Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
 ...
PMID:Analysis of carbohydrates on IgG preparations. 789 Dec 93

Bovine zona pellucida (ZP) glycoproteins from ovarian egg emerged as three bands with molecular mass of 78 kDa, 64 kDa and 21 kDa in SDS-PAGE under reducing conditions. Endo-beta-galactosidase (E beta G) digestion of the glycoproteins yielded five products with molecular mass of 76 kDa (E beta G-76), 68 kDa (E beta G-68), 63 kDa (E beta G-63), 47 kDa (E beta G-47) and 21 kDa (E beta G-21) under the same conditions. The N-terminal amino acid sequences of E beta G-76 and E beta G-21 were identical. This fact together with the results of diagonal SDS-PAGE indicated that E beta G-21 (N-terminal region) is linked to E beta G-63 (C-terminal region) through disulfide bond to form E beta G-76. Immunoblot analysis using anti-pig ZP protein antibodies revealed that bovine E beta G-76, E beta G-68 and E beta G-47 correspond to pig PZP2, PZP3 alpha and PZP3 beta glycoproteins, respectively. The E beta G-76 and E beta G-68 components were shown to be specifically cleaved during fertilization.
 ...
PMID:Characterization of the zona pellucida glycoproteins from bovine ovarian and fertilized eggs. 791 85

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.
 ...
PMID:Kinetic mechanism and characterization of human beta-galactosidase precursor secreted by permanently transfected Chinese hamster ovary cells. 799 46

The coxsackievirus B3 (CVB3) nonstructural proteins 2B and 3AB were synthesized as beta-galactosidase fusion proteins in E. coli in order to generate specific polyclonal antisera. 2B and 3AB fusion proteins were purified by preparative SDS-polyacrylamide gel electrophoresis and inoculated into rabbits. Protein 2C-specific antiserum was produced using synthetic oligopeptides which were defined by computer based amino acid sequence analysis. Specificity of the generated antisera was analysed by immunoblotting, immunofluorescence, immunohistochemistry and immunoelectron microscopy. All antisera allowed specific detection of the viral proteins 2B, 2C, and 3AB in CVB3-infected cells as well as in myocardial and pancreatic tissue of CVB3-infected mice. In addition, the CVB3 2C-specific antiserum was shown to be highly cross-reactive with the analogous protein of other picornaviruses, including cardiotropic enterovirus serotypes such as coxsackievirus A9, coxsackievirus B (types 1-5), and echovirus 11. Moreover, the immunological detection of nonstructural proteins enables diagnosis of replicating virus in infected tissue. These results demonstrate that the generated antisera are valuable tools for diagnostic approaches. Furthermore, they may help to elucidate the role of the nonstructural proteins 2B, 2C, and 3AB in enteroviral replication and pathogenesis.
 ...
PMID:Investigation of the coxsackievirus B3 nonstructural proteins 2B, 2C, and 3AB: generation of specific polyclonal antisera and detection of replicating virus in infected tissue. 807 17

The target molecules on the cell surface of Trypanosoma cruzi trypomastigotes reacting with lytic anti-alpha-galactosyl antibodies from chronic patients with Chagas' disease (Ch anti-Gal) have been purified by solvent extraction and identified as glycoconjugates migrating in the 74-96-kDa range (F2 antigen) and in the 120-200-kDa range (F3 antigen) on SDS-PAGE. The F3 antigen was tested for binding to Ch and normal human serum (NHS) anti-Gal and to MoAb 3C9. We observed that Ch anti-Gal and MoAb 3C9, but not NHS anti-Gal, bind strongly to the trypomastigote glycoconjugates. These antibodies, however, did not compete with each other for binding to F3 molecules, indicating that they are recognizing different epitopes. Binding of Ch anti-Gal to F3 antigen is abolished by treatment of these molecules with alpha- but not beta-galactosidase. Binding of 3C9 MoAb is abolished by treatment of F3 with sialidase. F2/F3 antigens absorbed Ch anti-Gal as well as lytic antibodies from total chagasic sera. These antigens also specifically discriminate between the serum reactivity of patients with active Chagas' disease and those of sera from cured patients, drug-treated patients with dissociated serology (positive conventional serology, negative trypanolytic activity), healthy individuals, and patients with several other infectious diseases. We also observed that F2/F3 antigens are anchored to the parasite membrane via glycosylphosphatidylinositol (GPI). The alpha-galactosyl epitopes recognized by Ch anti-Gal are present in a series of O-linked oligosaccharide chains in the mucin-like glycoprotein component of the complex.
 ...
PMID:GPI-anchored glycoconjugates from Trypanosoma cruzi trypomastigotes are recognized by lytic anti-alpha-galactosyl antibodies isolated from patients with chronic Chagas' disease. 808 Dec 63

Cocksfoot/orchard grass (Dactylis glomerata) anther cDNA clones encoding the group II allergen Dac g II were previously isolated on the basis of immunoreactivity of human, rabbit, and murine antibodies with a 24-kDa protein expressed as a fusion protein with beta-galactosidase. Nucleotide sequencing reveals an open reading frame predicting expression of a 98-amino-acid (11-kDa) polypeptide exhibiting > 90% homology with the group II allergen of Lolium perenne, Lol p II. In vitro translation of different sized clone fragments generated by polymerase chain amplification confirms eukaryotic expression of a 10-12-kDa polypeptide by SDS-PAGE and the position of a translational stop apparently unrecognized during expression of lambda gt11 in E. coli. The unusual characteristics of the prokaryote-expressed fusion proteins may be exerting conformational alterations in Dac g II, as reflected by previous demonstrations of differences in human IgE immunoreactivity. Northern blot analysis using PCR-generated partial and full-length probes suggests that group II allergens may be encoded by a different family or families of temporally expressed genes from those encoding group I major allergens, although a group I gene may have been the progenitor.
 ...
PMID:Nucleotide sequence of cDNA encoding the group II allergen of cocksfoot/orchard grass (Dactylis glomerata), Dac g II. 811 60

The nucleotide (nt) sequence of a 2.57-kb Sau3A fragment carrying the Rhizobium meliloti beta-galactosidase (beta Gal)-encoding gene (RmlacZ) was determined. An open reading frame (ORF) of 2.26 kb was identified which encoded a 755-amino-acid (aa) polypeptide with a calculated molecular mass of 84,141 Da, in fair agreement with the value of 88 kDa determined by SDS-PAGE. The deduced N-terminal aa sequence was confirmed by direct sequencing of electrophoretically purified R. meliloti beta Gal. The size of the native R. meliloti beta Gal was approx. 174 kDa. Similarities were found between the aa sequence of the R. meliloti beta Gal and those from Clostridium thermosulfurogenes EM1 and Agrobacterium radiobacter, as well as human beta-glucuronidase (beta Glu). Comparisons with beta Gal from Escherichia coli, Klebsiella pneumoniae, Lactobacillus bulgaricus and Kluyveromyces lactis found only weak similarities; however, the putative active site residues appear to be conserved. The RmlacZ sequence is flanked by two partially sequenced ORFs, which show aa sequence and organisational similarities to the previously reported lac operon in A. radiobacter.
 ...
PMID:Nucleotide and deduced amino acid sequences of Rhizobium meliloti 102F34 lacZ gene: comparison with prokaryotic beta-galactosidases and human beta-glucuronidase. 816 82

Following heat shock the expression of heat shock genes is regulated by the heat shock transcription factor, HSF, known to bind to arrays of the heat shock element, NGAAN, upstream of the heat shock genes. Phosphorylation of HSF is necessary for its activation. We report that the treatment of Chinese hamster HA-1 cells with 250 nM of okadaic acid (OA), a ser/thr phosphatase inhibitor, leads to an increase in activated HSF after heat shock. This is followed by the activation of the transcription of heat shock genes as assayed by the increase in the synthesis of beta-galactosidase in an HA-1 cell line containing the heat shock promoter ligated to the beta-galactosidase gene. To investigate the specificity of OA, we used other phosphatase inhibitors. We found that treatment of HA-1 cells with 500 microM of sodium vanadate, an inhibitor of tyr/phosphatases, resulted in a three to fivefold reduction in HSF activation and binding to the heat shock element following heat shock. Such reduction in HSF activation virtually abolished beta-galactosidase induction. Reduced HSP synthesis was further confirmed by SDS-PAGE and Western blot analysis using anti-HSP-70 and 28 antibodies. Sodium vanadate treatment of heat shocked cells greatly reduced levels of thermotolerance. These results show that ser/thr and specifically tyr/phosphatase inhibitors modulate the signal transduction pathway of HSF activation.
 ...
PMID:Inhibitors of tyrosine and Ser/Thr phosphatases modulate the heat shock response. 817 93

The sequences of two previously known tail genes, R and S, of the temperate bacteriophage P2 and the sequence of an additional open reading frame (orf-30) located between S and V, were determined. Amber mutations mapping within R and S, Ram3, Ram42, Ram23, Sam75, and Sam89 were sequenced and found to be within their corresponding open reading frames. We constructed overproducing plasmids for R and S and identified these proteins by SDS-PAGE of whole-cell lysates and Coomassie blue staining. The predicted molecular masses of proteins R and S were M(r) 17,400 and 17,300, respectively, although both polypeptides migrated more slowly during gel electrophoresis than would be expected from the sequence data. orf-30 occupies the strand opposite from RS and V and is preceded by several weak potential sigma 70-RNA polymerase promoters, some of which overlap with the V promoter. A construct that had the putative orf-30 promoter region upstream of the lacZ gene produced low levels of beta-galactosidase activity in vivo. Expression from the orf-30 promoter was not stimulated by the phage P4 transcriptional activator protein, delta, which acts at all the known P2 and P4 late promoters. Insertion mutagenesis showed that orf-30 was not an essential gene for P2 growth in Escherichia coli. None of the gene or protein sequences exhibited extensive homology to sequences in the nucleic acid and protein databases. However, the R protein contains a small region homologous to one in the phage T4 tail protein gp15, which is required for T4 tails to bind heads. We propose that R and S are tail completion proteins that are essential for stable head joining.
 ...
PMID:Molecular cloning and characterization of bacteriophage P2 genes R and S involved in tail completion. 817 26

A recombinant plasmid for expression of full-length human DNA ligase I (phLig-I) was constructed in a plasmid/phage chimeric vector, pTD-T7N, which was derived from pUC118 by oligonucleotide-directed mutagenesis. The insert contained a 2757-base pair coding sequence for a whole human DNA ligase I and an extra ACC codon adjacent to the ATG initiation codon. This ACC codon was required for achieving high levels of expression of full-length DNA ligase I in Escherichia coli strain BL21. The recombinant plasmid, which was designed to exploit the T7 late promoter and the ATG initiation codon for beta-galactosidase was transfected into E. coli BL21 cells that express T7 RNA polymerase. The recombinant clone produced relatively high levels of DNA ligase I with a molecular mass of 130 kDa, as estimated by SDS-polyacrylamide gel electrophoresis. The DNA ligase was purified to near-homogeneity by the two-step column chromatographic procedure from BLphLig-I cells that had been induced with isopropyl beta-D-thiogalactoside. The specific activity, chromatographic behavior, kinetic properties, molecular mass, and antigenicity of the recombinant human DNA ligase I were indistinguishable from those of purified mammalian DNA ligase I. Metabolically labeling experiments with 32P(i) indicate that the recombinant DNA ligase I was present as an enzyme-AMP reaction intermediate, but not as a phosphoprotein, in the E. coli cells.
 ...
PMID:Expression of active human DNA ligase I in Escherichia coli cells that harbor a full-length DNA ligase I cDNA construct. 822 62


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>