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)

Interaction between a peptide hormone and extracellular domains of its receptor is a crucial step for initiation of hormone action. We have developed a modification of the yeast two-hybrid system to study this interaction and have used it to characterize the interaction of insulin-like growth factor 1 (IGF-1) with its receptor by using GAL4 transcriptional regulation with a beta-galactosidase assay as readout. In this system, IGF-1 and proIGF-1 bound to the cysteine-rich domain, extracellular domain, or entire IGF-1 proreceptor. This interaction was specific. Thus, proinsulin showed no significant interaction with the IGF-1 receptor, while a chimeric proinsulin containing the C-peptide of IGF-1 had an intermediate interaction, consistent with its affinity for the IGF-1 receptor. Over 2000 IGF-1 mutants were generated by PCR and screened for interaction with the color assay. About 40% showed a strong interaction, 20% showed an intermediate interaction, and 40% give little or no signal. Of 50 mutants that were sequenced, several (Leu-5 --> His, Glu-9 --> Val, Arg-37 --> Gly, and Met-59 --> Leu) appeared to enhance receptor association, others resulted in weaker receptor interaction (Tyr-31 --> Phe and Ile-43 --> Phe), and two gave no detectable signal (Leu-14 --> Arg and Glu-46 --> Ala). Using PCR-based mutagenesis with proinsulin, we also identified a gain of function mutant (proinsulin Leu-17 --> Pro) that allowed for a strong IGF-1-receptor interaction. These data demonstrate that the specificity of the interaction between a hormone and its receptor can be characterized with high efficiency in the two-hybrid system and that novel hormone analogues may be found by this method.
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PMID:Analysis of a peptide hormone-receptor interaction in the yeast two-hybrid system. 937

The FhuA protein of Escherichia coli K-12 transports ferrichrome, the antibiotic albomycin, colicin M, and microcin 25 across the outer membrane and serves as a receptor for the phages T1, T5, phi80, and UC-1. FhuA is activated by the electrochemical potential of the cytoplasmic membrane, which probably opens a channel in FhuA. It is thought that the proteins TonB, ExbB, and ExbD function as a coupling device between the cytoplasmic membrane and the outer membrane. Excision of 34 residues from FhuA, tentatively designated the gating loop, converts FhuA into a permanently open channel. FhuA contains two disulfide bridges, one in the gating loop and one close to the C-terminal end. Reduction of the disulfide bridges results in a low in vivo reaction of the cysteines in the gating loop and no reaction of the C-terminal cysteines with biotin-maleimide, as determined by streptavidin-beta-galactosidase bound to biotin. In this study we show that a cysteine residue introduced into the gating loop by replacement of Asp-336 displayed a rather high reactivity and was used to monitor structural changes in FhuA upon binding of ferrichrome. Flow cytometric analysis revealed fluorescence quenching by ferrichrome and albomycin of fluorescein-maleimide bound to FhuA. Ferrichrome did not inhibit Cys-336 labeling. In contrast, labeling of Cys-347, obtained by replacing Val-347 in the gating loop, was inhibited by ferrichrome, but ferrichrome quenching was negligible. It is concluded that binding of ferrichrome causes a conformational change of the gating loop and that Cys-347 is part of or close to the ferrichrome binding site. Fluorescence quenching was independent of the TonB activity. The newly introduced cysteines and the replacement of the existing cysteines by serine did not alter sensitivity of cells to the FhuA ligands tested (T5, phi80, T1, colicin M, and albomycin) and fully supported growth on ferrichrome as the sole iron source. Since cells of E. coli K-12 display no reactivity to thiol reagents, newly introduced cysteines can be used to determine surface-exposed regions of outer membrane proteins and to monitor conformational changes during their function.
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PMID:Specific in vivo labeling of cell surface-exposed protein loops: reactive cysteines in the predicted gating loop mark a ferrichrome binding site and a ligand-induced conformational change of the Escherichia coli FhuA protein. 945 64

A study of proteolysis effects on recombinant protein yield was completed using the insect cell (Sf-9)-baculovirus (AcNPV) expression system. Activities of protease and beta-galactosidase (beta-gal), a marker heterologous protein, were assayed at various multiplicities of infection (MOI = 1, 5, and 20) on a time course postinfection. Also, several protein-substrate gel electrophoresis assays were run using gelatin, beta-gal, and bovine serum albumin (BSA) in the gel matrix, to determine the protein specificity of the proteases. The most abundant protease activity (cysteine), found at 49 kDa, degraded all three substrates, pre- and post-infection. Two other protease activities (40 and 36 kDa) appeared on polyacrylamide gel electrophoresis (PAGE) gels after 72 hpi (hours postinfection). In addition, the culture with the highest MOI had the highest beta-gal activity until 72 hpi, when the activity dramatically decreased coincidentally with a 2.5-fold increase in protease activity. This result and the electrophoresis evidence that the protease is specific to beta-gal, indicate that there is a negative correlation between protease activity and recombinant protein yield. These results guide efforts to control product-degrading proteolysis in insect cell-baculovirus expression systems by harvest timing and the addition of protease inhibitors.
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PMID:Appearance of protease activities coincides with p10 and polyhedrin-driven protein production in the baculovirus expression system: effects on yield. 954 73

A 5 kb region upstream of katA at 82 degrees on the Bacillus subtilis chromosome contains five ORFs organized in an operon-like structure. Based on sequence similarity, three of the ORFs are likely to encode an ABC transport system (ssuBAC) and another to encode a monooxygenase (ssuD). The deduced amino acid sequence of the last ORF (ygaN) shows no similarity to any known protein. B. subtilis can utilize a range of aliphatic sulfonates such as alkanesulfonates, taurine, isethionate and sulfoacetate as a source of sulfur, but not when ssuA and ssuC are disrupted by insertion of a neomycin-resistance gene. Utilization of aliphatic sulfonates was not affected in a strain lacking 3'-phosphoadenosine 5'-phosphosulfate (PAPS) sulfotransferase, indicating that sulfate is not an intermediate in the assimilation of sulfonate-sulfur. Sulfate or cysteine prevented expression of beta-galactosidase from a transcriptional ssuD::lacZ fusion. It is proposed that ssuBACD encode a system for ATP-dependent transport of alkanesulfonates and an oxygenase required for their desulfonation.
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PMID:Bacillus subtilis genes for the utilization of sulfur from aliphatic sulfonates. 978 4

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. The underlying ubiquitin-dependent proteolytic system, called the N-end rule pathway, is organized hierarchically: N-terminal aspartate and glutamate (and also cysteine in metazoans) are secondary destabilizing residues, in that they function through their conjugation, by arginyl-tRNA-protein transferase (R-transferase), to arginine, a primary destabilizing residue. We isolated cDNA encoding the 516-residue mouse R-transferase, ATE1p, and found two species, termed Ate1-1 and Ate1-2. The Ate1 mRNAs are produced through a most unusual alternative splicing that retains one or the other of the two homologous 129-bp exons, which are adjacent in the mouse Ate1 gene. Human ATE1 also contains the alternative 129-bp exons, whereas the plant (Arabidopsis thaliana) and fly (Drosophila melanogaster) Ate1 genes encode a single form of ATE1p. A fusion of ATE1-1p with green fluorescent protein (GFP) is present in both the nucleus and the cytosol, whereas ATE1-2p-GFP is exclusively cytosolic. Mouse ATE1-1p and ATE1-2p were examined by expressing them in ate1Delta Saccharomyces cerevisiae in the presence of test substrates that included Asp-betagal (beta-galactosidase) and Cys-betagal. Both forms of the mouse R-transferase conferred instability on Asp-betagal (but not on Cys-betagal) through the arginylation of its N-terminal Asp, the ATE1-1p enzyme being more active than ATE1-2p. The ratio of Ate1-1 to Ate1-2 mRNA varies greatly among the mouse tissues; it is approximately 0.1 in the skeletal muscle, approximately 0.25 in the spleen, approximately 3.3 in the liver and brain, and approximately 10 in the testis, suggesting that the two R-transferases are functionally distinct.
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PMID:Alternative splicing results in differential expression, activity, and localization of the two forms of arginyl-tRNA-protein transferase, a component of the N-end rule pathway. 985 43

Lactobacillus (Lb.) gasseri JCM1031, which is classified into the B1 subgroup of the Lb. acidophilus group of lactic acid bacteria, characteristically produces two different phospho-beta-galactosidases (P-beta-gal) I and II in the same cytosol as reported in our previous papers [Biosci. Biotech. Biochem., 60, 139-141, 708-710 (1996)]. To clarify the functional and genetic properties of the two enzymes, the structural genes of P-beta-gal I and II were cloned and sequenced. The structural gene of P-beta-gal I had 1,446 bp, encoding a polypeptide of 482 amino acid residues. The structural gene of P-beta-gal II had 1,473 bp, encoding a polypeptide of 491 amino acid residues. The deduced relative molecular masses of 55,188 and 56,243 agreed well with the previous value obtained from the purified P-beta-gal I and II protein, respectively. Multiple alignment of the protein sequence of P-beta-gal I and II with those of P-beta-gals from 5 microorganisms had 30-35% identity on the amino acid level, but those with phospho-beta-glucosidases from 5 microorganisms had the relatively high identity of about 50%. Considering that this strain grows on lactose medium and shows no beta-galactosidase activity, and that purified P-beta-gal I and II can obviously hydrolyze o-nitrophenyl-beta-D-galactopyranoside 6-phosphate (substrate), and also the conservation of a cysteine residue in the molecule, the P-beta-gal I and II were each confirmed as a novel P-beta-gal enzyme.
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PMID:Molecular cloning and sequencing of two phospho-beta-galactosidase I and II genes of Lactobacillus gasseri JCM1031 isolated from human intestine. 997 58

In cultured human endothelial cells, physiological levels of NO prevent apoptosis and interfere with the activation of the caspase cascade. In vitro data have demonstrated that NO inhibits the activity of caspase-3 by S-nitrosation of the enzyme. Here we present evidence for the in vivo occurrence and functional relevance of this novel antiapoptotic mechanism. To demonstrate that the cysteine residue Cys-163 of caspase-3 is S-nitrosated, cells were transfected with the Myc-tagged p17 subunit of caspase-3. After incubation of the transfected cells with different NO donors, Myc-tagged p17 was immunoprecipitated with anti-Myc antibody. S-Nitrosothiol was detected in the immunoprecipitate by electron spin resonance spectroscopy after liberation and spin trapping of NO by N-methyl-D-glucamine-dithiocarbamate-iron complex. Transfection of cells with a p17 mutant, where the essential Cys-163 was mutated into alanine, completely prevented S-nitrosation of the enzyme. As a functional correlate, in human umbilical vein endothelial cells the NO donors sodium nitroprusside or PAPA NONOate (50 microM) significantly reduced the increase in caspase-3-like activity induced by overexpressing caspase-3 by 75 and 70%, respectively. When human umbilical vein endothelial cells were cotransfected with beta-galactosidase, morphological analysis of stained cells revealed that cell death induction by overexpression of caspase-3 was completely suppressed in the presence of sodium nitroprusside, PAPA NONOate, or S-nitroso-L-cysteine (50 microM). Thus, NO supplied by exogenous NO donors serves in vivo as an antiapoptotic regulator of caspase activity via S-nitrosation of the Cys-163 residue of caspase-3.
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PMID:Nitric oxide inhibits caspase-3 by S-nitrosation in vivo. 1006 32

Phosphatases, C4 and C8 esterases, leucine and valine aminopeptidases, N-acetyl-beta-glucosaminidase, beta-glucosidase, beta-galactosidase and beta-glucuronidase were detected in extracts of the parasitic mite Psoroptes cuniculi. Lipase, trypsin-like and chymotrypsin-like activities were not present. Haemoglobin was hydrolysed by a detergent-soluble fraction of the mite extracts with a maximum hydrolysis between pH 3 and 5. Acid proteinase activity was greater against haemoglobin than bovine serum albumin. Inhibitors of cysteine, serine and metallo-proteinases failed to inhibit the hydrolysis of H-Pro-Thr-Glu-Phe-Phe(NO2)-Arg-Leu-OH while pepstatin A inhibited its hydrolysis in a dose-dependent manner (IC50 8.02 x 10(-11) M (+/- 0.30 x 10(-11). Thermal inactivation of the proteolytic activity followed an exponential decay pattern. Typical K(m) and Vmax values were 7.2 x 10(-5) (+/- 0.7 x 10(-5) M-1 and 1.13 x 10(-3) (+/- 0.05 x 10(-3) OD unit-1 min-1 respectively. Acid proteinase activity eluted from a size exclusion column in a single, major peak representing a molecular weight range of 21-24.5 kDa. The major endoproteinase of P. cuniculi therefore appears to be a cathepsin D-like aspartic proteinase.
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PMID:Hydrolytic enzymes of Psoroptes cuniculi (Delafond). 1007 Jul 42

Hypoxia-inducible factor 1 alpha (HIF1alpha) and its related factor, HLF, activate expression of a group of genes such as erythropoietin in response to low oxygen. Transfection analysis using fusion genes of GAL4DBD with various fragments of the two factors delineated two transcription activation domains which are inducible in response to hypoxia and are localized in the C-terminal half. Their sequences are conserved between HLF and HIF1alpha. One is designated NAD (N-terminal activation domain), while the other is CAD (C-terminal activation domain). Immunoblot analysis revealed that NADs, which were rarely detectable at normoxia, became stabilized and accumulated at hypoxia, whereas CADs were constitutively expressed. In the mammalian two-hybrid system, CAD and NAD baits enhanced the luciferase expression from a reporter gene by co-transfection with CREB-binding protein (CBP) prey, whereas CAD, but not NAD, enhanced beta-galactosidase expression in yeast by CBP co-expression, suggesting that NAD and CAD interact with CBP/p300 by a different mechanism. Co-transfection experiments revealed that expression of Ref-1 and thioredoxin further enhanced the luciferase activity expressed by CAD, but not by NAD. Amino acid replacement in the sequences of CADs revealed a specific cysteine to be essential for their hypoxia-inducible interaction with CBP. Nuclear translocation of thioredoxin from cytoplasm was observed upon reducing O2 concentrations.
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PMID:Molecular mechanisms of transcription activation by HLF and HIF1alpha in response to hypoxia: their stabilization and redox signal-induced interaction with CBP/p300. 1020 54

5-(Pentafluorobenzoylamino)fluorescein (PFB-F), a new thiol-reactive molecule was synthesized to improve the detection limits and specificity of the assays for glutathione S-transferase (GST) activity and glutathione (GSH). A rapid assay method to measure GSH concentration or GST activity and the simultaneous analysis of multiple samples is possible because the glutathione adduct, GS-TFB-F, is separated from PFB-F by thin-layer chromatography (TLC) and can be quantitated by a fluorescence scanner. The detection limits for GSH and for GST activity using TLC were found to be as low as 10 pmol/microl and 1 ng/microl using equine liver GST, respectively. Determination of GSH concentration or GST activity in bovine pulmonary artery endothelial (BPAE) cell lysates gave a linear response for samples corresponding to 500-2500 cells. PFB-F could also measure GST activities of GST fusion proteins and prove to be a suitable substrate for determining the activities of human GST isozymes and other sources of mammalian GST. The selectivity of PFB-F with GSH was proven by comparing trace amount of the adducts that formed with cysteine and beta-galactosidase to that formed with GSH. The HPLC profile of a reaction mixture where cell lysate was used in place of purified GST, also shows only two main peaks, corresponding to GS-TFB-F and unreacted PFB-F. The selectivity of PFB-F for GSH was further confirmed by exposing BPAE cells to dl-buthionine-[S,R]-sulfoximine (BSO). Our results of GS-TFB-F determination indicate that 12-, 24-, or 36-h incubations with BSO caused 2-, 6-, or 7.6-fold reductions in GSH levels, respectively.
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PMID:5-(Pentafluorobenzoylamino)fluorescein: A selective substrate for the determination of glutathione concentration and glutathione S-transferase activity. 1022 18


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