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)

GM1 ganglioside beta-galactosidase (beta-Gal) is deficient in the autosomal recessive disorder GM1 gangliosidosis. A portion of the enzyme occurs in a complex with neuraminidase and an additional glycoprotein, protective protein, but the nature of the interactions conferring the stability of the complex is unknown. Affinity chromatography of beta-Gal on p-aminophenylthiogalactose-Sepharose (PATG-Sepharose) at pH 4.3, the pH optimum of beta-Gal, resulted in a 260-fold enrichment of beta-Gal, but the major protein in the fraction had an M(r) value of 74,000. Affinity chromatography on PATG-Sepharose at pH 5.2 showed substantial enrichment (4000-fold) of beta-Gal, and the mature form of the enzyme (M(r) 64,000) was the major protein in the preparation. Using h.p.l.c. molecular-sieve chromatography, we found that about 15% of the total beta-Gal occurred in a high-M(r) form (greater than 600,000), the presumptive complex, with 85% eluting at M(r) 150,000, suggestive of a dimer. This distribution was independent of both high (60 mg/ml) and low (5 mg/ml) protein concentration and the pH (pH 4.3 or 5.2) of the sample applied to the column. Furthermore, incubation for 90 min at 37 degrees C, conditions which had previously been suggested as optimal for formation of the complex, had no effect on this distribution. Further fractionation by anion-exchange chromatography and a second affinity column step yielded a beta-Gal preparation that contained a single polypeptide chain (M(r) 64,000), was devoid of neuraminidase and protective protein (absent carboxypeptidase activity), and when injected into rabbits gave rise to monospecific rabbit antisera. We conclude that the protein composition of the complex is variable (i.e. it is different when isolated at pH 4.3 and 5.2) and that the amount of beta-Gal tightly associated with the complex constitutes a small fraction of the total beta-Gal activity. The more prevalent form of the enzyme is a beta-Gal homodimer that is stable and devoid of either neuraminidase activity or protective protein.
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PMID:Human placental beta-galactosidase. Characterization of the dimer and complex forms of the enzyme. 149 20

Treatment of exponentially growing cells of Escherichia coli with membrane-binding drugs such as chlorpromazine (CPZ) and procaine resulted in an induction of manganese-superoxide dismutase (Mn-SOD). A slight decrease was observed in the amount of Fe-SOD. The induction of Mn-SOD required de novo synthesis of this enzyme, since it was suppressed by rifampin. The treatment did not cause the induction of Mn-SOD when performed under anaerobic conditions. In E. coli cells with a sodA-lacZ operon fusion, CPZ and procaine induced beta-galactosidase in the presence of oxygen, whereas it was not expressed and was not induced by CPZ and procaine under anaerobic conditions. Although CPZ reduced the ability of cell suspensions to take up oxygen, it increased the cyanide-resistant fraction of the total respiration. Therefore, it appeared likely that the induction of the sodA gene was a response to an increase in superoxide radical production mediated by these membrane-binding drugs in E. coli cells, possibly by disruption of the electron transport systems in the cell membranes.
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PMID:Induction of manganese-superoxide dismutase by membrane-binding drugs in Escherichia coli. 204 68

The structural requirements for oligomerization and the generation of a functional mannose 6-phosphate (Man-6-P) binding site of the cation-dependent mannose 6-phosphate receptor (CD-MPR) were analyzed. Chemical cross-linking studies on affinity-purified CD-MPR and on solubilized membranes containing the receptor indicate that the CD-MPR exists as a homodimer. To determine whether dimer formation is necessary for the generation of a Man-6-P binding site, a cDNA coding for a truncated receptor consisting of only the signal sequence and the extracytoplasmic domain was constructed and expressed in Xenopus laevis oocytes. The expressed protein was completely soluble, monomeric in structure, and capable of binding phosphomannosyl residues. Like the dimeric native receptor, the truncated receptor can release its ligand at low pH. Ligand blot analysis using bovine testes beta-galactosidase showed that the monomeric form of the CD-MPR from bovine liver and testes is capable of binding Man-6-P. These results indicate that the extracytoplasmic domain of the receptor contains all the information necessary for ligand binding as well as for acid-dependent ligand dissociation and that oligomerization is not required for the formation of a functional Man-6-P binding site. Several different mutant CD-MPRs were generated and expressed in X. laevis oocytes to determine what region of the receptor is involved in oligomerization. Chemical cross-linking analyses of these mutant proteins indicate that the transmembrane domain is important for establishing the quaternary structure of the CD-MPR.
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PMID:The cation-dependent mannose 6-phosphate receptor. Structural requirements for mannose 6-phosphate binding and oligomerization. 254 94

Induction of Drosophila hsp70 protein was detected during aging in flight muscle and leg muscle in the absence of heat shock, using an hsp70-specific monoclonal antibody, and in transgenic flies containing hsp70-beta-galactosidase fusion protein reporter constructs. While hsp70 and reporter proteins were induced during aging, hsp70 message levels were not, indicating that aging-specific induction is primarily posttranscriptional. In contrast, hsp22 and hsp23 were found to be induced during aging at the RNA level and with a broader tissue distribution. The same muscle-specific hsp70 reporter expression pattern was observed in young flies mutant for catalase (H2O2:H2O2 oxidoreductase, EC 1.11.1.6). In catalase (cat) hypomorphic lines where flies survived to older ages, the time course of hsp70 reporter expression during aging was accelerated, and the initial and ultimate levels of expression were increased. The hsp70 reporter was also induced in young flies mutant for copper/zinc superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1). Taken together, the results suggest that aging-specific hsp70 expression may be a result of oxidative damage.
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PMID:Muscle-specific expression of Drosophila hsp70 in response to aging and oxidative stress. 747 94

Homodimers of immunoglobulin VL domains are minimal models of antibodies in that they display an ensemble of six hypervariable loops. Bence Jones protein REI is a mixture of a complete kappa light chain and the corresponding variable domain (REIV). The known three-dimensional structure of the REIV dimer (Epp et al., 1975, Biochemistry 14, 4943-4952) provides a basis for studying dimer stabilization by protein engineering. Mutant REIV-L94H was constructed and shown to have an equilibrium constant of dimerization about one order of magnitude higher than wildtype REIV. By fusing REIV and variants to the aminoterminal part of the Vibrio cholerae ToxR regulator protein (Miller et al., 1987, Cell 48, 271-279), a transcriptional signal in E. coli can be derived from REIV homodimer formation constant. The system senses dimerization of the immunoglobulin part of the fusion protein, located in the periplasmatic space, and transduces the signal as transcriptional activation to a ctx::lacZ gene construct integrated into the E. coli chromosome. There is positive correlation between the propensities of homodimer formation and the rate of transcriptional initiation at the ctx promoter. Since beta-galactosidase levels can easily be measured colorimetrically in crude cell lysates of a large number of clones using an ELISA reader, this procedure constitutes all elements required for a genetic screen in E. coli for immunoglobulin variants with altered association constants.
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PMID:Dimerization of Bence Jones proteins: linking the rate of transcription from an Escherichia coli promoter to the association constant of REIV. 800 58

We report on a general strategy for engineering dominant negative mutations that, in principle, requires neither extensive structural or functional knowledge of the targeted protein. The approach consists of fusing the lysosomal protease cathepsin B (CB) to a subunit of a multimeric protein. The CB fusion polypeptide can proteolytically digest the multimer and/or detour the multimer from its usual subcellular destination to the lysosome. We first demonstrate the general validity of the approach with CB fusion to E. coli lacZ, encoding tetrameric beta-galactosidase. Cotransfection of NIH 3T3 cells with a vector expressing a CB-lacZ fusion inhibits the beta-galactosidase activity produced by transfection of lacZ alone. We infer that the dominant negative inhibition results from both direct proteolysis of the beta-galactosidase tetramer by the fusion subunit and detour of the tetramer to the lysosome. In a specific application of this strategy, we have fused CB to the dimeric bHLH skeletal muscle transcription factor MyoD. The CB-MyoD fusion protein localizes to the cytoplasm, presumably the lysosome, demonstrating the dominance of lysosomal localization to nuclear localization. The CB-MyoD fusion appears to divert homodimerizing native MyoD from its usual nuclear destination, consequently inhibiting MyoD-mediated transactivation and in vitro differentiation of C2C12 myoblasts. Surprisingly, the CB-MyoD fusion fails to interact with the bHLH heterodimerization partners, E12 and E47, suggesting preferential MyoD homodimer formation, at least in the prenuclear cellular compartments.
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PMID:Preferential MyoD homodimer formation demonstrated by a general method of dominant negative mutation employing fusion with a lysosomal protease. 892 85

The mouse glucocorticoid receptor-interacting protein (GRIP1) is a member of the ERAP160 family of nuclear receptor (NR) coactivators (including SRC-1 and TIF2) which function as bridging proteins between ligand-activated NRs bound to cognate hormone-response elements (HREs) and the transcription initiation apparatus (TIA). Although these coactivators bind to several NRs, studies overexpressing these coactivators with these NRs in mammalian cells have not uniformly observed a corresponding enhancement of ligand-dependent transactivation. Here, we show that GRIP1 interacts in vitro in a ligand-dependent manner with thyroid receptor, retinoic acid receptor, and retinoid X receptor. Additionally, in yeast (Saccharomyces cerevisiae) GRIP1 coactivator protein markedly increased the ability of these full-length class II NRs to transactivate beta-galactosidase reporter genes containing cognate HREs. The magnitude of GRIP1 enhancement of liganded NR homodimer was dependent upon NR subtype and HRE configuration. For most HRE configurations, thyroid receptor and retinoic acid receptor homodimers were essentially unresponsive or very weakly active in the absence of GRIP1, but GRIP1 dramatically restored the ligand-dependent function of these NRs. Although GRIP1 exerted no significant effect on NR homodimers in the absence of their cognate ligands, it increased the transactivation of unliganded NR heterodimers. Whether GRIP1 increased ligand-dependent transactivation of a heterodimer to levels greater than that of the cognate homodimer was determined by HRE configuration and copy number. Compared with the limitations of yeast two-hybrid and mammalian coexpression systems, the yeast HRE-assay systems described in this report facilitated both the detection of putative mammalian NR coactivator function and the elucidation of their mechanisms of transactivational enhancement.
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PMID:Yeast hormone response element assays detect and characterize GRIP1 coactivator-dependent activation of transcription by thyroid and retinoid nuclear receptors. 910 40

In Escherichia coli, fatty acid synthesis and degradation are coordinately controlled at the level of transcription by FadR. FadR represses transcription of at least eight genes required for fatty acid transport and beta-oxidation and activates transcription of at least two genes required for unsaturated fatty acid biosynthesis and the gene encoding the transcriptional regulator of the aceBAK operon encoding the glyoxylate shunt enzymes, IclR. FadR-dependent DNA binding and transcriptional activation is prevented by long chain fatty acyl-CoA. In the present work, we provide physical and genetic evidence that FadR exists as a homodimer in solution and in vivo. Native polyacrylamide gel electrophoresis and glycerol gradient ultracentrifugation of the purified protein show that native FadR was a homodimer in solution with an apparent molecular mass of 53.5 and 57.8 kDa, respectively. Dominant negative mutations in fadR were generated by random and site-directed mutagenesis. Each mutation mapped to the amino terminus of the protein (residues 1-66) and resulted in a decrease in DNA binding in vitro. In an effort to separate domains of FadR required for DNA binding, dimerization, and ligand binding, chimeric protein fusions between the DNA binding domain of LexA and different regions of FadR were constructed. One fusion, LexA1-87-FadR102-239, was able to repress the LexA reporter sulA-lacZ, and beta-galactosidase activities were derepressed by fatty acids, suggesting that the fusion protein had determinants both for dimerization and ligand binding. These studies support the conclusion that native FadR exists as a stable homo-dimer in solution and that determinants for DNA binding and acyl-CoA binding are found within the amino terminus and carboxyl terminus, respectively.
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PMID:Characterization of the fatty acid-responsive transcription factor FadR. Biochemical and genetic analyses of the native conformation and functional domains. 938 99

Increased production of oxygen free radicals is an important mechanism of endothelial dysfunction in diabetes mellitus. Our goal was to test whether adenovirus (Ad)-mediated gene transfer of copper/zinc (CuZn) or manganese superoxide dismutase (Mn SOD) improves relaxation of diabetic vessels. The aortas from 9 alloxan-induced diabetic mellitus (DM) and 16 control rabbits were used. Control and DM rings were transduced ex vivo with Ad vectors encoding Mn SOD (AdMn SOD), CuZn SOD (AdCuZn SOD), beta-galactosidase (Ad(beta)gal), or diluents. In the absence of gene transfer, SOD activity was significantly increased in DM aortas. Transgene expression in DM AdCuZn SOD and DM AdMn SOD-transduced vessels was confirmed by Western blot analysis and by increased SOD activity (DM AdCuZn SOD, 76.2 +/- 9.3; DM AdMn SOD, 65.2 +/- 4.8; P < 0.05 vs. DM Ad(beta)gal; 50.9 +/- 4.4 U/mg protein). Superoxide production was increased in DM Ad(beta)gal-transduced aorta and relaxations to acetylcholine were impaired in these vessels. Gene transfer of CuZn SOD and Mn SOD corrected both of these defects. Thus Ad-mediated gene transfer CuZn and Mn SOD to the diabetic aorta improves endothelium-dependent relaxation.
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PMID:Gene transfer of superoxide dismutase isoforms reverses endothelial dysfunction in diabetic rabbit aorta. 1135 6

Mitochondria are thought to play a major role in hepatic oxidative stress associated with alcohol-induced liver injury. Thus, the hypothesis that delivery of the mitochondrial isoform of superoxide dismutase (Mn-SOD) via recombinant adenovirus would reduce alcohol-induced liver injury was tested. Rats were given recombinant adenovirus containing Mn-SOD (Ad.SOD2) or beta-galactosidase (Ad.lacZ) and then fed alcohol enterally for 4 weeks. Mn-SOD expression and activity of Ad.SOD2 in liver mitochondria of infected animals was increased nearly 3-fold compared with Ad.lacZ-infected controls. Mitochondrial glutathione levels in Ad.lacZ-infected animals were decreased after 4 weeks of chronic ethanol, as expected, but were unchanged in Ad.SOD2-infected animals. Alanine aminotransferase was elevated significantly by ethanol, an effect that was prevented by Ad.SOD2. Moreover, pathology (e.g. the sum of steatosis, inflammation, and necrosis) was elevated dramatically by ethanol in Ad.lacZ-treated rats. This effect was also blunted in animals infected with Ad.SOD2. Neutrophil infiltration was increased about 3-fold in livers from both Ad.lacZ- and Ad.SOD2-infected rats by ethanol treatment. Moreover, ESR-detectable free radical adducts in bile were increased about 8-fold by ethanol. Using (13)C-labeled ethanol, it was determined that nearly 60% of total adducts were due to the alpha-hydroxyethyl radical adduct. This increase in radical formation was blocked completely by Ad.SOD2 infection. Furthermore, apoptosis of hepatocytes was increased about 5-fold by ethanol, an effect also blocked by Ad.SOD2. Interestingly, tumor necrosis factor-alpha mRNA was elevated to the same extent in both Ad.lacZ- and Ad.SOD2-infected animals follows ethanol exposure. These data suggest that hepatocyte mitochondrial oxidative stress is involved in alcohol-induced liver damage and likely follows Kupffer cell activation, cytokine production, and neutrophil infiltration. These results also support the hypothesis that mitochondrial oxidant production is a critical factor in parenchymal cell death caused by alcohol.
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PMID:Overexpression of manganese superoxide dismutase prevents alcohol-induced liver injury in the rat. 1147 87


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