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)

Synthesis of the poliovirus polypeptide 3AB in bacterial cells results in an increase in membrane permeability. The alterations observed resemble those elicited by bacteriophage lytic proteins, which are presumed to cause pore formation in biological membranes. This property has been exploited in the development of an in vivo screening system that allows morphological differentiation of Escherichia coli clones expressing either wild-type 3AB or variant 3AB proteins lacking the ability to permeabilize bacteria. Expression of the wild-type 3AB gene in the presence of a chromogenic beta-galactosidase substrate causes E. coli clones to stain dark blue. In contrast, bacterial mutants that synthesize 3AB proteins with alterations in the hydrophobic domain lack pore-forming activity and stain to a light blue colour, allowing differentiation from wild-type clones. This phenotypic property correlates with the rate of entry of the beta-galactosidase substrate into the bacteria. The method developed here was used to screen more than 8000 E. coli clones after random PCR mutagenesis of the poliovirus 3AB gene. Our results show the existence of three different domains involved in the permeabilizing activity of 3AB protein. Twenty individual amino acid substitutions were identified in clones that showed the mutant phenotype and such bacteria displayed different reduced levels of permeability towards ONPG, hygromycin B, lysozyme and uridine. The procedure reported here may be of general interest to understand structure-function relationships in other eukaryotic proteins known to form pores.
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PMID:Screening for membrane-permeabilizing mutants of the poliovirus protein 3AB. 881 Oct 10

The nuclear matrix plays a critical role in DNA replication, gene transcription and RNA processing. Transcriptionally active genes are usually associated with the nuclear matrix through DNA sequences, matrix attachment regions or MARs, which tether looped DNA to the matrix. In stable transfection and in transgenic mice MAR elements placed at the flanks of genic constructs may enhance expression and insulate against position effect variability, suggesting that independent units of transcription are established insulated from the regulatory controls of their neighbors. Herpes simplex virus type 1 (HSV-1) establishes lifelong latency in the infected host. Latency repression of viral genes extends to foreign genes incorporated into the viral genome. We report here a test of the hypothesis that MAR elements, flanking a foreign gene in the HSV-1 genome, would act to insulate it from latency repression, achieving long-term expression. A recombinant virus was produced which has an expression construct inserted into the HSV-1 genome at the Us3 locus. The expression construct consists of the A MAR element on one flank, an HIV-LRT driving the lacZ gene and the B MAR element on the other flank. The A MAR element is a 3 kb pair fragment of the 5' portion of the chicken lysozyme gene and the B MAR element is a 2.6 kb pair fragment from the 5' end of the human beta-globin gene locus control region. The LTR is derived from a human immunodeficiency virus isolated from the brain of an AIDS patient. Virus was stereotactically injected in the hippocampus, olfactory bulb and striatum of rat brains. Intense blue reaction product indicating beta-galactosidase activity was found in cells in each injected area at 2 days after injection. At 14 days after injection beta-galactosidase activity was no longer detected at any of the injected sites. We conclude that the MAR element construct did not escape latency repression.
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PMID:Incorporation of nuclear matrix attachment regions into the herpes simplex virus type 1 genome does not induce long-term expression of a foreign gene during latency. 887 33

Helicobacter pylori adhere to Kato III and Hela S3 cells in monolayer cultures. To explore whether cell surface glycoconjugates on these two cell lines mediate binding of H. pylori, various carbohydrates, glycoproteins, and glycolipids were tested to inhibit H.pylori cell adhesion. The adhesion was measured (i) with a urease-based assay and (ii) by cells stained with fluorescein. Sodium periodate and sialidase treatment (but not alpha- or beta-galactosidase, heparitinase,lysozyme, or trypsin) inhibited H. pylori binding to both cell lines. Sulfatides and sulfated glycoconjugates (50 microg/ml) but not heparin or a number of simple carbohydrates inhibited binding (1 mg/ml). The two H.pylori strains studied (CCUG 17874 and strain 25) showed high binding of soluble 125I-labeled heparin and other sulfated carbohydrate compounds.
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PMID:Sulfatides inhibit binding of Helicobacter pylori to the gastric cancer Kato III cell line. 909 25

The effect of typical contaminants in inclusion body preparations such as DNA, ribosomal RNA, phospholipids, lipopolysaccharides, and other proteins on renaturation rate and yield of hen egg white lysozyme was investigated. Separate experiments were conducted in which known amounts of individual contaminants were added to test their effect on renaturation kinetics. On the basis of a simplified model for the kinetic competition between folding and aggregation, it was found that none of the above contaminants had an effect on the rate of the folding reaction, but some of them significantly affected the rate of the aggregation reaction and, thus, the overall renaturation yield. While ribosomal RNA did not seem to affect the aggregation reaction, plasmid DNA and lipopolysaccharides increased the aggregation rate, resulting in a decrease of about 10% in the overall renaturation yield. Phospholipids were found to improve refolding yields by about 15% by decreasing the overall rate of the aggregation reaction without affecting the rate of the folding reaction. Proteinaceous contaminants which aggregate upon folding, such as beta-galactosidase and bovine serum albumin, were found to significantly decrease renaturation yields by promoting aggregation. This effect was strongly dependent on the concentration of the proteinaceous impurity. On the other hand, the presence of refolding ribonuclease A, which does not significantly aggregate upon folding under the conditions tested in this work, did not affect the renaturation kinetics of lysozyme, even at concentrations as high as 0.7 mg/mL.
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PMID:Effect of inclusion body contaminants on the oxidative renaturation of hen egg white lysozyme. 910 38

N-Acetyl-D-glucosaminylpyrophosphorylundecaprenol (GlcNAc-P-P-Und), an intermediate in the biosynthesis of the enterobacterial common antigen in E.coli and some O-antigen chains in gram-negative bacteria, is formed by the transfer of GlcNAc 1-P from UDP-GlcNAc to Und-P, analogous to the reaction forming GlcNAc-P-P-dolichol (GlcNAc-P-P-Dol) in mammalian cells. Since the microsomal enzyme from animal cells exhibits a strong preference for Dol-P, which contains a saturated alpha-isoprene unit, the polyisoprenyl phosphate specificity of the homologous bacterial enzyme was characterized. The enzyme remained bound to the membrane fraction when spheroplasts, formed by lysozyme-EDTA treatment, were lysed in hypotonic buffer. GlcNAc-P-P-Und synthase (GPT) activity was elevated in a strain of E.coli bearing the rfe gene, which encodes GPT on a multicopy plasmid, and virtually absent from rfe null mutants. GPT actively utilized fully unsaturated polyprenyl phosphate (Poly-P) substrates with maximal activity seen with (C55) Und-P, but was unable to utilize (C55)Dol-P. This substrate specificity contrasts with the microsomal GPT from pig brain, which actively utilized (C55)Dol-P, but not Und-P, as substrate. GPT activity bound to particulate fractions from three strains of bacilli also exhibited a strict preference for fully unsaturated Poly-P substrates. Unexpectedly, E.coli GPT activity cofractionated with the cytosolic marker enzyme, beta-galactosidase, and not the membrane-bound enzyme, D-lactate dehydrogenase, in cells disrupted in a French pressure cell. The properties and polyisoprenyl phosphate specificity of the soluble form of GPT were identical to the activity associated with the membrane preparations obtained from spheroplasts. The evolutionary and functional significance of the use of polyisoprenyl glycosyl carrier lipids with saturated alpha-isoprene units in eukaryotes remains uncertain.
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PMID:Polyisoprenyl phosphate specificity of UDP-GlcNAc:undecaprenyl phosphate N-acetylglucosaminyl 1-P transferase from E.coli. 913 38

Hydrophobically modified dextrans, benzoyl dextran and valeryl dextran, have been used to study the interactions between tryptophan residues and benzoyl or valeryl groups by partitioning of tryptophan, tryptophan-tryptophan, (tryptophan)3, poly(lysine, tryptophan), beta-galactosidase and lysozyme in polymer aqueous two-phase systems. The two-phase systems used were polyethylene glycol (PEG)-benzoyl dextran, PEG-valeryl dextran, dextran-benzoyl dextran and dextran-valeryl dextran. Interaction between tryptophan residues and benzoyl or valeryl groups was observed by partitioning of tryptophan containing compounds to the phase containing hydrophobically modified dextran. At a certain phase composition the interactions were increased with increasing number of tryptophan per molecule. In a PEG-dextran system the partitioning of tryptophan peptides to the PEG phase was increased with increased number of tryptophan. In a PEG-benzoyl dextran system the opposite effect was obtained. At similar conditions benzoyl groups showed stronger interactions with tryptophans compared to valeryl groups. The partition coefficient of salts (sodium phosphate, NaCl, Nal and NaClO4) was determined in PEG-benzoyl dextran and PEG-valeryl dextran aqueous two-phase systems. The effect of addition of these salts on partitioning of poly(lysine, tryptophan), beta-galactosidase and lysozyme was studied. Salt effects on partitioning could be explained by the relative affinities of the ions for the polymers in the system. Charged molecules containing tryptophan were to an increasing degree partitioned to the phase for which the counterions had highest affinity. Strong effects on the partitioning of positively charged poly(lysine, tryptophan) and lysozyme were obtained with the ions I- and ClO4-.
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PMID:Interaction between tryptophan residues and hydrophobically modified dextran. Effect on partitioning of peptides and proteins in aqueous two-phase systems. 913 30

We summarize in this communication the data supporting the two functions of ribosome recycling factor (RRF, originally called ribosome releasing factor). The first described role involves the disassembly of the termination complex which consists of mRNA, tRNA and the ribosome bound to the mRNA at the termination codon. This process is catalyzed by two factors, elongation factor G (EF-G) and RRF. RRF stimulated protein synthesis as much as eight-fold in the in vitro lysozyme synthesis system, when ribosomes were limiting. In the absence of RRF, ribosomes remain mRNA-bound at the termination codon and translate downstream codons. In the in vitro system, the site of reinitiation is the triplet codon 3' to the termination codon. RRF is an essential protein for bacterial life. Temperature sensitive (ts) RRF mutants were isolated and in vivo translational reinitiation due to inactivation of ts RRF was demonstrated using the beta-galactosidase reporter gene placed downstream from the termination codon. A second function of RRF involves preventing errors in translation. In polyphenylalanine synthesis programmed by polyuridylic acid, misincorporation of isoleucine, leucine or a mixture of amino acids was stimulated upto 17-fold when RRF was omitted from the in vitro system. RRF did not influence the large error (10-fold increase) induced by streptomycin. This means that RRF participates not only in the disassembly of the termination complex but also in peptide elongation. Extending this concept and its conventional role for releasing ribosomes from mRNA, involvement of RRF in the reinitiation in the 3A' system (a construct using S aureus protein A, a collaborative work with Dr Isaksson), in programmed frame shifting, in trans-translation with 10Sa RNA (collaborative work with Dr Muto), and in the reinitiation downstream from the ORF A of the IS 3 (insertion sequence of a transposon, collaborative work with Dr Sekine) are discussed on the basis of preliminary data to be published elsewhere. Finally, we review the known RRF sequences from various organisms including eukaryotes and discuss the possible mechanism for disassembly of the eukaryotic termination complex.
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PMID:Dual functions of ribosome recycling factor in protein biosynthesis: disassembling the termination complex and preventing translational errors. 915 Aug 73

Polymorphonuclear cells kill microorganisms by the stock of antibiotic proteins and peptides stored in their lysosomal granules and have the ability to produce reactive oxygen intermediates (ROI) such as H2O2, O2-, and HOCl. Since the components involved in the microbicidal functions of buffalo (Bos bubalis) polymorphonuclear cells (PMN) have not been characterized, an assessment was made of the levels of various enzymes, the extent of extracellular release of these enzymes, and also their ability to produce H2O2/O2- upon activation with opsonized zymosan (OZ) or lipopolysaccharide (LPS). Using GPC-HPLC, OZ was shown to be a more potent secretagogue than LPS, causing a significantly greater release of low-molecular-weight components. Varying levels of the enzymes (myeloperoxidase, lactate dehydrogenase, acid and alkaline phosphatases, beta-galactosidase, beta-D-glucuronidase, elastase and lysozyme) were recorded in the buffalo PMN and both the activators (OZ and LPS) caused significant release of all the enzymes except alkaline phosphatase. Both the activators also caused a significant increase in H2O2/O2- production by the PMN. However, OZ caused a more pronounced activation than LPS. The studies revealed the presence of oxygen-dependent and oxygen-independent microbicidal systems with buffalo PMN, which responded more effectively to zymosan activation.
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PMID:The effect of activation of granulocytes on enzyme release and hydrogen peroxide and superoxide production in buffaloes. 915 8

Gene duplication with divergence to new functions has been an important mechanism in protein evolution. However, the questions of how many new functions can arise from a particular ancestral gene and how many mutational steps are typically required to generate new functions have been difficult to approach experimentally. We have addressed these questions using T4 lysozyme as a model system by synthesizing two combinatorial libraries of > 10(7) mutant T4 lysozyme genes: one library with an average of 14 missense mutations spread throughout the gene and one library in which 13 active site residues have been simultaneously randomized. These libraries were placed under selection in lacZ or pheA deficient strains of E. coli to investigate whether they sample sufficient diversity to contain mutants with acquired beta-galactosidase or prephenate dehydratase activities. Although neither selection yielded T4 lysozyme mutants with these new activities, a novel E. coli locus was cloned that weakly complements these mutants, allowing them to form 1 mm colonies in 4-6 weeks. This growth rate corresponds to a turnover number of approximately 1000 or 25 min-1 for the lacZ or pheA complementation systems, respectively, thus defining the limits of evolved enzymatic activity detectable in these selections. Thus, the strong selective pressure uncovered an unexpected solution to the biochemical blocks, a frequently observed phenomenon in selection experiments. The characterization of this locus will allow its elimination from future E. coli complementation schemes.
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PMID:Directed evolution studies with combinatorial libraries of T4 lysozyme mutants. 923 98

A cDNA encoding a new ubiquitin-specific protease, UBP41, in chick skeletal muscle was cloned using an Escherichia coli-based in vivo screening method. Nucleotide sequence analysis of the cDNA containing an open reading frame of 1,071 base pairs revealed that the protease consists of 357 residues with a calculated molecular mass of 40,847 Da, and is related to members of the UBP family containing highly conserved Cys and His domains. Chick UBP41 was expressed in E. coli and purified from the cells to apparent homogeneity, using 125I-labeled ubiquitin-alphaNH-MHISPPEPESEEEEEHYC as a substrate. The purified enzyme behaved as an approximately 43-kDa protein under both denaturing and nondenaturing conditions, suggesting that it consists of a single polypeptide chain. Like other deubiquitinating enzymes, it was sensitive to inhibition by ubiquitin-aldehyde and sulfhydryl blocking agents, such as N-ethylmaleimide. The UBP41 protease cleaved at the C terminus of the ubiquitin moiety in natural and engineered fusions irrespective of their sizes; thus, it is active against ubiquitin-beta-galactosidase as well as ubiquitin C-terminal extension protein of 80 amino acids. UBP41 also released free ubiquitin from poly-His-tagged di-ubiquitin. Moreover, it converted poly-ubiquitinated lysozyme conjugates to mono-ubiquitinated forms of about 24 kDa, although the latter molecules were not further degraded to free ubiquitin and lysozyme. These results suggest that UBP41 may play an important role in the recycling of ubiquitin by hydrolysis of branched poly-ubiquitin chains generated by the action of 26 S proteasome on poly-ubiquitinated protein substrates, as well as in the production of free ubiquitin from linear poly-ubiquitin chains and of certain ribosomal proteins from ubiquitin fusion proteins.
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PMID:Molecular cloning of a novel ubiquitin-specific protease, UBP41, with isopeptidase activity in chick skeletal muscle. 932 73


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