-dalton form comprised approximately 84% of the total alpha-galactosidase activity in crude extracts from mature leaves and was purified 132-fold. The partially purified 25,000-molecular weight form rapidly lost activity unless stabilized with 0.2% albumin and accounted for 16% of the total alpha-galactosidase activity in the crude extract. The smaller molecular weight form was not found in older leaves.The two forms were similar in several ways including their pH optima which were 5.2 and 5.5 for the 50,000- and 25,000-dalton form, respectively, and activation energies, which were 15.4 and 18.9 kilocalories per mole for the larger and smaller forms. Both enzymes were inhibited by galactose as well as by excess concentrations of p-nitrophenyl-alpha-d-galactoside sub-strate. K(m) values with this substrate and with raffinose and melibiose were different for each substrate, but similar for both forms of the enzyme. With stachyose, K(m) values were 10 and 30 millimolar for the 50,000- and 25,000- molecular weight forms, respectively.

Journal Article↗
PubMed1982

An alpha-Galactosidase with Hemagglutinin Properties from Soybean Seeds.

Soybean (Glycine max L.) seeds contain a galactose-binding protein which displays two activities: (a) an alpha-galactosidase activity and (b) a hemagglutinin activity. The alpha-galactosidase-hemagglutinin was purified to homogeneity by conventional protein purification procedures and also by affinity chromatography. This protein can be easily separated from soybean agglutinin, the N-acetyl-d-galactosamine-specific lectin in soybean. Further, these two agglutinins show no immunological relatedness. The alpha-galactosidase-hemagglutinin can be reversibly converted by pH changes from a tetrameric form which displays both enzymic and hemagglutinin activities to a monomeric form which displays enzymic activity only. Although both the monomeric and tetrameric forms are enzymically active, they display different pH optima and carbohydrate specificities.

Journal Article↗
PubMed1983

Partial Purification and Properties of an Alkaline alpha-Galactosidase from Mature Leaves of Cucurbita pepo.

A fourth molecular from of alpha-galactosidase, designated L(IV), an alkaline alpha-galactosidase, was isolated from leaves of Cucurbita pepo and purified 165-fold. It was active over a narrow pH range with optimal hydrolysis of p-nitrophenyl-alpha-d-galactoside and stachyose at pH 7.5. The rate of stachyose hydrolysis was 10 times that of raffinose. K(m) determinations in McIlvaine buffer (200 millimolar Na(2)-phosphate, 100 millimolar citric acid, pH 7.5) for p-nitrophenyl-alpha-d-galactoside, stachyose, and raffinose were 1.40, 4.5, and 36.4 millimolar, respectively. L(IV) was partially inhibited by Ca(2+), Mg(2+), and Mn(2+), more so by Ni(2+), Zn(2+), and Co(2+), and highly so by Cu(2+), Ag(2+), Hg(2+) and by p-chloromercuribenzoate. It was not inhibited by high concentrations of the substrate p-nitrophenyl-alpha-d-galactoside or by myo-inositol, but alpha-d-galactose was a strong inhibitor. As observed for most other forms of alpha-galactosidase, L(IV) only catalyzed the hydrolysis of glycosides possessing the alpha-d-galactose configuration at C(1), C(2), and C(4), and did not hydrolyze p-nitrophenyl-alpha-d-fucoside (alpha-d-galactose substituted at C(6)). The enzyme was highly sensitive to buffers and chelating agents. Maximum hydrolytic activity for p-nitrophenyl-alpha-d-galactoside was obtained in McIlvaine buffer (pH 7.5). In 10 millimolar triethanolaminehydrochloride-NaOH (pH 7.5) or 10 millimolar Hepes-NaOH (pH 7.5), hydrolytic activity was virtually eliminated, but the addition of low concentrations of either ethylenediaminetetraacetate or citrate to these buffers restored activity almost completely. Partial restoration of activity was also observed, but at higher concentrations, with pyruvate and malate. Similar effects were found for stachyose hydrolysis, but in addition some inhibition of L(IV) in McIlvaine buffer, possibly due to the high phosphate concentration, was observed with this substrate. It is questionable whether the organic acid anions possess any regulatory control of L(IV)in vivo. It was possible that the results reflected the ability of these anions, and ethylene-diaminetetraacetate, to restore L(IV) activity through coordination with some toxic cation introduced as a buffer contaminant.

Journal Article↗
PubMed1985

Accumulation and Subcellular Localization of alpha-Galactosidase-Hemagglutinin in Developing Soybean Cotyledons.

We have investigated the accumulation and intracellular localization of soybean (Glycine max [L.] Merr. cv Forrest) alpha-galactosidase-hemagglutinin during seed development. Cotyledon tissue was embedded in Lowicryl K4M and immunocytochemical localization was accomplished through treating thin sections with alpha-galactosidase antisera followed by indirect labeling with protein A coupled to colloidal gold. Gold particles were localized on the Golgi apparatus and protein bodies. We interpret this to indicate that alpha-galactosidase-hemagglutinin is transferred to and transported through the Golgi apparatus and finally deposited within the protein body by a Golgi apparatus-mediated process.

Journal Article↗
PubMed1985

High Production of Thermostable beta-Galactosidase of Bacillus stearothermophilus in Bacillus subtilis.

By cloning the beta-galactosidase gene of Bacillus stearothermophilus IAM11001 (ATCC 8005) into Bacillus subtilis, enzyme production was enhanced 50 times. beta-Galactosidase could be purified to 80% homogeneity by incubating the cell extract of B. subtilis at 70 degrees C for 15 min, followed by centrifugation to remove the denatured proteins. Because of its heat stability and ease of production, beta-galactosidase is suitable for application in industrial processes.

Journal Article↗
PubMed1989

Simple Method To Detect beta-Galactosidase.

A simple and rapid method was developed to detect beta-galactosidase by using alpha- or beta-naphthyl-beta-d-galactopyranoside as substrate and fast garnet GBC as a dye coupler following polyacrylamide gel electrophoresis. This method was specific for beta-galactosidase but not for phospho-beta-galactosidase.

Journal Article↗
PubMed2004

Directed evolution of single-chain Fv for cytoplasmic expression using the beta-galactosidase complementation assay results in proteins highly susceptible to protease degradation and aggregation.

BACKGROUND: Antibody fragments are molecules widely used for diagnosis and therapy. A large amount of protein is frequently required for such applications. New approaches using folding reporter enzymes have recently been proposed to increase soluble expression of foreign proteins in Escherichia coli. To date, these methods have only been used to screen for proteins with better folding properties but have never been used to select from a large library of mutants. In this paper we apply one of these methods to select mutations that increase the soluble expression of two antibody fragments in the cytoplasm of E. coli. RESULTS: We used the beta-galactosidase alpha-complementation system to monitor and evolve two antibody fragments for high expression levels in E. coli cytoplasm. After four rounds of mutagenesis and selection from large library repertoires (>107 clones), clones exhibiting high levels of beta-galactosidase activity were isolated. These clones expressed a higher amount of soluble fusion protein than the wild type in the cytoplasm, particularly in a strain deficient in the cytoplasmic Lon protease. The increase in the soluble expression level of the unfused scFv was, however, much less pronounced, and the unfused proteins proved to be more aggregation prone than the wild type. In addition, the soluble expression levels were not correlated with the beta-galactosidase activity present in the cells. CONCLUSION: This is the first report of a selection for soluble protein expression using a fusion reporter method. Contrary to anticipated results, high enzymatic activity did not correlate with the soluble protein expression level. This was presumably due to free alpha-peptide released from the protein fusion by the host proteases. This means that the alpha-complementation assay does not sense the fusion expression level, as hypothesized, but rather the amount of free released alpha-peptide. Thus, the system does not select, in our case, for higher soluble protein expression level but rather for higher protease susceptibility of the fusion protein.

Journal Article↗
PubMed1997

Verapamil inhibition of enzymatic product efflux leads to improved detection of beta-galactosidase activity in lacZ-transfected cells.

The beta-galactosidase activity encoded by the lacZ gene of Escherichia coli is widely used to monitor successful expression of transfected genes. Fluorogenic substrates allow detection of enzyme activity in viable cells, which, subsequently, can be selected for further study on the basis of fluorescence emission. We analyzed three fluorogenic substrates (FDG, C12FDG, and CMFDG), all of which incorporate fluorescein as their fluorophore, regarding intensity of fluorescent signal and selectivity toward the transfected beta-galactosidase activity vs. lysosomal enzyme activity. Among these substrates, 5-chloromethylfluoresecein di-beta-D-galactopyranoside (CMFDG) showed the strongest selectivity toward the lacZ-encoded enzyme activity. An attempt to improve this selectivity by alkalinization of the lysosomal pH with chloroquine, such that the endogenous enzyme would be exposed to a suboptimal pH, led to significant cell death. In contrast, inhibition of dye efflux with verapamil enhanced the selectivity of CMFDG toward the lacZ-encoded enzyme activity by approximately threefold. Incubation with probenecid, on the other hand, showed little effect.

3T3 Cells↗
PubMed1985

Galactosialidosis: low beta-galactosidase activity in serum after long-term clotting.

Serum beta-galactosidase activity was found to be markedly increased in clotting blood from normal subjects, but patients with galactosialidosis showed only a slight increase of this enzyme activity. Consequently, the beta-galactosidase activity was low in serum after long-term clotting in patients with this disease. The mechanism of the enzyme activation is unknown. The use of anticoagulants completely inhibited the activation.

Galactosidases↗
PubMed1987

Inactivation of GM1-ganglioside beta-galactosidase by a specific inhibitor: a model for ganglioside storage disease.

This study was designed to establish an in vitro model with biochemical and morphological similarities to the human neurodegenerative disease GM1 gangliosidosis. Utilizing a specific inactivator of the lysosomal enzyme GM1-ganglioside beta-galactosidase (beta-D-galactopyranosylmethyl-p-nitrophenyltriazene [beta-GalMNT]) and neuroblastoma X glioma hybrid cells (NG108-15), we suppressed beta-galactosidase activity for up to 72 hours. Coincidental with suppression of this enzyme to levels less than 1% of control, we found up to a nine-fold accumulation of its substrate, the GM1-ganglioside, and the ultrastructural appearance of membranous cytoplasmic bodies. beta-GalMNT treatment suppressed growth but had little effect on the specific activity of choline acetyltransferase, lactate dehydrogenase, or other lysosomal enzymes including galactosylceramidase. This model should permit studies of the neurophysiological effects of increased ganglioside accumulation and their reversibility.

Brain↗
PubMed1989

Rapid assay of beta-galactosidase and sialyltransferase by lectin affinity high performance liquid chromatography with fluorescence detection.

Fluorescein isothiocyanate (FITC)-labelled asialotransferrin and pyridyl aminated oligosaccharides were prepared from asialotransferrin and human milk using affinity chromatography and high performance liquid chromatography (HPLC), respectively. These substances were incubated with galactosidase or sialyltransferase and then examined by lectin affinity HPLC. The elution patterns changed according to the period of incubation and amount of enzyme. This analytical method using lectin affinity HPLC with fluorescence labelled glycoprotein or oligosaccharides as the substrates has great value for detecting these enzyme under the same chromatographic conditions. In addition, differences were noted in the activity of beta-galactosidase toward oligosaccharides having the Gal beta(1----3)GlcNAc or Gal beta(1----4)GlcNAc structure at reducing termini.

Chromatography, Affinity↗
PubMed1988

A single-cell assay of beta-galactosidase activity in Saccharomyces cerevisiae.

A novel assay of single-cell exogenous beta-galactosidase activity in Saccharomyces cerevisiae has been developed. Intracellular fluorescence due to the hydrolysis of resorufin-beta-D-galactopyranoside attains a steady state between production of resorufin and its subsequent leakage from the cell. The cells are permeabilized with Triton X-100, and the assay is performed at 0 degrees C. These conditions were chosen to minimize intercellular fluorescence communication. Free resorufin in the extracellular space is bound by bovine serum albumin to prevent its uptake by cells. Two regimes of fluorescence accumulation are observed, one limited by the rate of diffusion of substrate into the cell, and one limited by the rate of enzymatic cleavage of the substrate. A quantitative correlation between fluorescence and beta-galactosidase activity is obtained under optimized assay conditions.

Cell Separation↗
PubMed1987

[Intestinal beta-galactosidase in gnotobiotic animals following monoassociation with various E. coli strains].

Germ-free rats were monoassociated by E. coli germs not utilizing (L-) or utilizing lactose (L+) on endo-medium. There was no influence of germ status on the beta-galactosidase activity in the mucosa of the small and large intestine. With standard food, beta-galactosidase activity were to be measured in the chymus of all intestinal segments and in the faeces of germ-free as well as of monoassociated rats. In the chymus of caecum and colon and in the faeces of E. coli(L+)-animals, only, the short-time (12-16 days) application of lactose containing food resulted in an increase of the enzyme activity. Compared with the E. coli(L-)-animals, the lactose content in the chymus of all intestinal segments of this test group was decreased.

Animals↗
PubMed1984

Mutagenesis of the three bases preceding the start codon of the beta-galactosidase mRNA and its effect on translation in Escherichia coli.

The effect on the translation efficiency of various mutations in the three bases (the -1 triplet) that precede the AUG start codon of the beta-galactosidase mRNA in Escherichia coli was studied. Of the 39 mutants examined, the level of expression varies over a 20-fold range. The most favorable combinations of bases in the -1 triplet are UAU and CUU. The expression levels in the mutants with UUC, UCA or AGG as the -1 triplet are 20-fold lower than those with UAU or CUU. In general, a U residue immediately preceding the start codon is more favorable for expression than any other base; furthermore, an A residue at the -2 position enhances the translation efficiency in most instances. In both cases, however, the degree of enhancement depends on its context, i.e. the neighboring bases. Although the rules derived from this study are complex, the results show that mutations in any of the three bases preceding the start codon can strongly affect the translational efficiency of the beta-galactosidase mRNA.

Base Sequence↗
PubMed1985

A Tn3 lacZ transposon for the random generation of beta-galactosidase gene fusions: application to the analysis of gene expression in Agrobacterium.

The construction and use of a Tn3-lac transposon, Tn3-HoHo1, is described. Tn3-HoHo1 can serve as a transposon mutagen and provides a new and useful system for the random generation of both transcriptional and translational lacZ gene fusions. In these fusions the production of beta-galactosidase, the lacZ gene product, is placed under the control of the gene into which Tn3-HoHo1 has inserted. The expression of the gene can thus be analyzed by monitoring beta-galactosidase activity. Tn3-HoHo1 carries a non-functional transposase gene; consequently, it can transpose only if transposase activity is supplied in trans, and is stable in the absence of this activity. A system for the insertion of Tn3-HoHo1 into sequences specifically contained within plasmids is described. The applicability of Tn3-HoHo1 was demonstrated studying three functional regions of the Agrobacterium tumefaciens A6 Ti plasmid. These regions code for octopine catabolism, virulence and plant tumor phenotype. The regulated expression of genes contained within each of these regions was analyzed in Agrobacterium employing Tn3-HoHo1 generated lac fusions.

Amino Acid Sequence↗
PubMed1981

Construction and characterization of a tufA-lacZ fusion coding for an E. coli EF-Tu-beta-galactosidase chimeric protein.

A new phage lambda cloning vector was constructed that has a single EcoRI site upstream from weakly expressed lacI-Z gene isolated by Müller-Hill and Kania (1974). An EcoRI fragment containing the complete tufA gene of E. coli was cloned on the vector and the recombinant phage was crossed into the str operon that has tufA as its last gene. Subsequent selection gave rise to a tufA-lacZ fusion that codes for a chimeric peptide. The fused peptide has a molecular weight of 148,000 and contains 40% of the N-terminal of EF-Tu followed by part of the lac repressor-beta-galactosidase fusion. The specific activity of the fused peptide is about half of the activity of normal beta-galactosidase.

Bacterial Proteins↗
PubMed1986

Adrenoleukodystrophy and beta-galactosidase deficiency: patient and carrier.

A patient with adrenoleukodystrophy and his mother, a carrier, showed an elevated ratio of very long-chain fatty acids to long-chain fatty acids and decreased beta-galactosidase activity. Other lysosomal enzyme activities were normal except for the borderline level of arylsulfatase-A activity. However, the father and other patients with variant forms of adrenoleukodystrophy showed normal beta-galactosidase and other lysosomal enzyme activities.

Adrenoleukodystrophy↗