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)

The interactions between Na+ (and K+) and Asp-201 of beta-galactosidase were studied. Analysis of the changes in Km and Vmax showed that the Kd for Na+ of wild type beta-galactosidase (0.36 +/- 0.09 mM) was about 10x lower than for K+ (3.9 +/- 0.6 mM). The difference is probably because of the size and other physical properties of the ions and the binding pocket. Decreases of Km as functions of Na+ and K+ for oNPG and pNPG and decreases of the Ki of both shallow and deep mode inhibitors were similar, whereas the Km and Ki of substrates and inhibitors without C6 hydroxyls remained constant. Thus, Na+ and K+ are important for binding galactosyl moieties via the C6 hydroxyl throughout catalysis. Na+ and K+ had lesser effects on the Vmax. The Vmax of pNPF and pNPA (substrates that lack a C6 hydroxyl) did not change upon addition of Na+ or K+, showing that the catalytic effects are also mediated via the C6 hydroxyl. Arrhenius plots indicated that Na+, but not K+, caused k3 (degalactosylation) to increase. Na+ also caused the k2 (galactosylation) with oNPG, but not with pNPG, to increase. In contrast, K+ caused the k2 values with both oNPG and pNPG to increase. Na+ and K+ mainly altered the entropies of activation of k2 and k3 with only small effects on the enthalpies of activation. This strongly suggests that only the positioning of the substrate, transition states, and covalent intermediate are altered by Na+ and K+. Further evidence that positioning is important was that substitution of Asp-201 with a Glu caused the Km and Ki values to increase significantly. In addition, the Kd values for Na+ or K+ were 5 to 8 fold higher. The negative charge of Asp-201 was shown to be vital for Na+ and K+ binding. Large amounts of Na+ or K+ had no effect on the very large Km and Ki values of D201N-beta-galactosidase and the Vmax values changed minimally and in a linear rather than hyperbolic way. D201F-beta-galactosidase, with a very bulky hydrophobic side chain in place of Asp, essentially obliterated all binding and catalysis.
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PMID:A study of the relationships of interactions between Asp-201, Na+ or K+, and galactosyl C6 hydroxyl and their effects on binding and reactivity of beta-galactosidase. 1506 Jun 22

Oral induction of a disseminated mucosal immune response with polyplex-based DNA vaccines requires the delivery of intact polyplexes (polyelectrolyte complexes formed by self-assembly of plasmid DNA with a cationic polymer) to subepithelial lymphoid tissue (e.g. Peyer's patches) within the gastrointestinal tract. This work describes the formulation of a microparticle polyplex carrier allowing the potential of this approach to be realised. PEGylated PEI/DNA polyplexes (DNA concentration 20 microg/ml) formed at N/P 5:0 (defined as the ratio of polycation amino groups to DNA phosphates) were stable to salt-induced aggregation and could be concentrated to a final DNA concentration of 1 mg/ml without polyplex size increase. Polyplexes containing 1:1 polyethylene glycol (PEG)/polyethylenimine (PEI) ratio (mass/mass) gave similar levels of luciferase gene expression in B16F10 cells compared to non-PEG complexes. Poly-(D,L-lactide-co-glycolide) (PLGA) microparticles containing PEGylated polyplexes (approximately 17% DNA encapsulation efficiency) were formulated using a modified double emulsion solvent evaporation method. The microencapsulation and release of intact polyplexes from the microparticle carrier was demonstrated using polyanion (heparin sulfate and poly(aspartic acid) (PAA)) displacement techniques and electron microscopy. Microparticles containing PEGylated polyplexes (24 microg beta-galactosidase DNA) were given orally to Wistar rats. Significant transgene expression (compared to background) was found in peripheral tissue (spleen) 72 h after administration. This work demonstrates the potential application of microparticle carriers for mucosal polyplex-based vaccination.
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PMID:Formulation of a microparticle carrier for oral polyplex-based DNA vaccines. 1537 19

Exosomes are vesicles of endocytic origin secreted spontaneously by dendritic cells (DCs). We have shown previously that exosomes can transfer antigen or MHC-peptide complexes between DCs, thus potentially amplifying the immune response. We had also identified milk fat globule EGF/factor VIII (MFG-E8), also called lactadherin, as one of the major exosomal proteins. MFG-E8 has two domains: an Arg-Gly-Asp sequence that binds integrins alphavbeta3 and alphavbeta5 (expressed by human DCs and macrophages) and a phosphatidyl-serine (PS) binding sequence through which it associates to PS-containing membranes (among which exosomes). MFG-E8 is thus a good candidate molecule to address exosomes to DCs. Here, we show that MFG-E8 is expressed by immature bone-marrow-derived DCs (BMDCs) and secreted in association with exosomes in vitro. We have generated mice expressing an inactive form of MFG-E8, fused to beta-galactosidase. Analyzing these mice, we demonstrate that MFG-E8 is expressed in vivo in splenic DCs. In a mouse DC-dependent, antigen-specific, CD4 T cell-stimulation assay, exosomes produced by MFG-E8-deficient BMDCs were barely less efficient than exosomes bearing MFG-E8. We conclude that MFG-E8 is efficiently targeted to exosomes but is not essential to address exosomes to mouse BMDCs. Involvement of MFG-E8/lactadherin in exosome targeting to other DC subpopulations, or to human DCs, is still possible.
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PMID:Accumulation of MFG-E8/lactadherin on exosomes from immature dendritic cells. 1598 8

Infection of cells by the highly anemogenic feline leukemia virus subgroup C (FeLV-C) is mediated by the heme exporter FLVCR1, a cell surface protein containing 12 potential transmembrane segments with six presumptive extracellular loops (ECLs). To identify FLVCR1 residues critical for mediating FeLV-C infection, we first independently isolated a human cDNA encoding the FLVCR2 protein that shares 52% identity to human FLVCR1, and we show that FLVCR2 does not function as a receptor for FeLV-C. Then, by generating specific hybrids between FLVCR1 and FLVCR2 and testing susceptibility of mouse cells expressing these hybrids to beta-galactosidase encoding FeLV-C, we identify FLVCR1 ECLs 1 and 6 as critical for mediating FeLV-C infection. Mouse cells expressing a hybrid protein containing FLVCR2 backbone with the ECL6 sequence from FLVCR1 were highly susceptible to FeLV-C infection. Using site-directed mutagenesis, we show that a single mutation of Asn463 in FLVCR2 ECL6 to an acidic Asp residue (a residue present in the corresponding position 487 in FLVCR1 ECL6) is sufficient to render FLVCR2 functional as an FeLV-C receptor. However, an Asp487Asn mutation in FLVCR1 ECL6 or substitution of the entire FLVCR1 ECL6 sequence for FLVCR2 ECL6 sequence does not disrupt receptor function. Subsequent substitutions show that residues within FLVCR1 ECL1 also contribute to mediating FeLV-C infection. Furthermore, our results suggest that FLVCR1 regions that mediate FeLV-C surface unit binding are distinct from ECL1 and ECL6. Our results are consistent with previous conclusions that infection of cells by gammaretroviruses involves interaction of virus with multiple receptor regions.
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PMID:Comprehensive mapping of receptor-functioning domains in feline leukemia virus subgroup C receptor FLVCR1. 1643 31

We find that peptides containing -Asn-Gly- sequences typically show approximately 70-80% degree of deamidation after standard overnight (approximately 12 h) tryptic digestion at 37 degrees C. This emphasizes the need for more detailed information about the deamidation reaction in -Asn-Gly- sequences, in which two deamidated species are produced, one containing an aspartic acid (-Asp-Gly-) residue and the other containing an isoaspartic acid (-betaAsp-Gly-) residue. For the peptide SLNGEWR (54-60 beta-galactosidase, E. coli), all three components of the reaction mixture were separated by HPLC on C18 300-A sorbent, with trifluoroacetic acid as an ion-pairing modifier. Their intensity ratios suggested the elution order -betaAsp-/-Asn-/-Asp-, which was subsequently confirmed by MALDI MS and MS/MS analysis. The kinetics of the deamidation was studied in detail for the synthetic SLNGEWR parent using RP HPLC with UV detection. The half-life of this peptide was found to be approximately 8 h under digestion conditions. Analysis of a large pool of peptide retention data shows that the -betaAsp-/-Asn-/ -Asp- retention order is normally observed under the above conditions, especially if the original -NG- sequence is surrounded by hydrophobic amino acids. However, changing chromatographic conditions to 100-A pore size sorbents, or using formic acid as a modifier, increases the retention time of -betaAsp- relative to the -Asn-/-Asp- pair, so the order can sometimes be different.
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PMID:Deamidation of -Asn-Gly- sequences during sample preparation for proteomics: Consequences for MALDI and HPLC-MALDI analysis. 1697 Mar 46

Global proteome analysis of protein glycosylation is a major challenge due to the inherent heterogeneous and diverse nature of this post-translational modification. It is therefore common to enzymatically remove glycans attached to protein or peptide chains prior to mass spectrometric analysis, thereby reducing the complexity and facilitating glycosylation site determinations. Here, we have used two different enzymatic deglycosylation strategies for N-glycosylation site analysis. (1) Removal of entire N-glycan chains by peptide-N-glycosidase (PNGase) digestion, with concomitant deamidation of the released asparagine residue. The reaction is carried out in H218O to facilitate identification of the formerly glycosylated peptide by incorporatation of 18O into the formed aspartic acid residue. (2) Digestion with two endo-beta-N-acetylglucosaminidases (Endo D and Endo H) that cleave the glycosidic bond between the two N-acetylglucosamine (GlcNAc) residues in the conserved N-glycan core structure, leaving single GlcNAc residues with putative fucosyl side chains attached to the peptide. To enable digestion of complex and hybrid type N-glycans, a number of exoglycosidases (beta-galactosidase, neuraminidase and N-acetyl-beta-glucosaminidase) are also included. The two strategies were here applied to identify 103 N-glycosylation sites in the Cohn IV fraction of human plasma. In addition, Endo D/H digestion uniquely enabled identification of 23 fucosylated N-glycosylation sites. Several O-glycosylated peptides were also identified with a single N-acetylhexosamine attached, arguably due to partial deglycosylation of O-glycan structures by the exoglycosidases used together with Endo D/H.
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PMID:An enzymatic deglycosylation scheme enabling identification of core fucosylated N-glycans and O-glycosylation site mapping of human plasma proteins. 1763 88

Purification of recombinant proteins is often a challenging process involving several chromatographic steps that must be optimized for each target protein. Here, we developed a self-excising module allowing single-step affinity chromatography purification of untagged recombinant proteins. It consists of a 250-residue-long self-processing module of the Neisseria meningitidis FrpC protein with a C-terminal affinity tag. The N terminus of the module is fused to the C terminus of a target protein of interest. Upon binding of the fusion protein to an affinity matrix from cell lysate and washing out contaminating proteins, site-specific cleavage of the Asp-Pro bond linking the target protein to the self-excising module is induced by calcium ions. This results in the release of the target protein with only a single aspartic acid residue added at the C terminus, while the self-excising affinity module remains trapped on the affinity matrix. The system was successfully tested with several target proteins, including glutathione-S-transferase, maltose-binding protein, beta-galactosidase, chloramphenicol acetyltransferase, and adenylate cyclase, and two different affinity tags, chitin-binding domain or poly-His. Moreover, it was demonstrated that it can be applied as an alternative to two currently existing systems, based on the self-splicing intein of Saccharomyces cerevisiae and sortase A of Staphylococcus aureus.
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PMID:Single-step affinity purification of recombinant proteins using a self-excising module from Neisseria meningitidis FrpC. 1866 6

The therapeutic usefulness of the quinoxaline derivatives XK469 (2-{4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid) and SH80 (2-{4-[(7-bromo-2-quinolinyl)oxy]phenoxy}propionic acid) has been attributed to their abilities to induce G(2)/M arrest and apoptotic or autophagic cell death. Concentrations of XK469 or SH80 > or = 5 microM were cytostatic to cultures of the normal murine melanocyte cell line Melan-a. Higher concentrations caused dose-dependent cytotoxicity. Concentrations > or =10 microM provoked dramatic morphological changes typified by marked increases in cell size and granularity. XK469/SH80-treated cultures accumulated tetraploid (4N) DNA-containing cells within 24 h of treatment, an 8N population within 3 days, and a 16N population within 5 days. Increases in ploidy correlated with the appearance of multinucleated cells. Under no circumstances did cells exhibit evidence of furrow formation. Both drugs suppressed cytokinesis in additional mammalian cell lines. Cytotoxic concentrations of XK469 elevated DEVDase activities (a measure of procaspase-3/7 activation) and enhanced cellular staining by a fluorescent analog of the pan caspase inhibitor valine-alanine-aspartic acid-fluoromethyl ketone within 48 to 96 h of treatment. Within 48 h of treatment, cytostatic and cytotoxic concentrations of XK469 elevated p21 contents, reduced Bcl-2 and Bcl-XL contents, and induced autophagy, as monitored by the accumulation of phosphatidylethanolamine-modified cleavage product of microtubule-associated protein light chain 3 (LC3-II). Cultures treated with > or =10 microM XK469 or SH80 for 5 days could not be induced to divide upon removal of drugs. Such cultures maintained high LC3-II contents, exhibited reduced cyclin E and D1 contents, and extensively expressed senescence-associated beta-galactosidase within 14 to 17 days of cessation of drug treatment. Hence, XK469 and SH80 inhibit cytokinesis, promote polyploidy, and induce senescence in Melan-a cells.
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PMID:The chemotherapeutic agents XK469 (2-{4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid) and SH80 (2-{4-[(7-bromo-2-quinolinyl)oxy]phenoxy}propionic acid) inhibit cytokinesis and promote polyploidy and induce senescence. 1906 41

It has long been believed that amino acids comprising proteins of all living organisms are only of the L-configuration, except for Gly. However, peptidyl D-amino acids were observed in hydrolysates of soluble high molecular weight fractions extracted from cells or tissues of various organisms. This strongly suggests that significant amounts of D-amino acids are naturally present in usual proteins. Thus we analyzed the D-amino acid contents of His-tag-purified beta-galactosidase and human urocortin, which were synthesized by Escherichia coli grown in controlled synthetic media. After acidic hydrolysis for various times at 110 degrees C, samples were derivatized with 4-fluoro-7-nitro-2, 1, 3-benzoxadiazole (NBD-F) and separated on a reverse-phase column followed by a chiral column into D- and L-enantiomers. The contents of D-enantiomers of Ala, Leu, Phe, Val, Asp, and Glu were determined by plotting index D/(D + L) against the incubation time for hydrolysis and extrapolating the linear regression line to 0 h to eliminate the effect of racemization of amino acids during the incubation. Significant contents of D-amino acids were reproducibly detected, the D-amino acid profile being specific to an individual protein. This finding indicated the likelihood that D-amino acids are in fact present in the purified proteins. On the other hand, the D-amino acid contents of proteins were hardly influenced by the addition of D- or L-amino acids to the cultivation medium, whereas intracellular free D-amino acids sensitively varied according to the extracellular conditions. The origin of these D-amino acids detected in proteins was discussed.
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PMID:Detection of D-amino acids in purified proteins synthesized in Escherichia coli. 1976 21

Apoptosis is an essential process for the maintenance of normal physiology. The ability to noninvasively image apoptosis in living animals would provide unique insights into its role in normal and disease processes. Herein, a recombinant reporter consisting of beta-galactosidase gene flanked by two estrogen receptor regulatory domains and intervening Asp-Glu-Val-Glu sequences was constructed to serve as a tool for in vivo assessment of apoptotic activity. The results demonstrate that when expressed in its intact form, the hybrid reporter had undetectable beta-galactosidase activity. Caspase 3 activation in response to an apoptotic stimulus resulted in cleavage of the reporter, and thereby reconstitution of beta-galactosidase activity. Enzymatic activation of the reporter during an apoptotic event enabled noninvasive measurement of beta-galactosidase activity in living cells, which correlated with traditional measures of apoptosis in a dose- and time-dependent manner. Using a near-infrared fluorescent substrate of beta-galactosidase (9H-{1,3-dichloro-9,9-dimethylacridin-2-one-7-yl} beta-D-galactopyranoside), noninvasive in vivo imaging of apoptosis was achieved in a xenograft tumor model in response to proapoptotic therapy. Finally, a transgenic mouse model was developed expressing the ER-LACZ-ER reporter within the skin. This reporter and transgenic mouse could serve as a unique tool for the study of apoptosis in living cells and animals, especially in the context of skin biology.
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PMID:A transgenic mouse for imaging caspase-dependent apoptosis within the skin. 2035 19


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