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)

A set of plasmid expression vectors for cloning of DNA fragments containing open reading frames has been obtained. The plasmids carry the strong leftward promoter of bacteriophage lambda and the translation initiation signals from either the gene ner of bacteriophage Mu or the gene 4 of bacteriophage phi 29. The vectors could overexpress the cloned sequences as fusion peptides at the N terminus with the N-terminal segment of the phi 29 protein p4 or at the C terminus with the Escherichia coli beta-galactosidase from its 8th residue, or both. Alternatively, the cloned sequences could be directed to overproduce proteins in an unfused form. DNA fragments of the hemagglutinin gene from human influenza A virus, have been cloned in one of the plasmid vectors and some potential antigenic determinants have been characterized using monoclonal antibodies.
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PMID:A set of expression plasmids for the synthesis of fused and unfused polypeptides in Escherichia coli. 331 85

Antigenic determinants of influenza virus hemagglutinin were expressed in Escherichia coli. DNA coding for presequences of hemagglutinin were removed and an ATG codon was placed before DNA coding for mature hemagglutinin. A number of expression plasmids were constructed in which various segments of this reconstructed hemagglutinin DNA were fused to DNA coding for bacterial beta-galactosidase. The fusion proteins exhibited specific binding to antiviral antibodies. This binding could be competitively inhibited by excess viral hemagglutinin, demonstrating that these fusion proteins contained antigenic determinants of hemagglutinin.
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PMID:Expression of antigenic determinants of the hemagglutinin gene of a human influenza virus in Escherichia coli. 617 Sep 82

A new procedure for a sialidase assay, by bioluminescence, has been developed. The substrate, N- acetylneuraminyllactose (sialyllactose), hydrolysed by the sialidase activity, releases lactose. This lactose is hydrolysed with beta-galactosidase. The released galactose is oxidized with galactose dehydrogenase and NAD. The NADH produced in the last step is measured by a luminescence system, coupling two enzymes, NAD(P)H dehydrogenase (FMN) and luciferase. This microassay, which is specific, rapid, simple and ultra-sensitive, is a measure for amounts as little as (at least) 5 pmol of N-acetylneuraminic acid (corresponding to 0.15 ng of the released sialic acid). It uses commercialized reagents (non-radioisotopic) and avoids interferences common in other procedures. This method has been used for measuring sialidase activity directly on intact virus, avoiding inconvenient modifications produced in the extraction of the enzyme. The specific activity of sialidase of influenza virus X31 (H3N2), determined by this procedure, is 0.65 U/mg of total virus protein.
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PMID:Sialidase assay by luminescence in the low picomole-range of sialic acid. Its application to the measurement of this activity in influenza virus. 673 52

Most of the carbohydrate was removed from influenza virus MRC II (H3N2) and its purified hemagglutinin (HA) on treatment with glycosidases, including alpha-mannosidase, beta-N-acetylglucosaminidase, beta-galactosidase and alpha-fucosidase. The release of 50 per cent of the carbohydrate from intact virus particles significantly affected hemagglutinating activity. The ability of untreated and glycosidase-treated virus to inhibit the binding of antibodies directed against the hemagglutinin was almost indistinguishable by competitive radioimmunoassay (RIA). Up to 60 per cent of the carbohydrate from the purified HA of influenza virus could be removed. The antigenicity of glycosidase treated HA molecules decreased 8-fold compared to intact HAs as measured by competitive RIA. In addition, glycosidase digestion of 125I-labeled HA resulted in a decrease in its reactivity in direct RIA. We conclude that the carbohydrate portion of the HA of influenza virus is not of major importance in defining the antigenicity of HA.
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PMID:The role of carbohydrate in determining the immunochemical properties of the hemagglutinin of influenza A virus. 678 4

Swainsonine, an indolizidine alkaloid, inhibits the alpha-mannosidase that is involved in glycoprotein processing. Thus, in cultured animal cells, this alkaloid causes an increase in the surface content of high mannose glycoproteins and a decrease in the amount of complex type glycoproteins (Elbein, A. D., Solf, R., Dorling, P. R., and Vosbeck, K. (1982) Proc. Natl. Acad. Sci. U. S. A., 78, 7393-7397). In this report, the effect of swainsonine on the synthesis virus hemagglutinins was examined. Primary calf kidney cultures were infected with influenza virus and viral replication was allowed to proceed in the absence or presence of swainsonine. Several hours after the addition of swainsonine, [2-3H]mannose or [6-3H]glucosamine were added to label the hemagglutinins and the mature virus particles were isolated. Virus particles raised in the presence of this alkaloid had the same infectivity and hemagglutination titer as virus particles from control cells. However, when the hemagglutinins were examined on sodium dodecyl sulfate gels, the major hemagglutinin (HA0) and its subunits, HA1 and HA2, from swainsonine-treated cells, migrated faster, indicating that they were of lower molecular weights. The labeled hemagglutinins were digested with pronase and the resulting glycopeptides were chromatographed on Bio-Gel P-4. Both the mannose-labeled and glucosamine-labeled glycopeptides from swainsonine-treated virus migrated more slowly on these columns than those of controls cells, suggesting that they were altered in structure. Furthermore, when the glycopeptides were digested with endoglucosaminidase H, 90% of the glycopeptides from swainsonine-treated cells were susceptible to this enzyme, whereas only 30% of those from control cells were digested. The major oligosaccharide released from inhibited cells by endoglucosaminidase H was digestible with alpha-mannosidase, whereas that of control cells was resistant to this enzyme. However, the control cell glycopeptide was digested by a combination of neuraminidase, beta-galactosidase, beta-N-acetylhexosaminidase, and alpha-mannosidase. These data show that swainsonine prevents the formation of complex glycoproteins and gives rise to increased amounts of high-mannose glycoproteins.
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PMID:Swainsonine prevents the processing of the oligosaccharide chains of influenza virus hemagglutinin. 679 7

Neuraminidase or sialidase (EC 3.2.1.18, acylneuraminyl hydrolase) from a strain of the influenza virus A (H3N2), identical to the A/Hong Kong/68 (H3N2) strain, has been purified and characterized by electrofocusing; only about 20% of the previous enzymic activity was lost after electrofocusing. The enzyme activity was measured by the peryodate-thiobarbiturate procedure, by the methoxyphenol-antipyrine method, and by spectrophotometry at 340 nm of the NADH produced in the oxidation of the beta-galactose + NAD+; this beta-galactose was released from lactose by beta-galactosidase; and lactose was liberated from N-acetylneuraminyl-lactose by the neuraminidase activity. The results of the interference by some chemical compounds, which are not true inhibitory agents for the enzyme, on the peryodate-thiobarbiturate reaction are indicated, as well as the detection of other compounds which are true inhibitors of this enzyme in vitro. This neuraminidase was able to release sialic acid with linkages alpha 2-3, alpha 2-6 and alpha 2-8 from several substrates, but with very different efficiency. Natural substrates such as the oligosaccharide N-acetylneuraminyl-lactose, glycoproteins (fetuin, bovine horse brain, colominic acid, and synthetic substrates such as 5-N-acetyl-2-O-(3-methoxyphenyl)-alpha-D-neuraminic acid and 2'-(4-methyl umbellyferil)-alpha-D-N-acetylneuraminic acid were hydrolyzed by this enzyme. Finally, the finding of neuraminidase in ovine, equine and porcine platelet is summarized.
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PMID:[Neuraminidase of influenza virus]. 714 96

Neuraminidase (acylneuraminyl hydrolase, EC 3.2.1.18) from the influenza virus A/Hong Kong/68 (H3N2) was purified after treatment of the purified virus with sarcosyl (sodium laurylsarcosinate), centrifugation at 110 000 x g, and chromatography on DEAE-Sephadex and Sephadex G-200. It migrated as a single component during electrophoresis on polyacrylamide gel, and its molecular weight was estimated about 270 000. The enzyme was thermolabile, the activity being reduced to 60% in 10 min at 50 degrees C. The purified neuraminidase had an apparent Km value of 4.1 . 10(-3) M for 5-N-acetyl-2-O-(3-methoxyphenyl)-alpha-D-neuraminic acid and was able to release sialic acid with linkages alpha 2-3, alpha 2-6 and alpha 2-8 (with very different efficiency) from fetuin, gangliosides, colominic acid, and bovine and porcine submaxillary mucins. The enzymic activity was measured by several procedures: (A) spectrophotometric determination at 340 nm of the NADH produced in the reaction catalysed by beta-galactose dehydrogenase on beta-galactose + NAD+, this beta-galactose was the product released from lactose by beta-galactosidase and lactose was the product of the neuraminidase activity on N-acetylneuraminyl-lactose; (B) determination of the colored quinone yielded by the liberated methoxyphenol with 4-aminoantipyrine (Santer, U.V., Yee-Foon, J. and Glick, M.C. (1978) Biochim. Biophys. Acta 523, 435-442); (C) periodate-thiobarbiturate procedures (Warren, L. (1959) J. Biol. Chem 234, 1971-1975 or Aminoff, D. (1961) Biochem. J. 81, 384-391). Some peculiarities of these methods are discussed.
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PMID:Neuraminidase from influenza virus A (H3N2): specificity towards several substrates and procedure of activity determination. 721 37

To compare the requirements for paramyxovirus-mediated cell fusion, the fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins of simian virus 5 (SV5), human parainfluenza virus 3 (HPIV-3), and Newcastle disease virus (NDV) were expressed individually or coexpressed in either homologous or heterologous combinations in CV-1 or HeLa-T4 cells, using the vaccinia virus-T7 polymerase transient expression system. The contribution of individual glycoproteins in virus-induced membrane fusion was examined by using a quantitative assay for lipid mixing based on the relief of self-quenching (dequenching) of fluorescence of the lipid probe octadecyl rhodamine (R18) and a quantitative assay for content mixing based on the cytoplasmic activation of a reporter gene, beta-galactosidase. In these assays, expression of the individual F glycoproteins did not induce significant levels of cell fusion and no cell fusion was observed in experiments when cells individually expressing homologous F or HN proteins were mixed. However, coexpression of homologous F and HN glycoproteins resulted in extensive cell fusion. The kinetics of fusion were found to be very similar for all three paramyxoviruses studied. With NDV and HPIV-3, no cell fusion was detected when F proteins were coexpressed with heterologous HN proteins or influenza virus hemagglutinin (HA). In contrast, SV5 F protein exhibited a considerable degree of fusion activity when coexpressed with either NDV or HPIV-3 HN or with influenza virus HA, although the kinetics of fusion were two- to threefold higher when the homologous SV5 F and HN proteins were coexpressed. Thus, these data indicate that among the paramyxoviruses tested, SV5 has different requirements for cell fusion.
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PMID:Quantitative measurement of paramyxovirus fusion: differences in requirements of glycoproteins between simian virus 5 and human parainfluenza virus 3 or Newcastle disease virus. 747 81

The ompA genes of Escherichia coli and Shigella dysenteriae have been used to construct a group of enterobacterial surface expression vectors for foreign genes. Linker oligonucleotides were inserted into the sequence corresponding to the third or fourth outer domain to allow in-frame sandwich fusion of foreign genes or epitopes into ompA. Influenza haemagglutinin was inserted without its leader peptide and anchor sequences and shown to be transferred as an ompA fusion protein to the bacterial surface in large amounts. The stability of this system depends on the stem structure (i.e. the bottom part) of the haemagglutinin unit which apparently initiates the folding process that extends into the ompA segment. This fusion construct can be used as a vector system and has been used to transfer to the bacterial surface several other proteins inserted into it, including beta-galactosidase, foot-and-mouth disease virus (FMDV) and malaria antigens. All are exported from the cytoplasm across both the inner and outer membranes to become exposed on the bacterial surface. Very hydrophobic segments or inserts with distinct secondary structures, such as the capsid protein, VP1 of FMDV, will, however, block this process.
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PMID:OmpA fusion proteins for presentation of foreign antigens on the bacterial outer membrane. 779 62

The protein regions involved in the nuclear translocation of the influenza virus PA polymerase subunit have been identified by deletion analysis of the protein expressed from a recombinant simian virus 40. Two regions seem to play a role in the process: region I (amino acids 124 to 139) and region II (amino acids 186 to 247). A nucleoplasmin-like nuclear translocation signal (NLS) has been identified in region I and an additional NLS appears to be present in region II, although no consensus targeting sequence can be detected. Alteration in any of the regions identified by short deletions completely prevented nuclear transport, whereas elimination of the regions I or II by large amino- or carboxy-terminal deletions did not prevent nuclear targeting of the truncated protein. In addition, a point mutation at position 154 completely eliminated nuclear transport. A beta-galactosidase fusion protein containing the 280 amino acid terminal region of the PA protein was partially transported to the nucleus and mutant PA proteins with a cytoplasmic phenotype could not be rescued by superinfection with influenza virus. These results suggest that the PA protein contains a functional nuclear targeting region which is required in influenza virus infection, with two independent NLSs, one in region I and the other in region II.
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PMID:Complex structure of the nuclear translocation signal of influenza virus polymerase PA subunit. 811 37


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