Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Production of the two phospholipases C (PLCs) in Pseudomonas aeruginosa PAO1 is induced under conditions of phosphate limitation, or by the osmoprotectants choline or glycine betaine. Tn5 mutagenesis was performed on strain PAO1 to isolate mutants deficient in choline-dependent induction of PLC. Two mutants, Tn5T1 and Tn5G19, were identified which produce decreased levels of PLC in phosphate-replete media supplemented with choline. A total of 136 and 496 bp of flanking DNA from Tn5G19 and Tn5T1 was cloned by an inverse polymerase chain reaction (PCR) and sequenced. The DNA flanking the Tn5T1 insertion contains an open reading frame predicted to encode a peptide that is approx. 60% identical to the N-terminus of a previously identified protein (P35) of unknown function from Escherichia coli. The P35 gene, which is located in the nusA-infB operon in E. coll, was designated orp (osmoprotectant regulator of PLC). Haemolytic titres, total PlcH protein and beta-galactosidase activity expressed from a chromosomally inserted plcH-lacZ operon fusion were reduced in strain Tn5T1 in comparison with the parental strain (PAO1) carrying the same fusion. However, this mutant expressed several-fold higher levels of plcH message than strain PAO1 in the presence of choline, while the phosphate-starvation-dependent transcript of plcH could not be detected in this mutant. The defects in Tn5T1 are complemented by a DNA fragment, isolated from a genomic library of PAO1, that carries the orp gene. The deduced amino acid sequence of the DNA fragment cloned from Tn5G19 exhibits 84% identity with the betB gene product of E. coli that has betaine aldehyde dehydrogenase activity. This enzyme catalyses the conversion of betaine aldehyde to glycine-betaine. Unlike the parental strain, the Tn5G19 mutant could not utilize choline as a sole carbon, nitrogen and energy source, and it was deficient in betaine aldehyde dehydrogenase activity. Also, consistent with a disruption of betB in Tn5G19, choline inhibited growth of this strain in media containing 0.7 M NaCl, while glycine-betaine restores growth to wild-type levels. The defects in Tn5G19 are complemented by a DNA fragment from PAO1 that carries the betB gene. The orp gene is located between 0.6 to 6.6 min while betB is located between 10.5 to 12.5 min on the chromosome of PAO1.
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PMID:Molecular characterization of mutants affected in the osmoprotectant-dependent induction of phospholipase C in Pseudomonas aeruginosa PAO1. 900 19

A series of reactive polymeric supports for the covalent binding of enzymes was prepared from methacrylamide in conjunction with various copolymerized monomers. In all cases the specific surface area of the prepared copolymer particles increased with the ratio of crosslinker to monomer. The polymers containing oxirane and aldehyde groups were promising for direct binding of beta-galactosidase, whereas the supports containing amino groups also qualified for enzyme immobilization with glutaraldehyde.
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PMID:Immobilization of enzymes on methacrylamide-based copolymers. 909 Jul 22

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

Forced overexpression of wild-type Alzheimer amyloid precursor protein (APP) causes postmitotic neurons to degenerate. Caspase-3 (CPP32) is a principal cell death protease involved in neuronal apoptosis during physiological development and under pathological conditions. Here, we investigated whether APP overexpression activates caspase-3 in human postmitotic neurons using adenovirus-mediated gene transfer. When a recombinant adenovirus vector expressing human wild-type APP695 was infected in vitro into neurally differentiated embryonal carcinoma NT2 cells, only postmitotic neurons underwent severe degeneration. Before neurodegeneration, full-length APP- and Abeta-immunoreactive peptides were accumulated in infected neurons, and caspase-3-like protease activity was markedly elevated. Western blot analysis revealed that activated caspase-3 subunits were generated in APP-accumulating neurons. Such neuronal caspase-3 activation was undetectable in NT2 neurons infected with beta-galactosidase-expressing adenovirus. Addition of the caspase-3 inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde to the culture medium significantly reduced the severity of degeneration exhibited by APP-overexpressing neurons. Immunocytochemical analyses revealed that some APP-accumulating neurons contained activated caspase-3 subunits and exhibited the characteristics of apoptosis, such as chromatin condensation and DNA fragmentation. Activation of caspase-3 was also observed in vivo in rat hippocampal neurons infected with the APP-expressing adenovirus. These results suggest that wild-type APP is an intrinsic activator of caspase-3-mediated death machinery in postmitotic neurons.
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PMID:Activation of neuronal caspase-3 by intracellular accumulation of wild-type Alzheimer amyloid precursor protein. 1043 52

5-Aminolevulinic acid (ALA) is a heme precursor that accumulates in some porphyric disorders and in lead poisoning which can undergo metal-catalyzed oxidation producing reactive oxygen species and the keto-aldehyde, 4,5-dioxovaleric acid (DOVA). Evidence in vitro of ALA-induced DNA lesions suggests that ALA and DOVA have mutagenic potential that could possibly contribute to an increased frequency of hepatocellular carcinoma (HCC) in patients with acute intermittent porphyria (AIP). In this study, we evaluated the genotoxic potential of ALA and DOVA. In the absence of exogenous metabolic activation, ALA and DOVA were mutagenic in Salmonella typhimurium tester strain TA104. ALA was also mutagenic in S. typhimurium TA102, but not in TA98, TA100, or TA1535, indicating an oxidative mechanism. Removal of H(2)O(2) with catalase gave only partial protection, suggesting generation of other mutagenic species. Both ALA and DOVA damaged the DNA of Escherichia coli PQ37, inducing the SOS response detected by an increase in beta-galactosidase activity. These results verified the potential mutagenic activity of ALA and DOVA and reinforce the hypothesis that DNA damage induced by ALA may be associated with the development of HCC in individuals suffering from AIP.
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PMID:Genotoxicity of 5-aminolevulinic and 4,5-dioxovaleric acids in the salmonella/microsuspension mutagenicity assay and SOS chromotest. 1221 Oct 78

This work exemplifies the advantages of using a battery of new heterofunctional epoxy supports to immobilize enzymes. We have compared the performance of a standard Sepabeads-epoxy support with other Sepabeads-epoxy supports partially modified with boronate, iminodiacetic, metal chelates, and ethylenediamine in the immobilization of the thermostable beta-galactosidase from Thermus sp. strain T2 as a model system. Immobilization yields depended on the support, ranging from 95% using Sepabeads-epoxy-chelate, Sepabeads-epoxy-amino, or Sepabeads-epoxy-boronic to 5% using Sepabeads-epoxy-IDA. Moreover, immobilization rates were also very different when using different supports. Remarkably, the immobilized beta-galactosidase derivatives showed very improved but different stabilities after favoring multipoint covalent attachment by long-term alkaline incubation, the enzyme immobilized on Sepabeads-epoxy-boronic being the most stable. This derivative had some subunits of the enzyme not covalently attached to the support (detected by SDS-PAGE). This is a problem if the biocatalysts were to be used in food technology. The optimization of the cross-linking with aldehyde-dextran permitted the full stabilization of the quaternary structure of the enzyme. The optimal derivative was very active in lactose hydrolysis even at 70 degrees C (over 1000 IU/g), maintaining its activity after long incubation times under these conditions and with no risk of product contamination with enzyme subunits.
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PMID:Stabilization of a multimeric beta-galactosidase from Thermus sp. strain T2 by immobilization on novel heterofunctional epoxy supports plus aldehyde-dextran cross-linking. 1476 68

The use of bioluminescence as a sensitive marker for detection of Pseudomonas spp. in the rhizosphere was investigated. Continuous expression of the luxCDABE genes, required for bioluminescence, was not detectable in the rhizosphere. However, when either a naphthalene-inducible luxCDABE construct or a constitutive luxAB construct (coding only for the luciferase) was introduced into the Pseudomonas cells, light emission could be initiated just prior to measurement by the addition of naphthalene or the substrate for luciferase, n-decyl aldehyde, respectively. These Pseudomonas cells could successfully be detected in the rhizosphere by using autophotography or optical fiber light measurement techniques. Detection required the presence of 10 to 10 CFU/cm of root, showing that the bioluminescence technique is at least 1,000-fold more sensitive than beta-galactosidase-based systems.
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PMID:Use of Bioluminescence Markers To Detect Pseudomonas spp. in the Rhizosphere. 1634 10

The nucleotide sequence of a 4936 bp Thermoanaerobacter ethanolicus genomic DNA fragment containing the thermostable beta-galactosidase gene lacA and two incomplete open reading frames has been determined. The product of the first frame is highly homologous to alpha-galactosidases (melibiases), the product of the third frame is homologous to the alpha-D-mannosidases. The terminal area of the lacA, immediately following the stop-codon, harbors presumably a transcription termination site. Based on the location of the putative alpha-galactosidase gene melA and of the beta-galactosidase gene lacA on the T. ethanolicus chromosome, their combined transcription could be presumed. The calculated molecular mass of LacA is 86 kDa. LacA belongs to GH family 2 (GH2). Maximal activity of the purified recombinant enzyme was observed between pH values of 5.7 and 6.0 and temperatures of 75-80 degrees C. The highest activity, 480 units mg(-1), was found on lactose (Km 30 mM), the activities on pNPhGal and oNPhGal amounting to 330 and 420 units mg(-1), respectively. Immobilization on aldehyde silochrome increases the thermostability of the enzyme and keeps its high activity.
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PMID:[Thermoanaerobacter ethanolicus gene cluster containing the alpha- and beta-galactosidases genes melA and lacA, and properties of recombinant lacA]. 1635 27

Novel aryl beta-d-galactopyranosides were synthesized employing phase-transfer catalysis, and assayed as potential galactose donors in the presence of beta-galactosidase from bovine testes using pNP-Gal as a reference. The aglycones were represented mainly by nitrophenols containing halogens, hydroxymethyl, aldehyde, carboxyl, ester or amino functions. An unusual intermolecular acetyl migration onto the benzylic alcohol group was observed during galactosylation of hydroxymethylnitrophenols. Pyridyl glycosides were obtained by reaction with the corresponding silver pyridinolates. Glycosides of halo-, hydroxymethyl- or methoxycarbonyl-nitrophenols as leaving groups gave virtually the same yields of transglycosylation products. A minor increase was achieved with nitrosalicylaldehyde as leaving group, whereas carboxy or amino derivatives gave very low or no yield of the transglycosylation product. Commercially available donors such as resorufinyl and 4-methylumbelliferyl beta-d-galactopyranosides exhibited a lower transglycosylation potential than these novel pNP-Gal derivatives.
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PMID:Synthesis and evaluation of glycosyl donors with novel leaving groups for transglycosylations employing beta-galactosidase from bovine testes. 1711 90

A study of the cross-linking of beta-galactosidase on magnetic beads is reported here. The magnetic beads were prepared from artemisia seed gum, chitosan, and magnetic fluid in the presence of a cross-linking regent (i.e., glutaraldehyde). The reactive aldehyde groups of the magnetic beads allowed the reaction of the amino groups of the enzymes. The animated magnetic beads were used for the covalent immobilization of beta-galactosidase. The effect of various preparation conditions on the activity of the immobilized beta-galactosidase, such as immobilizing time, amount of enzyme, and the concentration of glutaraldehyde, were investigated. The influence of pH and temperature on the activity and the stability of the enzyme, both free and immobilized, have been studied. And o-nitrophenyl-beta-D: -galactopyranoside (ONPG) was chosen as a substrate. The beta-galactosidase immobilized on the magnetic beads resulted in an increase in enzyme stability. Optimum operational temperature for immobilized enzyme was 10 degrees Celsius higher than that of free enzyme and was significantly broader.
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PMID:Immobilization of beta-galactosidase onto magnetic beads. 1928 68


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