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:2.3.1.28 (chloramphenicol acetyltransferase)
5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A reliable assay is reported for the detection of the marker gene aminoglycoside phosphotransferase activity in cells that express this enzyme transiently or as a result of stable genetic transformation. This method combines the simplicity of the dot assays with the reliability of the more elaborate and time consuming electrophoretic or chromatographic methods. Inhibition of phosphatases and protein kinases during the reaction reduces labeled ATP consumption by these enzymes. As a result, this assay allows the detection of approx 10 times lower levels of the enzyme than currently used methods. To detect the expression of reporter genes in transformed cells aminoglycoside phosphotransferase can be used as well as the widely used chloramphenicol acetyltransferase enzyme.
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PMID:Detection of the neor gene expression in animal cells after genetic transformation. 132 34

Sequence analysis upstream from the subtilin structural gene (spaS) in Bacillus subtilis ATCC 6633 revealed several open reading frames, SpaB, SpaC, and SpaD. SpaB, consisting of 599 amino acid residues, shows excellent homology with a variety of membrane translocator proteins, such as HlyB from Escherichia coli and some mammalian multidrug resistance proteins. When the spaB gene was interrupted by integration of a chloramphenicol acetyltransferase gene, the ability of the cell to produce subtilin, as determined by a halo assay, was lost. The homology of SpaB to translocator proteins, including transmembrane and ATP-binding regions, suggests that SpaB may play a role in subtilin secretion. The SpaB open reading frame overlaps with another open reading frame called SpaC, and the possibility that the SpaB and SpaC proteins become fused by frameshifting is considered. Regions of homology between SpaD (177 residues) and HlyD were also found, suggesting that SpaD may participate with SpaB in translocation of subtilin through the membrane. Although no readily interpretable homologies to SpaC (442 residues) were found, its sequence suggests that it is membrane associated. The absence of rho-independent transcription terminators between these open reading frames suggests that they are all part of the same operon.
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PMID:The subtilin gene of Bacillus subtilis ATCC 6633 is encoded in an operon that contains a homolog of the hemolysin B transport protein. 173 28

A consensus sequence in parvovirus nonstructural protein NS1 has been predicted to be an ATP-binding domain associated with an ATPase and a DNA helicase activity. To investigate the function of NS1 in viral gene expression, a site-directed mutagenesis converting NS1 lysine 405 to serine in parvovirus H-1 was carried out by the polymerase chain reaction. As shown previously, a parvovirus genome containing a deleted NS1 gene was excised from a bacterial plasmid and replicated when a wild-type NS1 gene was provided in trans but failed to be excised and replicate when the mutant NS1 gene was supplied. Interestingly, the serine 405 mutation totally lost the activity of trans activation on the virus late promoter (P38) in a chloramphenicol acetyltransferase (CAT) assay and it lost evidence for cytotoxicity in two tumor cell lines (HeLa Gey and NB324K). The serine 405 NS1 protein was translocated normally to the nucleus. These results suggest that the NS1 lysine 405 of H-1 in its putative purine nucleotide-binding site is essential for viral DNA replication and that this domain may be involved in the regulation of the P38 promoter by an unknown mechanism. The loss of NS1 cytotoxicity on tumor cells suggests that NS1 expression is the major cause of cell killing by parvoviruses, which may facilitate further study of the mechanism of oncosuppression by parvoviruses.
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PMID:Mutation of lysine 405 to serine in the parvovirus H-1 NS1 abolishes its functions for viral DNA replication, late promoter trans activation, and cytotoxicity. 214 94

The matrix-targeting signal of mitochondrial preornithine carbamyl transferase has been fused to either murine dihydrofolate reductase (pODHFR) or bacterial chloramphenicol acetyltransferase (pOCAT). Loosening of the tightly folded "native" structure of the two proteins following their synthesis in a rabbit reticulocyte lysate was assayed by the acquisition of protease sensitivity (pODHFR and pOCAT) or by the loss of enzyme activity (pOCAT). By these criteria, the bulk population of both precursor proteins was tightly folded following release from the ribosome, even in the presence of ATP and excess reticulocyte lysate. Neither protein unfolded as a consequence of binding to the surfaces of anionic liposomes or intact mitochondria. However, a non-native form of full-length pOCAT, exhibiting a loss of enzymatic activity and an enhanced protease sensitivity, was detected in association with a submitochondrial fraction that banded between the inner and outer mitochondrial membrane fractions on sucrose density gradients. Delivery of the precursor molecule to this position required ATP and a proteinaceous component on the surface of the organelle.
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PMID:Import of precursor proteins into mitochondria: site of polypeptide unfolding. 234 78

The chicken estrogen receptor exists in three interconvertible forms, two of which bind estradiol with high affinity and one which lacks the capacity to bind estradiol. Interconversion is regulated by reactions involving ATP/Mg2+. By cotransfecting into A431 cells estrogen receptor cDNA in an expression vector together with the pA2 (-821/-87) tk-CAT vitellogenin construct, we demonstrate that constitutive expression of chloramphenicol acetyltransferase (CAT) activity can be regulated either by selection of ligand or by modifying phosphorylation reactions in the recipient cells. In the presence of estrogen receptors, constitutive expression of CAT activity is inhibited in three situations: (i) in the absence of an estrogenic ligand; (ii) in the presence of an anti-estrogen; and (iii) in the presence of an estrogenic ligand together with 12-O-tetradecanoylphorbol 13-acetate (TPA). Estrogen receptor mediated repression of constitutive CAT activity is not observed with the pA2 (-331/-87) tk-CAT construct, indicating that DNA sequences required for repression are located between -821 and -331 base pairs upstream of the transcription initiation site.
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PMID:Receptor interconversion model of hormone action. 3. Estrogen receptor mediated repression of reporter gene activity in A431 cells. 234 42

It has been suggested that newly synthesized proteins are maintained in their unfolded state by cellular ATP-driven factors which may prevent or reverse the formation of misfolded structures or promote the correct assembly of oligomeric proteins or post-translational secretion. Using a photocross-linking approach, we have identified the 20S heat-shock GroEL protein as the major cytosolic component which forms a complex with the unfolded newly synthesized pre-beta-lactamase or chloramphenicol acetyltransferase in Escherichia coli. Dissociation of these complexes is ATP-dependent. The unfolded state of pre-beta-lactamase, maintained by the transient interaction with GroEL, may be essential for the secretion of this protein.
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PMID:Transient association of newly synthesized unfolded proteins with the heat-shock GroEL protein. 290 24

The uptake and expression by plastids isolated from dark-grown cucumber cotyledons (etioplasts) of two pUC derivatives, pCS75 and pUC9-CM, respectively carrying genes for the large small subunits of ribulose bisphosphate carboxylase/oxygenase of Anacystis nidulans or chloramphenicol acetyltransferase, is reported. Untreated etioplasts take up only 3% as much DNA as that taken up by EDTA-washed etioplasts after 2 hr of incubation with nick-translated [32P]-pCS75. The presence or absence of light does not affect DNA uptake, binding, or breakdown by etioplasts. Calcium or magnesium ions inhibit DNA uptake by 86% but enhance binding (23-200%) and breakdown (163-235%) of donor DNA by EDTA-treated etioplasts. Uncouplers that abolish membrane potential (delta psi), transmembrane proton gradient (delta pH), or both do not affect DNA uptake, binding, or breakdown by etioplast. However, both DNA uptake and binding are severely inhibited by ATP. Presumably this results from the hydrolysis of ATP, because the poorly hydrolyzable analog adenyl-5'-yl imidodiphosphate does not inhibit the uptake or binding of DNA by etioplasts. beta-Lactamase specified by the ampicillin resistance gene of pCS75 can be detected only in EDTA-treated etioplasts that have been incubated with the plasmid pCS75. After the incubation of EDTA-treated etioplasts with pCS75, immunoprecipitation using antiserum to the small subunit of ribulose bisphosphate carboxylase/oxygenase from A. nidulans reveals the synthesis of small subunits; these are smaller by 2 kDa than the cucumber small subunit encoded by the nuclear genome. Treatment of etioplasts with 10 mM EDTA shows a 10-min duration to be optimal for the expression of chloramphenicol acetyltransferase encoded by pUC9-CM. A progressive increase in the expression of this enzyme is observed with an increase in the concentration of pUC9-CM in the DNA uptake medium. The plasmid-dependent incorporation of [35S]methionine by EDTA-treated organelles declines markedly during cotyledon greening in vivo.
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PMID:Uptake and expression of bacterial and cyanobacterial genes by isolated cucumber etioplasts. 311 48

Vibrio anguillarum strains were isolated from chloramphenicol-resistant bacteria in diseased fish. Plasmid Rms418, which confers chloramphenicol resistance, was transferred from V. anguillarum GN11379 to Escherichia coli K12 by conjugation. The Rms418-encoded chloramphenicol acetyltransferase (CAT) [EC 2.3.1.99] was isolated and purified to homogeneity using affinity chromatography on immobilized p-amino-chloramphenicol or ATP. The general CAT could be adsorbed by a matrix with a chloramphenicol base ligand (Zaidenzaig, Y. & Shaw, W.V. (1976) FEBS Lett. 62,266-271), but the Rms418-encoded CAT was not bound under these conditions. The specific activity of the enzyme, when measured by the spectrophotometric assay, was 71.4 units/mg protein at 37 degrees C. The molecular weight of the enzyme treated with SDS and 2-mercaptoethanol was shown to be approximately 22,000. The molecular weight of the native enzyme, as determined by gel filtration, was approximately 69,000, and the optimal pH was 7.8. The Km values for chloramphenicol and CoASAc were 34.5 and 150 microM, respectively. Enzyme activity was inhibited by HgCl2, p-chloromercuribenzoate (p-CMB), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and ethylendiaminotetraacetic acid (EDTA). The half life at 53 degrees C was approximately 100 min.
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PMID:Purification and some properties of a chloramphenicol acetyltransferase mediated by plasmids from Vibrio anguillarum. 314 69

ATP-citrate lyase (CL) catalyzes the conversion of citrate and CoA to oxaloacetate (OA) and acetyl-CoA. As the coupled malic dehydrogenase (MDH) assay is not able either to study the effect of oxaloacetate (OA) on CL activity or to measure accurately CL activity in biological samples, a new assay was developed. The CL-citrate coupled CAT assay measures the amount of acetyl-CoA formed by transferring radiolabeled acetyl-CoA synthesized from [14C]citrate to chloramphenicol with chloramphenicol acetyltransferase (CAT). Employing this assay, the rate of increase in acetyl-CoA synthesis from citrate is linear with respect to added CL. Kinetic values for ATP, CoA and citrate are similar to those obtained using the MDH assay. The effect of CL phosphorylation on enzyme activity was determined. CL phosphorylated by cAMP-dependent protein kinase or by this kinase and glycogen synthase kinase-3 (GSK-3) decreases the apparent Vmax without changing the apparent Km. The effect of OA, a product of the enzyme reaction, on CL activity was also determined. Computational analysis of the data obtained without added OA and at three concentrations of OA indicate that the apparent Km for the substrate is not altered even though the apparent Vmax is decreased. The effect of OA on the activity of phosphorylated enzyme was also determined. OA decreases the apparent Vmax of the phosphorylated enzyme to the same extent as in control CL. This assay is able to measure CL activity in cytosol from 3T3-L1 adipocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effect of oxaloacetate and phosphorylation on ATP-citrate lyase activity. 766 53

Mice homozygous for chromosomal deletions at or around the albino locus on chromosome 7 express reduced levels of a group of liver genes. Here, we report the isolation and characterization of cDNA and genomic clones encoding one of the affected genes, the mouse adult liver S-adenosylmethionine (AdoMet) synthetase. This enzyme catalyzes the synthesis of AdoMet, which functions in transmethylation and transsulfuration. Mouse AdoMet synthetase cDNA is 3232 base pairs (bp) in length and contains an open reading frame that encodes an enzymatically active polypeptide of 396 amino acids. The mouse AdoMet synthetase shares 98 and 96% amino acid sequence identity with the adult liver enzyme in the rat and human, respectively. AdoMet synthetases possess the consensus ATP-binding motif Gly-X-Gly-X-X-Gly and a putative ATP-binding Lys residue at conserved locations. As an initial step toward understanding the control of AdoMet synthetase gene expression, we characterized the complete transcription unit of this gene. The AdoMet synthetase gene spans approximately 18 kilobases and consists of nine exons ranging from 78 to 1920 bp. The transcription initiation site was demonstrated by rapid amplification of cDNA ends and confirmed by primer extension studies. A putative TATA box is located at -28 to -23 bp upstream of the transcription start site. The cis-acting DNA elements in the 5'-flanking region of the AdoMet synthetase gene that drive chloramphenicol acetyltransferase gene expression in mouse hepatocytes were identified by transient expression assays. The -365 to -2-bp DNA region upstream of the transcription start site of the AdoMet synthetase gene contains promoter elements, and the -518 to -366-bp DNA region might be involved in negative gene regulation.
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PMID:Cloning and expression of murine S-adenosylmethionine synthetase. 831 64


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