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PubMed1981

beta-Galactosidase-linked immunoassay of prostaglandin F2 alpha.

A sensitive and specific enzyme immunoassay for prostaglandin F2 alpha was developed in which prostaglandin F2 alpha was labeled with beta-D-galactosidase. After competitive binding to antibody between enzyme-labeled and free prostaglandin F2 alpha, the immunoreactive product was precipitated by double antibody technique, and the enzyme activity of the precipitate was determined fluorometrically. The procedures allowed determination of 0.03-20 pmol of prostaglandin F2 alpha. The detection limit of 0.03 pmol was comparable with that of previously reported radioimmunoassays. Enzyme immunoassay and radioimmunoassay showed almost the same cross-reactivity with other prostaglandins and metabolites.

Binding, Competitive↗
PubMed1982

The interrelation between high- and low-molecular-weight forms of GM1-beta-galactosidase purified from porcine spleen.

1) Two forms of acid beta-galactosidase [EC 3.1.23] with different molecular weights catalyzing the hydrolysis of GM1-ganglioside and p-nitrophenyl-beta-D-galactoside were separated and purified from porcine spleen. 2) The apparent molecular weights were 400,000-600,000 and 70,000-74,000 for the high (termed Am form) and low (termed A1 form) molecular weight forms, respectively. 3) On examination by sodium dodecyl sulfate (SDS)/polyacrylamide gel electrophoresis, both forms of the enzyme had a common protein band of molecular weight 63,000, and the Am form showed three additional protein bands with molecular weights of 31,000, 21,000, and 20,000. 4) Both forms of the enzyme had similar catalytic functions with regard to pH-optimum, Km, substrate specificity and sensitivity to substrate analogues and other substances such as detergents, bovine serum albumin (BSA) and NaCl. 5) Both forms of the enzyme were fairly stable upon preincubation at 45 degrees C at acidic pH (pH 4.5), but lost their activities at neutral pH (pH 7.0). 6) The A1 form was a monomer at neutral pH (pH 7.0) and formed a dimer at acidic pH (pH 4.5). However, most of the Am form could not be converted to a dimeric form on gel filtration at acidic pH.

Animals↗
PubMed1986

Purification and characterization of alpha-galactosidase from watermelon.

An alpha-galactosidase [EC 3.2.1.22] was isolated from the fruit of the watermelon, Citrullus battich. The enzyme was purified by procedures including extraction, ammonium sulfate precipitation, and chromatographies on DEAE-Sephadex, CM-Sephadex and Sephadex G-100. The final preparation was found to be fairly homogeneous on disc and SDS-polyacrylamide gel electrophoresis, and sufficiently free from other glycosidase activities. The molecular weight of the enzyme was estimated to be 45,000 by Sephadex G-100 column chromatography and SDS-polyacrylamide gel electrophoresis. The enzyme was most active at pH 4.5 for natural substrates and at 5.9 for artificial substrates. The enzyme liberates the alpha-galactose units from oligosaccharides of the raffinose series and ceramide trihexoside, and the hemagglutination-inhibiting activities of human ovarian cyst B-glycoprotein and blood group B-type ghosts were abolished by the enzyme.

Chromatography, Gel↗
PubMed1988

Induction of helper and suppressor T cells by nonoverlapping determinants on the large protein antigen, beta-galactosidase.

The fine specificity of the T cell repertoire directed against T helper (Th)-inducing and T suppressor (Ts)-inducing determinants was examined with cyanogen bromide and tryptic peptides of Escherichia coli beta-galactosidase (GZ), a large tetrameric protein (monomer molecular weight = 116 kDa). Immunization with cyanogen bromide fragment 2 [CB-2, amino acids (a.a.) 3-92] induced both specific Th and Ts cells. Study of the induction of these functionally opposite T cell subpopulations with tryptic peptides of CB-2 indicated that Th and Ts were activated by separate, nonoverlapping determinants. Th-inducing activity resided in a nonapeptide, T6 (a.a. 44-52), whereas T4 (a.a. 27-37) induced Ts cells. The presence of distinct helper and suppressor determinants suggests that the specificity repertoire in these T cell subpopulations may differ, perhaps owing to the expression of antigen-recognizing receptors that are coded by unique gene families. Alternatively, antigen presentation structures may be physicochemically quite different, and bind to distinct parts of the peptide antigen.

Animals↗
PubMedDate not supplied

Hepatic beta galactosidase and feline GMI gangliosidosis.

This paper describes the clinical, morphological and biochemical features of three cats with a progressive neurological disorder. Clinical features were ataxia and progressive tremor. The morphological characteristics were those of lysosomal storage disease affecting neurones of the central nervous system and autonomic ganglia; membranous cytoplasmic bodies were demonstrated by electron microscopy in cerebral neurones. Chemical analysis of brain from two of the cats revealed an increased content of total gangliosides, sialic acid and a specific increase in GMI ganglioside. Enzyme analysis of homogenates of leucocytes, spleen and brain showed less than 5% or normal 4-methylumbelliferyl-beta galactosidase (4MU-beta gal) activity. In liver, activity was markedly reduced at pH values below 4.2, but there was considerable activity above this value. The properties of 4MU-beta gal in normal and diseased feline livers were investigated. Sephadex gel filtration of diseased liver homogenates showed an absence of two thermolabile "acid' components, and reduced activity of a third thermostable "neutral' component. The biochemical abnormalities found in the diseased cats are similar to those found in human juvenile GMI gangliosidosis (type 2).

Animals↗
PubMed1978

Dependence upon pH of steady-state parameters for the beta-galactosidase-catalysed hydrolyses of beta-D-galactopyranosyl derivatives of different chemical types.

The effect of pH upon the beta-galactosidase-catalyzed hydrolyses of aryl galactosides is essentially similar for each of the three steps of their hydrolysis. It differs markedly from that on the hydrolysis of galactosyl pyridinium salts; these proceed through a 'non-bottleneck' pathway. While pH increase abolishes the rate of every step of the reaction for aryl galactosides, it favors the first step of hydrolysis of the galactosyl pyridinium salts, which supports the hypothesis that catalysis of these compounds originates largely in non-covalent interactions.

Escherichia coli↗
PubMed1984

Specific cell-surface labeling of polyglycosyl chains in human erythrocytes and HL-60 cells using endo-beta-galactosidase and galactosyltransferase.

In order to identify the molecule components carrying polyglycosyl chains on cell surfaces a two-step enzymatic method was developed. In the first step, the cells were incubated with endo-beta-galactosidase to selectively expose terminal N-acetylglucosamine residues of the lactosamine backbone to the chains. In the second step these residues were glycosylated by incubation with galactosyltransferase and radioactive UDP-galactose. As many as 2.5-3.0 X 10(6) residues per cell could be transferred to human erythrocytes. Negligible amounts of labeling occurred if either of the enzymes was omitted from the incubations. Of the label 80% was found in glycoproteins. In accordance with previous observations, bands 3 and 4.5 were found to be the main carriers of polyglycosyl chains. In human promyelotic HL-60 leukemia cells, a major band of apparent molecular weight of 110000-140000 was labeled. In addition, bands of lower molecular weight which appear to have escaped detection by previous methods were also labeled. The novel labeling method was found to be simple to perform, uses commercially available reagents, and leads to the efficient and highly specific labeling of cell surface molecules carrying polyglycosyl chains.

Cell Line↗
PubMed1984

Characteristic features of an alpha-galactosidase from mung beans.

Two molecular forms, I and II (high and low molecular mass) of alpha-galactosidase were demonstrated in dry mung beans and a multi-step procedure was developed for isolating the tetrameric enzyme I in good yield. Two affinity chromatographic techniques were employed and an overall 10 000-fold purification was achieved. The enzyme was able to catalyse the hydrolysis of alpha-galactosidic linkages as well as agglutinate rabbit erythrocytes (clot formation). The clot was temporary and dissolved on longer incubation, yielding free galactose. The pH optima for both activities were similar. The enzyme also destroyed human-blood-group-B activity and increased H activity. The effect of pH on Km and Vmcx of the enzyme indicated the importance of carboxyl (pK approximately equal to 4.0) and histidine (pK approximately equal to 6.5) groups for activity. This was confirmed by amino acid modification experiments in the absence and presence of the substrate. The stoichiometry of enzyme inactivation showed the probable presence of 12 carboxyl groups and 9 histidine imidazole groups/molecule enzyme in the active site. The effects of modification of the groups on enzymic and hemagglutinating activities were parallel. A model explaining the display of both the activities by the enzyme, is presented in which hemagglutination is shown to be due to the formation of slightly stable enzyme-substrate complex. It is proposed that a true lectin should not alter the covalent status of the binding sugar.

Amino Acids↗
PubMed1986

1H-NMR studies at 500 MHz of a neutral disaccharide and sulphated di-, tetra-, hexa- and larger oligosaccharides obtained by endo-beta-galactosidase treatment of keratan sulphate.

In the preceding paper in this journal, the major oligosaccharides obtained by endo-beta-galactosidase digestion of bovine corneal keratan sulphate were identified as a neutral disaccharide, GlcNAc beta 1-3Gal, and sulphated di-, tetra-, hexa-, octa- and decasaccharides based on the sequence (-3/4GlcNAc beta 1-3Gal beta 1-)n having 1, 3, 5, 7 and 9 sulphate groups, respectively. In the present study, these oligosaccharides have been analysed by 500-MHz 1H-NMR spectroscopy using spin-decoupling and two-dimensional correlated spectroscopy experiments. The NMR data confirm the beta-configuration of all the interglycosidic linkages and are consistent with an alternating sequence of----4GlcNAc and----3Gal, a non-reducing-end N-acetylglucosamine residue and a reducing-end galactose residue. The NMR data have also established that a sulphate group is linked to the C6 position of all sugar residues except the reducing-end galactose as follows: (Formula: see text). The signals of the protons attached to the sulphated carbon atoms show marked downfield shifts (approximately 0.4 ppm from equivalent protons of non-sulphated carbon atoms), while the protons at C5 vicinal to sulphated atoms show a change of 0.1-0.2 ppm and other protons of the sulphated monosaccharides show smaller changes in chemical shift (0.01-0.1 ppm). The proton at C4 of the non-sulphated reducing-end galactose linked at C3 also shows a significant change in chemical shift (0.03 ppm).

Animals↗
PubMed1980

alpha-Galactosidase isozymes in normal individuals, and in Fabry hemizygotes and heterozygotes.

A method for staining of alpha-galactosidase with the synthetic substrate alpha-naphthyl-alpha-galactopyranoside after isoelectric focusing on gel slabs has been devised. Depending on the method used for cell extraction, at least seven isozymes could be detected in cell extracts of cultured fibroblasts from normal individuals. Thermal treatment revealed that both heat-stable and heat-labile isozymes occur in normal fibroblasts. The heat-labile isozymes were not detected in cells from Fabry hemizygotes and thus truly reflect products of the alpha-gal A locus. Three heat-stable isozymes observed in normal individuals were also found in Fabry heterozygotes and hemizygotes and are presumably determined by the alpha-gal B locus. The remaining isozymes were stained very weakly in the hemizygotes and were heat-stable. The relation of these isozymes to the A or B locus is uncertain. After treatment with neuraminidase the alpha-gal A isozymes could not be detected and one of the alpha-gal B isozymes appeared broader. The isozyme pattern observed in heterozygotes was almost identical to the normal one.

Child↗
PubMed1986

Incorporation of glycosylated beta-galactosidase into bovine brain synaptosomes.

Using a synthesized glycoprotein, beta-galactosidase modified with p-aminophenyl beta-D-galactopyranoside (beta-D-Gal beta-gal), the incorporation of the glycoprotein into bovine brain synaptosomes was studied. The uptake was mediated by a specific receptor to beta-D-galactoside, and was inhibited by GM1 ganglioside. The uptake was found to require energy and to be sensitive to metabolic inhibitors. Kinetic studies on beta-D-Gal beta-gal uptake indicated the presence of a saturable, carrier-mediated transport system in synaptosomes. By subcellular fractionation the beta-D-Gal beta-gal taken up was found in the fractions corresponding to the nucleus and membrane fragments, the soluble cytosomal fractions, and the mitochondria and lysosomes. The uptake was markedly increased by addition of Ca2+ to the incubation medium. The maximal uptake was obtained at pH 8.0 in the presence of 10 mM Ca2+ at 37 degrees C. By addition of a Ca2+ ionophore A23187, beta-D-Gal beta-gal uptake was increased in a dose-dependent way parallel to the increase in the intrasynaptosomal concentration of Ca2+. Preincubation of synaptosomes with calmodulin antagonists such as trifluoperazine and N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide (W-7) was found to inhibit the uptake markedly, and diazepam, an inhibitor of Ca2+/calmodulin-dependent protein kinase, also inhibited the uptake. At a concentration between 1 and 10 microM, 50% inhibition of the uptake was observed with either inhibitor. On the other hand, the addition of dibutyryl cyclic AMP did not affect the uptake of the glycoprotein into synaptosomes. These results suggest that the incorporation of this macromolecule is dependent on a Ca2+/calmodulin-dependent protein kinase.

Animals↗
PubMed1987

Modulation of beta-galactosidase activity in peritoneal macrophages from C57B1 mice after exposure to Proprionibacterium acnes.

Peritoneal macrophages (PM luminal diameter) from untreated C57B1 mice contain high levels of beta-galactosidase (beta-gal) and these PM luminal diameter are heterogeneous in their expression of this enzyme. Intraperitoneal (i.p.) injection of saline caused a transient depression in the level of enzyme activity in the PM luminal diameter whereas i.p. injection of Proprionibacterium acnes (P. acnes) gave rise to a marked decrease of beta-gal activity in these cells. This reduction in enzymatic activity persisted for as long as the PM luminal diameter were activated for cytotoxicity towards the L929 tumor cell line, up to 35 days after injection. beta-gal activity was present in the lavage fluid from day 2-21 after injection of P. acnes but none was detected in the lavage fluid after injection of saline. It is proposed that the enzymatic activity in the lavage fluid is derived from monocytes which migrate from the blood into the peritoneal cavity. There was an influx of granulocytes in the P. acnes group which persisted up to 35 days after injection. In contrast none were observed in the saline group after 2 days. PM luminal diameter harvested after 1-35 days were large, highly vacuolized and many contained bacteria; these PM luminal diameter had the typical morphology of activated cells. It is suggested that the processing of P. acnes by granulocytes may play a role in the activation of macrophages in the early inflammatory response, with concurrent loss in beta-gal activity. However, in the later stages, interferon-gamma and other induced lymphokines may be instrumental in causing a decrease in beta-gal activity.

Animals↗
PubMed1979

beta-Galactosidase and selective neutrality.

Three hypotheses to explain the amino acid composition of proteins are inconsistent (P congruent to 10(-9) with the experimental data for beta-galactosidase from Escherichia coli. The exceptional length of this protein, 1021 residues, permits rigorous tests of these hypotheses without complication from statistical artifacts. Either this protein is not at compositional equilibrium, which is unlikely from knowledge about other proteins, or the evolution of this protein and its coding gene have not been selectively neutral. However, the composition of approximately 60 percent of the molecule is consistent with either a selectively neutral or nonneutral evolutionary process.

Amino Acid Sequence↗
PubMed1988

A defective HSV-1 vector expresses Escherichia coli beta-galactosidase in cultured peripheral neurons.

A defective herpes simplex virus 1 (HSV-1) vector, pHSVlac, has been developed that contains a transcription unit that places the Escherichia coli lacZ gene under the control of the HSV-1 immediate early 4/5 promoter. The vector pHSVlac was propagated with the HSV-1 temperature-sensitive mutant ts K as helper virus. Infection of neurons from rat superior cervical ganglia and dorsal root ganglia in primary culture resulted in stable expression of high levels of beta-galactosidase without cell death. These HSV-1 vectors should be useful for introducing genes into postmitotic cells, such as neurons, in vitro and in vivo.

Animals↗
PubMed1977

Production of beta-galactosidase from Streptococcus thermophilus grown in whey.

beta-D-Galactosidase (EC 3.2.1.23) was extracted from Streptococcus thermophilus grown in deproteinized cheese whey. Cultural conditions optimum for maximum enzyme production were pH 7.0, 40 degrees C, and 24 h. Proteose peptone (2.0%, wt/vol) and corn steep liquor (2.8%, wt/vol) were highly stimulatory, increasing the enzyme units available in their absence from 660 U/liter of medium to 18,200 and 10,000 U/liter of medium, respectively, in their presence. There was an insignificant increase in the production of enzyme in the presence of added inorganic nitrogen and phosphorus sources. Enzymatic hydrolysis for recuction of lactose content in aqueous solution and in skim milk was studied.

Culture Media↗
PubMed1978

Rapid enumeration of Fecal Coliforms in water by a colorimetric beta-galactosidase assay.

The colorimetric beta-galactosidase assay is based upon the enzymatic hydrolysis of the substrate o-nitrophenyl-beta-D-galactoside (ONPG) by fecal coliforms. This technique provides an estimate of the fecal coliform concentration within 8 to 20 h. A 100-ml portion of test sample was passed through a 0.45 micrometer membrane filter. This filter was then incubated at 37 degrees C for 1 h in EC medium followed by the addition of filter-sterilized ONPG. The incubation was continued at 44.5 degrees C until a half-maximum absorbance (at 420 nm) was reached. The time between the start of incubation and the half-maximum absorbance was proportional to the concentration of fecal coliforms present. Escherichia coli (K-12) was used to measure the kinetics of substrate hydrolysis and the response time of different cell concentrations. High cell densities produced an immediate response, whereas 1 cell/ml will produce a response in less than 20 h. In field studies in which samples were taken from a range of grossly polluted streams to relatively clean lake water, a linear correlation between ONPG hydrolysis times and fecal coliform most-probable-number values was established. A total of 302 isolates randomly selected from positive ONPG-EC media, which were derived from 11 different habitats, were identified as E. coli (96.69 percent), Enterobacter cloacae (2.32 percent), Klebsiella pneumoniae (0.66 percent), and Citrobacter freundii (0.33 percent).

Bacteriological Techniques↗
PubMed1982

Regulation of beta-D-galactosidase synthesis in Candida pseudotropicalis.

Regulation of lactose (beta-D-galactosidase) synthesis in the lactose-utilizing yeast Candida pseudotropicalis was studied. The enzyme was inducible by lactose and galactose. When grown on these sugars the enzyme level of the yeast was 20 times or higher than when grown on glycerol. The Km and optimal pH were similar for the lactase induced either by lactose or galactose. The hydrolysis of o-nitrophenyl-beta-D-galactopyranoside by the lactase was inhibited by galactose and several analogs and galactosides, but not by glucose. Lactose uptake activity observed in lactose-grown cells was very reduced in cells grown on glucose or galactose. Glucose repressed the induction of lactase, but not the metabolic system for galactose utilization. In continuous culture on lactose medium at dilution rates below 0.2 h-1 the specific lactase activity was higher than in batch cultures and decreased with increases in dilution rate. Lactase was induced by pulses of lactose and galactose in cells growing on glucose, but only at low dilution rates were the steady-state concentration of glucose was very low.

Candida↗