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 convenient and sensitive enzyme-linked immunosorbent assay (ELISA) for the STb heat-stable enterotoxin of Escherichia coli was developed and used to quantify STb production by strains with a high level of expression. Based on an antigenic profile of the secreted form of STb, a synthetic peptide (STb3-27) spanning the major predicted epitope was synthesized, coupled to keyhole limpet hemocyanin, and used to immunize rabbits. Anti-STb3-27 antibodies were affinity purified on a synthetic peptide-Sepharose 4B column and used in a direct-binding STb ELISA. Based on a highly purified form of toxin as a standard, the ELISA detected as little as 1 to 2 ng of STb from crude culture filtrates. ELISA data revealed that natural STb-producing strains elaborate little STb in defined-medium cultures relative to that elaborated by a recombinant strain harboring a cloned copy of the estB gene. Replacement of the endogenous STb promoter with any of several highly active promoters, including a bacteriophage T7 promoter, a beta-galactosidase promoter, and a tryptophan-beta-galactosidase hybrid (tac) promoter, increased the yield of STb 10- to 20-fold over levels obtained by an E. coli strain harboring the recombinant estB gene. The high level of STb antigen detected by the ELISA correlated with intestinal secretory activity. The combination of a convenient assay and effective hyperproduction of STb will serve as a basis for a large-scale toxin purification strategy.
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PMID:High-level production of Escherichia coli STb heat-stable enterotoxin and quantification by a direct enzyme-linked immunosorbent assay. 225 13

Transcription of the trp operon of Bacillus subtilis is regulated in response to the availability of tryptophan. The first structural gene of the operon is preceded by a 204-base-pair transcribed leader region that contains a segment with the features of a procaryotic termination site. Transcription of the leader region was analyzed in vivo and in vitro to determine whether this putative termination site was used to regulate operon expression. When RNA was isolated from wild-type cells grown in the presence of excess tryptophan, transcripts of the operon ended at the putative termination site. In contrast, RNA isolated from cells grown in the absence of tryptophan or from a mutant strain which is constitutive for trp operon expression contained trp transcripts that extended beyond the termination site into the structural genes. To assess termination quantitatively in vivo, a trpE-lacZ fusion was constructed in which the trp promoter and leader region controls hybrid beta-galactosidase formation. The effects on hybrid beta-galactosidase levels of point mutations and deletions introduced into this leader region were determined. The results obtained establish that transcription of the trp operon structural genes is regulated in the leader region. This regulation appears to be mediated by the formation of alternative secondary structures of the leader transcript. In vitro transcription studies with wild-type and mutant templates provided additional evidence that the identified alternative RNA secondary structures regulate transcription termination. We hypothesize that binding of a tryptophan-activated regulatory protein to a specific segment of the nascent leader transcript prevents formation of one of the alternative secondary structures, thereby directing RNA polymerase to terminate transcription.
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PMID:Novel form of transcription attenuation regulates expression the Bacillus subtilis tryptophan operon. 242 55

Hypertonic NaCl administered to rats or mice has been demonstrated to induce in the liver a rapid disaggregation of polyribosomes and inhibition of protein synthesis. This study was concerned with whether hypertonic NaCl would affect nucleocytoplasmic translocation of RNA in the livers of rats. The effect of tube-feeding a hypertonic (10.7%) NaCl solution (321 mg in 3 ml/100 g body wt) for 10 minutes on in vitro release of 14C-orotate-labeled nuclear RNA was assayed. Although the combination of nuclei and cytosols of livers of hypertonic NaCl-treated rats revealed diminished in vitro labeled nuclear RNA release in comparison with hepatic nuclei and cytosols of control (water-treated) rats, each of the two components varied in activity. Even though the overall effect was an inhibitory one, cytosols of livers of hypertonic NaCl-treated rats stimulated in vitro release of labeled nuclear RNA, whereas nuclei of livers of hypertonic NaCl-treated rats revealed diminished in vitro release of labeled nuclear RNA in comparison with cytosols and nuclei of livers of control rats. The stimulatory effect of the hepatic cytosols of the hypertonic NaCl-treated rats was essentially unaffected by pretreatment of the rats with puromycin or cycloheximide, but was abolished by pretreatment of the cytosols in vitro with alpha-mannosidase or beta-galactosidase. Passage of cytosols of control and experimental livers through concanavalin A-agarose columns concentrated the activities of the eluates in stimulating in vitro labeled nuclear RNA release. In vivo 14C-orotate labeling of hepatic nuclear RNA for 30 minutes was increased by hypertonic NaCl treatment in comparison with water treatment of control animals. In vivo 14C-glucosamine incorporation into hepatic proteins of nuclei and nuclear envelopes was increased in hypertonic NaCl-treated rats in comparison with controls. In vitro 3H-tryptophan binding to proteins (trichloracetic acid-precipitable) to cytosols of livers of hypertonic NaCl-treated rats was increased in comparison with binding of controls. The results suggest that the administration of hypertonic NaCl rapidly leads to a change in hepatic cytosol whereby the activity to stimulate in vitro labeled nuclear RNA release is enhanced. This occurs without new protein synthesis, and the effect is probably mediated through a glycoprotein. In contrast, the hepatic nuclei of the rats treated with hypertonic NaCl show a decreased ability to release in vitro labeled nuclear RNA, possibly because of the development of a nuclear lesion.
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PMID:The influence of hypertonic NaCl on nucleocytoplasmic translocation of RNA in the rat liver. 242 14

Aspergillus niger tryptophan auxotrophic mutants have been isolated after UV irradiation of conidiospores. The mutants belong to two different complementation groups, trpA and trpB, which complement each other in heterokaryons. Neither of the mutations could be complemented with the cloned A. niger trpC gene. To obtain A. niger trpC mutants in a direct way, gene inactivation by cotransformation was performed. For this purpose an in-frame gene fusion between the A. niger trpC and Escherichia coli lacZ genes was constructed and shown to be functionally expressed after introduction into A. niger by cotransformation with the pyrA gene as selective marker. Among the beta-galactosidase expressing cotransformants, obtained with either circular or linearized vectors, no trpC mutants were detected, even after enrichment. Such mutants, however, could be obtained by cotransformation of A. niger with specific fragments of the fusion gene. Biochemical analysis of the cotransformants indicated that in nearly all cases the fusion gene had replaced the wild-type trpC gene. Genetic analysis showed that the trpC mutation is not linked to any of the A. niger loci described so far. The trpC mutants can be complemented by the cloned A. niger trpC gene as well as by the A. nidulans trpC gene.
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PMID:Tryptophan auxotrophic mutants in Aspergillus niger: inactivation of the trpC gene by cotransformation mutagenesis. 261 62

The beta-galactosidase-based assay for lysine developed by Tuffnell & Payne was used to measure the bioavailabilities of cyst(e)ine, methionine, threonine and tryptophan in pronase digests of 17 foods. The digests were assayed by estimating the beta-galactosidase synthesis responses of five Escherichia coli mutants, each requiring one of the respective amino acids for protein synthesis. Deletion mutants were used whenever possible in order to ensure strain stability. Single digests of each food were assayed with 3 or 4 separate cultures of each mutant and the results were compared with those from the corresponding chemical assay. Omitting the anomalously low values for one food, the rank correlation coefficients of the bio- and chemo-assay values were 0.61 (cysteine), 0.91 (lysine), 0.95 (methionine), 0.64 (threonine) and 0.85 (tryptophan). Mean (+/- S.D.) relative amino acid bioavailabilities (casein = 100%) for the 17 foods were: cysteine, 53 +/- 23; lysine, 90 +/- 10; methionine, 95 +/- 18; threonine, 89 +/- 13; and tryptophan, 89 +/- 25. The cysteine mutant appeared to give unusually low bioavailability values except for the milk products. These amino acid mutants afford a rapid method for assaying the bioavailabilities of at least four of the five amino acids studied.
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PMID:The use of Escherichia coli mutants to measure the bioavailability of essential amino acids in foods. 265 34

Defective transducing phages carrying aroG, the structural gene for phenylalanine (phe)-inhibitable phospho-2-keto-heptonate aldolase (EC 4.1.2.15; previously known as 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase[phe]), have been isolated, and DNA from two of these phages has been used to construct a restriction map of the region from att lambda to aroG. A 7.6-kb PstI-HindIII fragment from one of these phages was cloned into pBR322 and shown to contain aroG. The location of aroG within the 7.6 kb was established by subcloning and Tn3 transpositional mutagenesis. A fragment carrying the aroG promoter and operator has been cloned into a high copy number promoter-cloning vector (pMC489), and the resulting aroGpo-LacZ' (alpha) fusion subcloned in a low copy number vector. Strains with this fusion on the low copy number vector exhibit negative regulation of beta-galactosidase expression by both phenylalanine and tryptophan and positive regulation by tyrosine in a tyrR+ background.
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PMID:Cloning of aroG, the gene coding for phospho-2-keto-3-deoxy-heptonate aldolase(phe), in Escherichia coli K-12, and subcloning of the aroG promoter and operator in a promoter-detecting plasmid. 286 Nov 43

A total of 32 strains of the family Leptospiraceae (23 strains of Leptospira interrogans, 6 strains of Leptospira biflexa, 2 strains of Leptonema and 1 strain of Leptospira parva) were examined for enzyme activities using 89 substrates (API ZYM system). More than 90% of the strains belonging to the family Leptospiraceae possessed strong activities of beta-D-galactosidase, beta-D-glucosidase and 5 esterases (C5, C6, C8, C9 and C10). More than 90% of the strains belonging to the genus Leptospira, except L. parva, had strong activities of L-lysine arylamidase and alpha-L-glutamate arylamidase. L. biflexa strains, except serovar andamana, were different from the other strains examined in that they possessed glycyl-glycine arylamidase, glycyl-phenylalanine arylamidase and L-tryptophan arylamidase. L. biflexa strains, except andamana, L. parva and Leptonema strains possessed strong activities of glycine arylamidase and leucyl-glycine arylamidase. Two strains of the genus Leptonema were different from the strains belonging to the genus Leptospira in that they possessed strong activities of beta-D-lactosidase. L. parva lacked alpha-D-galactosidase which other strains belonging to the family Leptospiraceae possessed. Dendrogram analysis revealed that strains belonging to the family Leptospiraceae were divided into 4 groups. The first group consisted of all strains belonging to L. interrogans and serovar andamana of L. biflexa; the second group consisted of the remaining 5 serovars of L. biflexa; the third group consisted of the genus Leptonema; and the fourth group consisted of only L. parva.
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PMID:Enzyme activities of the strains belonging to family Leptospiraceae detected by the API ZYM system. 289 26

Out of carcinogenic heterocyclic amines, which are produced by pyrolysis of tryptophan in food, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) were found to reduce the activity of enzymes related to catecholamine metabolism in clonal rat pheochromocytoma PC12h cells. By 6 days' culture in the presence of 10 nM to 10 microM Typ-P-1 and -2, these heterocyclic amines were accumulated in the cells, and activity of tyrosine hydroxylase (TH) and aromatic L-aminoacid decarboxylase (AADC) were reduced markedly. Reduction of these enzyme activity was observed with Trp-P-1 and -2 at the concentrations lower than 1 microM, while cell protein and enzyme activity of a non-specific enzyme, beta-galactosidase were reduced only with 10 microM Trp-P-1. These results show that these heterocyclic amines are neurotoxins specific for dopaminergic neurons.
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PMID:Reduction of enzyme activity of tyrosine hydroxylase and aromatic L-aminoacid decarboxylase in clonal pheochromocytoma PC12h cells by carcinogenic heterocyclic amines. 290 12

A single-copy integration vector was used for the in vitro construction of translational fusions to the lacZ gene of Escherichia coli. Insertion of a single copy of the lacZ fusion into the B. subtilis chromosome leads to an easily detected Amy- phenotype. A trpE-lacZ fusion was constructed in which the trp promoter directs hybrid beta-galactosidase (beta Gal) synthesis. The level of beta Gal in a wild-type strain carrying the trpE-lacZ fusion in the chromosome is regulated by exogenous tryptophan, while a 5-methyltryptophan-resistant mutant constitutively synthesizes betaGal. A trpF-lacZ fusion was constructed and used to determine the effect of a frameshift mutation in the trpE gene on expression of the trpF-lacZ fusion. The frameshift mutation in trpE led to a three-fold reduction in the levels of the trpF-lacZ fusion. The levels of the betaGal activity of these integrated lacZ fusions appear to provide a quantitative measure of the expression of B. subtilis genes under single-copy conditions.
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PMID:Construction of a single-copy integration vector and its use in analysis of regulation of the trp operon of Bacillus subtilis. 301 40

The cold agglutinin isolated from the albumin gland of the snail Achatina fulica was modified with various chemical reagents in order to detect the amino acids and/or carbohydrate residues present in its carbohydrate-binding sites. Treatment with reagents considered specific for modification of lysine, arginine and tryptophan residues of the cold agglutinin did not affect the carbohydrate-binding activity of the agglutinin. Modification of tyrosine residues showed some change. However, modification with carbodiimide followed by alpha-aminobutyric acid methyl ester causes almost complete loss of its binding activity, indicating the involvement of aspartic acid and glutamic acid in its carbohydrate-binding activity. The carbohydrate residues of the cold agglutinin were removed by beta-elimination reaction, indicating that the sugars are O-glycosidically linked to protein part of the molecule. Removal of galactose residues from the cold agglutinin by the action of beta-galactosidase indicated that the galactose molecules are beta-linked. These carbohydrate-modified glycoproteins showed a marked change in agglutination property, i.e. they agglutinated rabbit erythrocytes at both 10 degrees C and 25 degrees C, indicating that the galactose residues of the glycoprotein play an important role in the cold-agglutination property of the glycoprotein. The c.d. data showed the presence of an almost identical type of random-coil conformation in the native cold agglutinin at 10 degrees C and in the carbohydrate-modified glycoprotein at 10 degrees C and 25 degrees C. This particular random-coil conformation is essential for carbohydrate-binding property of the agglutinin.
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PMID:Studies on chemical modification of cold agglutinin from the snail Achatina fulica. 311 67


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