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)

Carboxymethylated Escherichia coli beta-galactosidase EC 3.2.1.23 could be broken to polypeptides of fairly uniform size (average molecular weight about 22,000 daltons) by heating for less than or equal to 8 h at 100 degrees C and pH 7.5 IN 8 M-urea. Using phosphocellulose chromatography in NaCl-urea gradients, the resulting polypeptide mixture could be resolved in three fractions essentially homogeneous by disc gel electrophoresis in urea at several pH values, and by isoelectric focusing. One of these fractions was active as alpha-donor in in vitro complementation of beta-galactosidase activity with Escherichia coli mutant M15; this activity was largely retained after CNBr cleavage. All three fractions carried arginine as carboxyl-terminal amino acid. No significant amount of any specific amino could be detected in NH2-terminal position.
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PMID:Thermal fragmentation of Escherichia coli beta-galactosidase. Isolation and characterization of an alpha-complementing and two non-complementing polypeptide fractions. 1 Nov 92

Hydrolysis with trypsin of citraconyl-carboxy-methyl-beta-galactosidase was carried out under limiting conditions. No Asp-Arg-X sequences were cleaved and many large peptides were produced. Butanol extraction from dilute acid proved very useful for separating the more hydrophobic fragments. Peptides were purified and sequenced. From this digest and two earlier preparations, all 80 theoretically possible tryptic fragments have been isolated and their structures determined.
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PMID:Amino acid sequence of beta-galactosidase. VI. Limited tryptic digestion of the citraconylated protein and sequences of tryptic peptides. 14 95

We have previously demonstrated the existence of two types of endopeptidase in Escherichia coli. A purification procedure is described for one of these, designated protease II. It has been purified about 13,500-fold with a recovery of 24%. The isolated enzyme appears homogeneous by electrophoresis and gel filtration. Its molecular weight is estimated by three different methods to be about 58,000. Its optimal pH is around 8. Protease II activity is unaffected by chelating agents and sulfhydryl reagents. Amidase and proteolytic activities are stimulated by calcium ion, which decreases the enzyme stability. Like pancreatic trypsin, this endopeptidase catalyses the hydrolysis of alpha-amino-substituted lysine and arginine esters. It appears distinct from the previously isolated protease I, which is a chymotrypsin-like enzyme. The apparent Michaelis constant for hydrolysis of N-benzoyl-L-arginine ethyl ester is 4.7 X 10(-4) M. The esterase activity is inhibited by diisopryopylphosphorofluoridate (Ki(app) equals 2.7 X 10(-3) M) and tosyl lysine chloromethyl ketone (Ki(app) equals 1.8 X 10(-5) M), indicating that serine and histidine residues may be present in the active site. However, protease II is insensitive to phenylmethanesulfonyl fluoride and several natural trypsin inhibitors. Its amidase and esterase activities are competitively inhibited by free arginine and aromatic amidines. The proteolytic activity measured on axocasein is very low. In contrast to trypsin, protease II is without effect on native beta-galactosidase. It easily degrades aspartokinase I and III. Nevertheless both enzymes are resistant to proteolysis in the presence of their respective allosteric effectors. These results provide further evidence that such differences in protease susceptibility can be related to the conformational state of the substrate. The possible implication of structural changes in the mechanism of preferential proteolysis in vivo, is discussed.
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PMID:Protease II from Escherichia coli. Purification and characterization. 24 Aug 39

The relative toxicities of several incorporated analogs of phenylalanine, methionine, arginine, and proline were assessed by a variety of criteria in a derivative of Escherichia coli 15 requiring the antagonized amino acids. Toxicity of the analog-substituted cell protein was most consistently indicated by its insolubility at graded temperatures, its increased breakdown, the relative suppression of further cell growth, and lethality. The relative toxicity of poorly utilized analogs could be judged clearly only by the first two criteria. Toxicity generally increased as follows: selenomethionine < 2,5-dihydrophenylalanine and m-fluorophenylalanine < o-fluorophenylalanine and norleucine < ethionine < p-fluorophenylalanine < azetidine-2-carboxylate < canavanine. The overall perturbation of cell protein structure indicated by the toxicity of the methionine and phenylalanine analogs correlated with their alteration of charge and bulk and was greatly modified by minor positional modifications of fluorine. Among the more specific functional impairments, the activity and heat stability of beta-galactosidase were lowered in parallel by substitutions of phenylalanine and methionine analogs, but not in the usual order of toxicity. Flagella were transiently motile with p-fluorophenylalanine, moderately motile with m-fluorophenylalanine, and fully motile with all methionine analogs. Usually the analog incorporations were no more than bacteriostatic in E. coli strains, canavanine killing only the E. coli 15 substrain extensively in minimal media. Selenomethionine supported indefinite growth of procaryotes such as Bacillus subtilis and certain E. coli strains, but only upon supplementation, at least initially, with many nonessential metabolites.
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PMID:Comparative physiological effects of incorporated amino acid analogs in Escherichia coli. 35 62

Regulation of argA operon expression in Escherichia coli K-12 was studied in a cell-free, deoxyribonucleic acid-dependent, enzyme-synthesizing system. lambdaAZ-7 deoxyribonucleic acid, which carries a fusion of the lacZ structural gene to the argA operon so that beta-galactosidase synthesis is under argA regulation, was used as the template. To eliminate extraneous readthrough from lambda promoters, lambda repressor was introduced into the synthesis mixtures by preparing the S-30 component from a strain (514X5a-12-29) that carries a multicopy hybrid plasmid (pKB252) containing the lambdacI gene. Under these conditions beta-galactosidase synthesis was repressed 90% by the arginine repressor when a sufficient concentration of L-arginine was present. This repression could be overcome by escape synthesis when the lambdaAZ-7 deoxyribonucleic acid concentration in the synthesis mixtures was increased. Guanosine 3'-diphosphate-5'-diphosphate stimulated beta-galactosidase synthesis from this template.
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PMID:Regulation of argA operon expression in Escherichia coli K-12: cell-free synthesis of beta-galactosidase under argA control. 41 Jul 86

Carboxymethylated beta-galactosidase from Escherichia coli was dissociated at 100 degrees C to form carboxymethylated fragments A and B. The mol.wts. of carboxymethylated fragments A and B were determined by gel filtration to be 64300 and 22400 respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of carboxymethylated fragments A and B that had been pretreated with 2-mercaptoethanol and sodium dodecyl sulphate yielded mol.wts. of 64000 and 22100 respectively. Carboxymethylated fragments A and B had arginine as their C-terminal amino acid. When a crude extract of E. coli M15 was filtered through a column of Sepharose 6B, it was found that carboxymethylated fragment B could restore beta-galactosidase activity when added to fractions having mol.wts. estimated to be 123000, 262000 and 506000. These fractions are referred to as ;complementable fractions'. Similarly, it was found that carboxymethylated fragment A could restore enzyme activity to tractions having mol.wts. estimated to be 63000, 253000 and 506000. Estimates of the molecular weights of the beta-galactosidase activity obtained by restoration with carboxymethylated fragments A and B were made by filtering the active enzyme through another column of Sepharose 6B. The enzyme obtained by complementation with carboxymethylated fragment B, i.e. the complemented enzyme, had mol.wt. 525000, and that obtained with carboxymethylated fragment A had mol.wts. of 525000, 646000 and 2000000. The latter finding suggests that multiple forms of complemented beta-galactosidase can exist.
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PMID:Restoration of beta-galactosidase to Escherichia coli M15. Complementation studies. 41 87

Intracistronic alpha-complementation between a cyanogen bromide digest of beta-galactosidase and an extract of the lac Zminus operator-proximal deletion mutant M15 was used to monitor the purification of a cyanogen bromide peptide (CB2) responsible for the complementation. Key steps in the purification were ion exchange chromatography on carboxymethylcellulose and sulfopropyl-Sephadex in the presence of urea, and Sephadex gel filtration. CB2 contains residues 3 to 92 of beta-galactosidase. Its sequence is: Ile-Thr-Asp-Ser-Leu-Ala-Val-Val-Leu-Gln-Arg-Arg-Asp-Trp-Glu-Asn-Pro-Gly-Val-Thr-Gln-Leu-Asn-Arg-Leu-Ala-Ala-His-Pro-Pro-Phe-Ala-Ser-Trp-Arg-Asn-Ser-Glu-Glu-Ala-Arg-Thr-Asp-Arg-Pro-Ser-Gln-Gln-Leu-Arg-Ser-Leu-Asn-Gly-Glu-Trp-Arg-Phe-Ala-Trp-Phe-Pro-Ala-Pro-Glu-Ala-Val-Pro-Glu-Ser-Trp-Leu-Glu-Cys-Asp-Leu-Pro-Glu-Ala-Asp-Thr-Val-Val-Val-Pro-Ser-Asn-Trp-Gln-Met. Thus no more than 1/13 of the beta-galactosidase polypeptide chain, starting 2 residues from the NH2 terminus, is necessary for alpha-complementation with M15 as alpha-acceptor.
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PMID:Amino acid sequence of beta-galactosidase. IV. Sequence of an alpha-complementing cyanogen bromide peptide, residues 3 to 92. 109 37

Culturing of Salmonella typhimurium or Escherichia coli cells in the presence of low concentrations (</=1 mug/ml) of chloramphenicol (CAP) permitted exponential growth, but at doubling times up to twice those of controls. When such cultures were subsequently starved for uracil or arginine, derepression of aspartate transcarbamylase (ATCase) or ornithine transcarbamylase, respectively, was enhanced three- to 10-fold as compared to cultures not exposed to CAP. Enhancement of beta-galactosidase synthesis by prior exposure to CAP was also observed in uracil-starved E. coli cultures. Stimulation of enzyme synthesis appeared to be a specific effect of CAP; low levels of erythromycin, puromycin, sparsomycin, tetracycline, and rifampin did not show such effects. Derepression of ATCase synthesis in exponentially growing cells in the presence of CAP did not result in stimulation of enzyme synthesis by CAP. A prior history of growth of a culture in the presence of CAP was shown to be necessary for enhancement of enzyme synthesis by CAP; furthermore, continued presence of CAP in the medium during starvation was not necessary for enhanced enzyme synthesis and inhibited it in some instances. Enhanced enzyme synthesis in starving, CAP-treated cultures could be blocked by rifampin, which suggested that CAP treatment allows prolonged or more extensive messenger ribonucleic acid synthesis.
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PMID:Stimulation of derepressed enzyme synthesis in bacteria by growth on sublethal concentrations of chloramphenicol. 114 88

A 0.9 kb cDNA for the foot and mouth disease virus (FMDV) type Asia 1 63/72, cloned in the plasmid pUR222 by dC/dG tailing method, was expressed into a protein which was immunogenic in guinea pigs and cattle. The protein purified to homogeneity was found to be basic and of 38 kDa. A sequence of 879 nucleotides of the inserted cDNA was obtained. The nucleotide sequence was 65% GC-rich and was homologous to the gene for VPI of FMDV types A5, OIK and C3 to the extent of 35-40%. From the nucleotide sequence, a sequence of 293 amino acids was derived which contained 43 arginine, 4 lysine, 7 glutamic acid and 18 aspartic acid residues making the protein highly basic. The molecular weight was calculated to be 31.6 kDa. The 38 kDa protein produced by the cloned cDNA is a fused protein composed of the 293 amino acids; 5 and 55 amino acids of the alpha-complementation protein of the beta-galactosidase at the N and C terminal, respectively, and 5 amino acid coded by the dG/dC tails used for cloning the cDNA.
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PMID:Nucleotide sequence of the cDNA and the derived amino acid sequence for the major antigenic protein of foot and mouth disease virus, type Asia 1 63/72. 131 47

Northern-blot analysis was used to demonstrate that an increase in extracellular glucose concentration increased the content of preproinsulin mRNA 2.3-fold in the beta-cell line HIT T15. A probe for the constitutively expressed glyceraldehyde-3-phosphate dehydrogenase was used as a control. Mannoheptulose blocked this effect of glucose. A stimulatory effect on preproinsulin mRNA levels was also observed in response to mannose and to 4-methyl-2-oxopentanoate. However, galactose and arginine were ineffective. Glucagon, forskolin and dibutyryl cyclic AMP also elicited an increase in HIT-cell preproinsulin mRNA. The ability of the 5' upstream region of the preproinsulin gene to mediate the effect of glucose and other metabolites on transcription was studied by using a bacterial reporter gene technique. HIT cells were transfected with a plasmid, pOK1, containing the upstream region of the rat insulin-1 gene (-345 to +1) linked to chloramphenicol acetyltransferase (CAT). Co-transfection with a plasmid pRSV beta-gal containing beta-galactosidase driven by the Rous sarcoma virus promoter was used as a control for the efficiency of transfection; expression of CAT activity in transfected HIT cells was normalized by reference to expression of beta-galactosidase. Glucose caused a dose-dependent increase in expression of CAT activity, with a half-maximal effect at 5.5 mM and a maximum response of 4-fold. Mannoheptulose blocked this effect of glucose. Other metabolites (mannose, 4-methyl-2-oxopentanoate and leucine plus glutamine) were also able to increase insulin promoter-driven CAT expression, but galactose and arginine were ineffective. The stimulatory effect of glucose on CAT expression was not blocked by verapamil and was inhibited by increasing extracellular Ca2+ from 0.4 to 5 mM. Both dibutyryl cyclic AMP and forskolin caused an increase in insulin promoter-driven gene expression in the presence of 1 mM-glucose, but neither agent further increased the level of expression occurring in the presence of a maximally stimulating glucose concentration. The phorbol ester phorbol 12-myristate 13-acetate (PMA) also increased insulin promoter-driven CAT expression in the presence of 1 mM-, but not 11 mM-glucose. Staurosporine blocked the stimulatory effect not only of PMA but also of glucose and of dibutyryl cyclic AMP. We conclude that the 5' upstream region of the insulin gene contains sequences responsible for mediating the stimulatory effect of glucose on insulin-gene transcription.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Control of insulin gene expression by glucose. 132 37


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