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)

To begin to assess the independent structural and functional characteristics of the mitochondrially encoded subunits of mammalian cytochrome c oxidase, we have converted the cloned mitochondrial gene for rat subunit II (coxII) into its universal codon equivalent (ucoxII) by oligonucleotide-directed, site-specific mutagenesis. This involved synthesizing 12 oligodeoxynucleotides to achieve the 13 ATA to ATG and the 5 TGA to TGG changes needed. To express ucoxII in Escherichia coli, we used a number of different expression vectors in which the promoters and ribosome-binding sequences of the messenger RNA were varied. While ucoxII alone was expressed at a low level, a striking increase in the level of expression resulted when the ucoxII gene was fused to other E. coli genes. The COXII peptide was identified by proteolytic digestion, partial sequencing, and reaction with specific antisera. A cro-beta-galactosidase-COXII fusion protein has been purified, characterized, and used to produce polyclonal antibodies to the COXII peptide. The ucoxII gene was also expressed in a cell-free translation system and in Xenopus oocytes, yielding a nondenatured, membrane-associated peptide with the same apparent molecular weight as authentic subunit II. In oocytes and in a reticulocyte lysate in vitro system supplemented with microsomal membranes, the protein is glycosylated and coisolates with the washed membrane fraction. In both cases, the COXII peptide is soluble under mild conditions in a nonionic detergent and is precipitable by antibodies to subunit II. The production of subunit II in the in vitro translation system is stimulated as strongly by addition of soybean phospholipid vesicles as by microsomal membranes, providing further evidence of membrane insertion and stabilization.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Conversion of a mitochondrial gene for mammalian cytochrome c oxidase subunit II into its universal codon equivalent and expression in vivo and in vitro. 184 93

We have used cDNA encoding the cellular receptor for poliovirus (PVR) to prepare polyclonal antisera against beta-galactosidase PVR fusion proteins. One of these antisera allowed identification of a glycoprotein doublet band of about 67 kDa in membrane preparations of HeLa cells and in a PVR cosmid-bearing mouse cell line. In vitro translation of PVR-specific transcripts gave rise to a protein of 46 kDa; the product had a molecular weight of 67 kDa when microsomal membranes were added to the cell-free extract. Overexpression of PVR cDNA in mouse L-cells by means of a recombinant vaccinia virus led to the synthesis of a glycoprotein having a molecular weight identical to that of the glycosylated in vitro product. The vaccinia virus-mediated protein was also recognized by a monoclonal antibody that blocks poliovirus infection. Its biological activity was demonstrated by poliovirus binding and infectivity assays. The data show that PVR is a glycoprotein of 67 kDa and that this protein is sufficient to confer poliovirus susceptibility to mouse cells.
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PMID:Vaccinia virus-mediated expression and identification of the human poliovirus receptor. 185 Sep 5

Since liver microsomal cytochrome b5 spontaneously associates with liposomes and membranes by means of its C-terminal hydrophobic domain (HP), chimeric proteins containing HP prepared by genetic fusion might also spontaneously associate with liposomes or cellular membranes. Synthetic DNA corresponding to the hydrophobic domain of cytochrome b5 was enzymatically fused in-frame to cloned DNA corresponding to the C-terminus of the Escherichia coli enzyme, beta-galactosidase. This protein, LacZ:HP, synthesized in E. coli and purified from a crude E. coli membrane extract, was shown to spontaneously associated with liposomes, as does cytochrome b5. Association is rapid and stable in the presence of salt and high pH and the fusion protein behaves as an integral membrane protein. LacZ:HP can be readily and extensively purified from crude extracts by association with liposomes and this procedure may provide a convenient purification scheme for proteins not otherwise readily purified, for example polypeptides from cloned gene fragments to be used for antibody production. These hybrid proteins may represent a new potentially useful class of polypeptides capable of hydrophobic interactions with membranes.
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PMID:Beta-galactosidase fused to the hydrophobic domain of cytochrome b5 spontaneously associates with liposomes. 189 1

The umu test system is a newly developed method to evaluate genotoxicities of a wide variety of environmental carcinogens and mutagens (Oda et al., 1985; Nakamura et al., 1987). In the present study, SOS-inducing activity of 142 synthetic dyes was investigated by the umu test using Salmonella typhimurium (TA1535/pSK1002) under the condition of absence and presence of rat liver microsomal fraction. The samples showing a beta-galactosidase activity of more than 1.5 fold over the background level were reexamined and the dose-response curves were prepared at various doses. Then, the samples showing beta-galactosidase activity of more than 1.5-fold of the background level were defined as genotoxic. Among the synthetic dyes examined, 11 compounds induced umu gene expression. The potent genotoxic compounds without metabolic activation were Acid Black 26, Acid Black 50, Acid Brown 2, Disperse Red 73, Disperse Red 145, Disperse Red 157, Disperse Violet 52, Reactive Red 110, Reactive Yellow 13 and Reactive Yellow 75, and in the presence of S9, Reactive Blue 147 was judged to be genotoxic. An evident dose-response relationship was observed between the doses of the dye and umu-gene expression in these 11 dyes.
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PMID:[Genotoxicity of synthetic dyes in umu test using Salmonella typhimurium TA1535/pSK1002 (1). Results of examination for acid dyes, direct dyes, disperse dyes and reactive dyes]. 211 88

We have shown that hybrid proteins composed of the yeast repressible acid phosphatase (PHO5) and bacterial beta-galactosidase (lacZ) interfere with secretion of native acid phosphatase (Wolfe, P. B. (1988) J. Biol. Chem. 263, 6908-6915). We now report that PHO5-LacZ hybrid proteins have a more general effect on secretion and prevent translocation of several secreted proteins. Translocation of both the mating pheromone alpha-factor and the vacuolar protease carboxypeptidase Y is partially blocked when PHO5-LacZ hybrids are expressed. Cell fractionation and protease sensitivity indicate that alpha-factor and carboxypeptidase Y accumulate in precursor form on the cytoplasmic surface of the endoplasmic reticulum. Indirect immunofluorescence with antibody directed against beta-galactosidase supports the localization of hybrid proteins to the endoplasmic reticulum. Analysis of the hybrid protein phenotype in vivo and in vitro suggests that the hybrid proteins deplete a soluble factor required for efficient translocation across the endoplasmic reticulum. First, a decrease in the expression of a hybrid protein in vivo decreases its effect on translocation. Second, an in vitro translation/translocation reaction, prepared from a hybrid-bearing strain, is deficient in its ability to translocate prepro-alpha-factor across yeast microsomal membranes. This deficiency is complemented by addition of cytosol prepared from wild type cells. Finally, the hybrid protein phenotype is shown to be independent of the requirement for SSA gene products.
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PMID:Expression of acid phosphatase-beta-galactosidase hybrid proteins prevents translocation by depleting a soluble factor. 212 90

Cyclophellitol [1S,2R,3S,4R,5R,6R)-5-hydroxymethyl-7-oxabicyclo[4,1,0] heptane-2,3,4-triol) was tested against 9 glycosidases and found to be a specific inhibitor of almond beta-glucosidase. Cyclophellitol inhibited almond beta-glucosidase activity by 50% at 0.8 micrograms/ml and was a competitive inhibitor of almond beta-glucosidase as revealed by Lineweaver-Burk plot. Cyclophellitol was inactive against yeast alpha-glucosidase, beta-galactosidase, beta-glucuronidase, alpha-L-fucosidase, end-beta-N-acetyl glucosaminidase, alpha-mannosidase, and cellulase. It was weakly active toward fungal beta-xylosidase. Cyclophellitol-treated almond beta-glucosidase was equally suppressed after dialysis; thus cyclophellitol is likely to bind to almond beta-glucosidase irreversibly. The inhibitor was found by fluorimetric assay to be active against beta-glucosidase but inactive toward alpha-glucosidase in Molt-4 microsomal fraction. It also inhibited Molt-4 beta-glucocerebrosidase completely at 2 micrograms/ml when the enzyme was assayed with a synthetic labeled substrate, and the inhibitory activity was more than one hundred times higher than that of nojirimycin, castanospermine, or of deoxynojirimycin. Mice administered 1 mg of cyclophellitol daily for 5 days began to exhibit severe abnormalities of nervous system similar to those found in Gaucher's mouse.
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PMID:Biological activities of cyclophellitol. 214 35

In the present study, SOS-inducing activity of 76 basic dyes was investigated by umu test using Salmonella typhimurium TA1535/pSK1002 under the condition of absence and presence of rat liver microsomal fraction. The test was carried out with five doses of basic dyes (400, 120, 40, 12, and 4 micrograms/ml). The samples showing beta-galactosidase activity more than 1.5-fold over the background level were reexamined and the dose-response curves were prepared at various doses. Thereafter, samples showing beta-galactosidase activity unit more than 1.5-fold of the background level were defined as genotoxic. Among the basic dyes examined, 13 compounds induced umu gene expression. The potent genotoxic compounds without metabolic activation were Blue 40, Blue 47, Brown 14, Orange 30, Red 24, Violet 30, Violet 31, Yellow 13(h), Yellow 19, Yellow 25, Yellow 67, and Yellow 73 and in the presence of S9, Orange 47 was judged as genotoxic in addition to the aforementioned dyes. An evident dose-response relationship between the doses of the dye and umu gene expression was observed in these 13 dyes.
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PMID:[Genotoxicity of synthetic dyes in the umu test using Salmonella typhimurium TA1535/pSK1002 (II). Results of examination of basic dyes]. 227 83

Fusion proteins constructed between beta-galactosidase and six different segments of either cytochrome P450IIB1 or cytochrome P450IIB2 (ranging from 18 to 33 amino acids in length) were expressed in Escherichia coli. Rabbit antibodies raised against these fusion proteins were first adsorbed through a beta-galactosidase column and then immunopurified on a second column containing the corresponding fusion protein. With the exception of the antibodies directed against the hydrophobic amino-terminal segment of cytochrome P450IIB1, all the antipeptide antibodies recognized the major phenobarbital-inducible cytochromes P450IIB1 and -IIB2 on immunoblots of liver microsomal proteins. Two of the antibodies were raised against regions where cytochromes P450IIB1 and -IIB2 differ in primary structure, and were differentially reactive toward these two highly homologous cytochromes. Several of the antipeptide antibodies were also reactive with a third phenobarbital-inducible microsomal protein expressed in livers of some individual Sprague-Dawley rats which was shown to be more highly related to P450IIB1 than P450IIB2. This P450IIB1-related P450, designated P450IIB1*, was purified to apparent homogeneity and shown to hydroxylate the steroid hormones testosterone and androstenedione with the well-defined regiospecificity and high catalytic activity characteristic of P450IIB1. A fourth microsomal protein detected using the antipeptide antibodies appeared to be more highly related to P450IIB2. Because the segments on the P450 molecules recognized by these antipeptide antibodies are known, it is possible to predict where P450IIB1* and the P450IIB2-related protein differ from cytochromes P450IIB2 and -IIB1, respectively. These studies demonstrate the utility of site-specific anti-P450 antibodies raised to fusion peptides for studies on the expression of structurally related P450s and polymorphic variants within the cytochrome P450 gene superfamily.
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PMID:Antibodies targeted against hypervariable and constant regions of cytochromes P450IIB1 and P450IIB2. 253 47

Flavonoids (103 species) were tested for inhibitory activity against mouse liver sialidase using sodium p-nitrophenyl-N-acetyl-alpha-D-neuraminate (PNP-NeuAc) as substrate. Isoscutellarein-8-O-glucuronide from the leaf of Scutellaria baicalensis showed most potent activity (IC50, 40 microM), and this flavone appeared to be a non-competitive inhibitor of the enzyme. This flavone inhibited the lysosomal solubilized sialidase against PNP-NeuAc and sialyllactose effectively, but not microsomal enzyme against gangliosides and colominic acid, whereas, negligible or weak inhibitory activities were observed for influenza virus sialidase, beta-galactosidase, alpha-mannosidase, and alpha-glucosidase tested. These results indicate that this flavone may be useful to elucidate the function of the lysosomal solubilized sialidase.
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PMID:Inhibition of mouse liver sialidase by plant flavonoids. 277 64

The molecular cloning of certain antigens to thyroid autoantibodies present in patients with autoimmune thyroid disease would be of great value in further understanding the pathogenesis of these diseases, and in devising new approaches for their treatment. The ideal method for molecular cloning is to first obtain a partial amino acid sequence of a purified protein and to construct an oligonucleotide probe for screening an appropriate cDNA library. Unfortunately in the case if thyroid autoimmunity, important antigens such as the TSH receptor and the microsomal antigen have not been purified. An alternate approach devised by Young & Davis (1983) is to construct a cDNA library in a vector that expresses the encoded proteins. The library can then be screened with an antibody as a probe. We have constructed cDNA libraries in gt11 using mRNA purified from human, pig and rat thyroid cells. Our experiences in constructing and screening these libraries will be described. The advantages of this system are 1) the protein does not have to be purified, 2) previously unknown antigens may be identified. The disadvantages are 1) lack of specificity with antibody selection, 2) because the cDNA is inserted in the beta-galactosidase gene in the vector the antigen is expressed as a fusion protein. This may disturb the tertiary structure of the antigen and alter its antigenicity, 3) cDNA inserts frequently only contain part of the antigen molecule, and may therefore lack important epitopes; polyclonal antibody may therefore be preferable to monoclonal, 4) only 1 in 6 cDNA inserts will be in the correct reading frame for antigen expression, 5) the expressed protein is not glycosylated.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Molecular cloning of antigens to thyroid autoantibodies using the expression vector lambda gt11. 295 18


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