Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Pharmacological and biochemical characteristics of the partially purified gamma-aminobutyric acid (GABA)B receptor using baclofen affinity column chromatography have been examined. The Scatchard analysis of [3H]GABA binding to the purified GABAB receptor showed a linear relationship and the KD and Bmax values were 60 nM and 118 pmol/mg of protein, respectively. Although GTP and Mg2+ did not affect on the GABAB receptor binding, Ca2+ significantly increased [3H]GABA binding to the purified GABAB receptor in a dose-dependent manner and showed its maximum effect at 2 mM. The enhancement of the binding by Ca2+ was found to be due to the increase of Bmax by the Scatchard analysis. The treatments with pronase and trypsin significantly decreased the binding of [3H]GABA, but phospholipase A2 had no significant effect on the binding. In addition, treatment with glycosidases such as glycopeptidase A and beta-galactosidase significantly decreased the binding of [3H]GABA to the purified GABAB receptor. These results suggest that purification of the solubilized GABAB receptor by the affinity column chromatography may result in the functional uncoupling of GABAB receptor with GTP-binding protein. Furthermore, the present results suggest that cerebral GABAB receptor may be a glycoprotein and membrane phospholipids susceptible to phospholipase A2 treatment may not be involved in the exhibition of the binding activity.
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PMID:Pharmacological and biochemical characteristics of partially purified GABAB receptor. 166 62

The 18 kDa and 32 kDa lectin binding proteins of Chlamydia trachomatis were characterized as glycoproteins by treatments with glycosidases. The proteins of the serovar L2 whole cell lysate were separated by SDS-PAGE and transferred to nitrocellulose paper. After treatment with an enzyme, the proteins were reacted with a biotinylated lectin. Each of the endoglycosidases tested affected the binding of the lectin to the protein. PNGase F inhibited the binding of Dolichos biflorus agglutinin (DBA), soybean agglutinin (SBA), and Ulex europaeus agglutinin I (UEAI) to both the 18 kDa and 32 kDa proteins. Endoglycosidase F and H inhibited the binding of these lectins to the 32 kDa protein completely and to the 18 kDa protein partially. In the exoglycosidase treatments, alpha-L-fucosidase prevented binding of only UEAI to the two proteins while beta-galactosidase inhibited the binding of SBA. Mannosidase abolished the binding of all the lectins tested. Neuraminidase had no effect. The proteins isolated by electroelution from the excised gels after SDS-PAGE were digested with an endoglycosidase. PNGase F-treated proteins showed a lower molecular weight mobility in which the lectin binding ability was destroyed. Endo-alpha-N-acetylgalactosaminidase had no effect. The polysaccharide stain of isolated proteins with p-phenylenediamine showed a positive reaction. Radiolabeling with [3H]glucosamine did not reveal the 18 kDa and 32 kDa proteins in autoradiography but [3H]galactose did.
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PMID:The characterization of lectin-binding proteins of Chlamydia trachomatis as glycoproteins. 172 47

A full-length cDNA encoding human acid beta-galactosidase was inserted into the baculovirus genome under transcriptional regulation of the viral polyhedrin gene promoter. The Spodoptera frugiperda cells infected with the recombinant virus expressed the beta-galactosidase activity 300-fold higher than human fibroblasts. Immunoblot analysis revealed an 82-kDa protein band, which was modified in molecular size by deglycosylating enzymes; an 80-kDa band appeared after N-glycanase digestion, and two bands (80-kDa and 81-kDa) appeared after endoglycosidase H digestion. This result suggested that the enzyme molecule was glycosylated, partly with high-mannose type oligosaccharides. The intracellular distribution of the enzyme observed by indirect immunofluorescence staining was perinuclear or diffusely cytoplasmic, and not characteristic of lysosomes; the enzyme was secreted to the culture medium in large quantities, and not translocated to lysosomes. Possible application of this expression system to the studies of the structure and function of normal and mutant human beta-galactosidases was discussed.
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PMID:Expression, glycosylation, and intracellular distribution of human beta-galactosidase in recombinant baculovirus-infected Spodoptera frugiperda cells. 210 71

Previous studies have shown that plasma membrane compounds are involved in the contact-dependent inhibition of growth of human diploid fibroblasts. The purification of the active plasma membrane glycoprotein is described in this report. The glycoprotein has an apparent molecular mass of 60-70 kD and, due to differential sialylation, isoelectric points between pH 5.5. and 6.2. Treatment with sialidase yielded one spot in two-dimensional gel electrophoresis with an isoelectric point of 6.3. After removal of the N-glycosidically linked oligosaccharide chains, the apparent molecular mass is reduced by approximately 22 kD. Treatment was diluted NaOH, which removes the O-glycosidically linked portion of oligosaccharides, resulted in a reduction of the apparent molecular mass by approximately 5 kD. The addition of 50 ng/ml of this glycoprotein-for which the term "contactinhibin" is proposed-in immobilized form to sparsely seeded human fibroblasts resulted in a reversible 70-80% inhibition of growth. The inhibition was not confined to human fibroblasts as other cells were also inhibited, with the exclusion of transformed cells, which are refractory to contactinhibin. The inhibitory activity was abolished by treatment with beta-galactosidase or glycopeptidase F, indicating that the glycan moiety is the biologically active part of the molecule. Confluent cultures treated with antibodies raised against contactinhibin were released from the contact-dependent inhibition of growth. In addition to enhanced saturation density, these cultures exhibited a crisscross growth pattern and the formation of foci. Immunocytochemical studies showed that contactinhibin was associated with vimentin. Furthermore, contactinhibin was found to be not expressed in a species- or organ-specific manner.
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PMID:Isolation and characterization of a 60-70-kD plasma membrane glycoprotein involved in the contact-dependent inhibition of growth. 227 80

The effect of treatments with various enzymes and chemically modifying agents on [3H]muscimol binding to a purified gamma-aminobutyric acid (GABA)/benzodiazepine receptor complex from the bovine cerebral cortex was examined. Treatments with pronase, trypsin, guanidine hydrochloride, and urea significantly decreased the binding of [3H]muscimol, but dithiothreitol, N-ethylmaleimide, reduced glutathione, oxidized glutathione, cysteine, and cystine had no significant effect. These results indicate that the GABA receptor indeed consists of protein, but -SH and -S-S- groups in the protein are not involved in the exhibition of the binding activity. On the other hand, column chromatography using concanavalin A-Sepharose eluted protein having [3H]muscimol binding activity and staining of glycoprotein using an electrophoresed slab gel indicated the existence of two bands originating from the subunits of the GABA/benzodiazepine receptor complex. Furthermore, treatments with various glycosidases such as glycopeptidase A, beta-galactosidase, and alpha-mannosidase significantly increased the binding of [3H]muscimol. These results strongly suggest that GABA/benzodiazepine receptor complex is a glycoprotein and that its carbohydrate chain may be a hybrid type. Treatment with beta-galactosidase resulted in the disappearance of the low-affinity site for [3H]muscimol binding and in an increase of Bmax of the high-affinity site, without changing the KD value. These results suggest that the carbohydrate chain in the receptor complex may have a role in exhibiting the low-affinity binding site for GABA. The observation that the enhancement of [3H]muscimol binding by treatments with beta-galactosidase and glycopeptidase A were much higher than that with alpha-mannosidase may also indicate a special importance of the beta-galactosyl residue in the inhibition of GABA receptor binding activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Glycoprotein as a constituent of purified gamma-aminobutyric acid/benzodiazepine receptor complex: structures and physiological roles of its carbohydrate chain. 303 54

Dopamine beta-hydroxylase (D beta H) (EC 1.14.17.1) from adrenal medulla is a glycoprotein with approximately 5% carbohydrate by weight. The oligosaccharide chains of this enzyme were enzymatically removed with various glycosidic enzymes (endoglycosidases D, F, and H; glycopeptidase F; alpha-mannosidase; neuraminidase; and beta-galactosidase). The time course of deglycosylation was monitored by polyacrylamide gel electrophoresis, and evidence for sugar removal was shown by a modification of the Western blot technique utilizing 125I-labeled concanavalin A and by amino acid analysis. Protein was detected in the gel by using specific antibodies and 125I-labeled protein A. Steady-state kinetic data of deglycosylated D beta H show minor differences between the native and the deglycosylated protein. The Km values for tyramine were 2.17 and 1.66 mM whereas the Km values for oxygen were 0.18 and 0.14 mM for the native and the deglycosylated protein, respectively. The Vmax values (pH 5.0) for the two forms of the enzyme were comparable, with the deglycosylated D beta H being 15% lower. These data indicate that the oligosaccharide moieties present on D beta H do not play a role in catalysis.
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PMID:Characterization and kinetic studies of deglycosylated dopamine beta-hydroxylase. 360 11

Characterization of monoclonal antibodies (MAbs) produced for therapeutic or diagnostic purposes increasingly includes an assessment of their carbohydrate content. Using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC/PAD), we have analyzed the PNGase F released oligosaccharides of several IgG preparations including human polyclonal IgG, a humanized monoclonal IgG (MAb M115), and a murine monoclonal IgG (MAb MY9-6) derived respectively from serum, hybridoma cultures, and ascites fluid. The N-linked oligosaccharides released by PNGase F treatment of the above IgGs were found to consist mainly of neutral, fucosylated, biantennary species. Comparison of glycosylation of human polyclonal IgG, MAb M115, and MAb MY9-6 revealed differences in the levels of galactosylation and in the levels as well as the form of sialic acid present. HPAEC/PAD oligosaccharide profiling, combined with the use of enzymes (PNGase F, endoglycosidase F2, endoglycosidase H, neuraminidase, beta-galactosidase, and beta-N-acetylhexosaminidase), and monosaccharide analysis allowed making of tentative structural assignments. By performing monosaccharide analysis directly on PVDF electroblotted heavy and light chain bands separated by SDS-PAGE, it was verified that IgGs used in this study were glycosylated predominantly in their heavy chain.
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PMID:Analysis of carbohydrates on IgG preparations. 789 Dec 93

Chinese hamster ovary cell clones permanently transfected with the cDNA for human lysosomal beta-galactosidase secrete the enzyme precursor into the cell medium, from which it is purified to apparent homogeneity in a single step by affinity chromatography. The purified precursor is fully active, displays the same pH optimum and Km values as the mature placental enzyme, and has an intact C-terminus. The intact enzyme when chromatographed on a Sephacryl S-200 molecular-sieve column elutes as a 105,500 Da monomer, whereas on SDS/PAGE gels the polypeptide migrates as an 88 kDa polypeptide. A time course of digestion with glycopeptide-N-glycanase shows the gradual conversion of the precursor from an 88 to a 72 kDa protein, suggesting the presence of five N-linked oligosaccharides in the protein. The precursor is readily taken up in a mannose-6-phosphate-dependent manner into beta-galactosidase-deficient, GM1-gangliosidosis fibroblasts, and the enzyme activity is returned to normal levels. We show that the stereochemical course of enzymic hydrolysis involves the retention of the beta-configuration at the anomeric centre, suggesting a double-displacement mechanism. Furthermore, the enzyme is rapidly and irreversibly inactivated in the presence of the mechanism-based inactivator 2,4-dinitrophenyl-2-deoxy-2-fluoro-beta-D-galactopyranoside, which implicates a covalent intermediate. The enzyme is also inactivated by 1-ethyl-3(3-dimethylamino-propyl)carbodi-imide and by phenylglyoxal, which implicates carboxylate and arginine residues respectively in the active site. We conclude that the beta-galactosidase precursor is functionally identical to the mature lysosomal form of the enzyme and serves as an excellent enzyme source for investigation of structure-function relationships in the protein.
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PMID:Kinetic mechanism and characterization of human beta-galactosidase precursor secreted by permanently transfected Chinese hamster ovary cells. 799 46

Antibodies against Leishmania major wheat germ agglutinin-binding glycoproteins were used to select from a genomic lambda gt11 expression library a clone coding for a L. major glycoprotein. The partial DNA sequence indicated the presence of a mosaic of repetitive sequences. Southern blot hybridisation on genomic DNA using the cloned gene as a probe at high stringency suggested a single gene, which was localised to chromosome band 18. Northern blot analysis of L. major mRNA detected a major transcript of 7.5 kb and a minor 4.0-kb transcript. Antibodies affinity-purified on the fusion protein identified a complex of two water-soluble cytoplasmic polypeptides of approximately 96 kDa and 92 kDa in L. major promastigotes and amastigotes. They also recognised polypeptides in other Leishmania species, in Crithidia lucilliae and very weakly in Leptomonas. The apparent molecular weight of these polypeptides, while conserved within each species, varied between species. A peptide map of the two polypeptides from L. major generated an identical pattern suggesting a close relatedness at the protein level. This protein complex was not hydrolysed by N-glycanase and was not affected by tunicamycin, but treatment with anhydrous hydrogen fluoride suggested that it is O-glycosylated. The glycan moiety appears to be N-acetylglucosamine, and N-acetylglucosamine beta-1,4-galactosyltransferase was capable of adding [3H]galactose to it. This was susceptible to beta elimination and beta-galactosidase treatment. Taken together, the data indicates that gp96/92 belongs to the newly described class of cytoplasmic and nuclear glycoproteins containing O-linked N-acetylglucosamine.
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PMID:Identification, characterisation and genomic cloning of a O-linked N-acetylglucosamine-containing cytoplasmic Leishmania glycoprotein. 811 27

During short incubations of a Golgi apparatus-enriched subcellular fraction from rat liver with UDP-[3H]GlcNAc, label is efficiently transferred to endogenous acceptors. Most of the macromolecular radioactivity is specifically released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase, indicating that it is mainly associated with N-linked oligosaccharides. The glycoprotein acceptors are resistant to proteases unless detergent is added in amounts greater than the critical micellar concentration. This shows that the acceptors are within the lumen of intact compartments, which have the correct topological orientation expected for the Golgi apparatus in intact cells. Structural characterization of the radiolabeled N-linked oligosaccharides shows a variety of distinct neutral and anionic species. The neutral chains include bi-, tri-, and tetra-antennary molecules with terminal beta-[3H] GlcNAc residues. In vitro sialylation shows that some of the tetra-antennary chains have beta 1,3-linked Gal residues on their unlabeled antennae. An unknown modification appears to block the action of beta-galactosidase on these galactosylated oligosaccharides. Chasing the labeling reaction with a mixtures of UDP-Gal, CMP-Neu5Ac, and adenosine 3'-phosphate,5'-phosphosulfate causes an increase in the percent of radiolabeled anionic oligosaccharides. Most of the negative charge is due to sialic acid (Sia), and some appears to be in phosphodiester-linked [3H]GlcNAc. The sialylated oligosaccharides are a mixture of bi-, tri-, and tetra-antennary species with 1-3-Sia residues, and some of the [3H]GlcNAc residues are directly covered with unlabeled Gal and Sia residues. This in vitro approach should recapitulate reactions that occur in the biosynthesis of N-linked oligosaccharides in the Golgi apparatus of the intact cell. Since the conditions during labeling do not permit inter-compartmental transport, the oligosaccharides produced should represent the biosynthetic capabilities of individual Golgi compartments. Evidence is presented for a functional association of GlcNAc transferases I, II, and alpha-mannosidase II, with separation from GlcNAc transferase IV and/or V. The structures also indicate co-compartmentalization of several GlcNAc transferase(s) with beta-galactosyltransferase(s) and sialyltransferase(s). The compartmental organization of the Golgi apparatus is discussed in light of these findings.
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PMID:Biosynthesis of oligosaccharides in intact Golgi preparations from rat liver. Analysis of N-linked glycans labeled by UDP-[6-3H]N-acetylglucosamine. 834 99


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