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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The efficiency of Escherichia coli nucleic acids samples: covalently closed circular DNA, linear chromosomal DNA, total RNA degradation mediated by the action of high oxygen pressure; hydrochloric hydroxylamine in alkaline conditions in the presence of cooper ions and in analogous conditions without cooper ions was studied. The nativity of nucleic acids was determined by means of fluorometric analysis of nucleic acids/ethidium bromide complexes. Experiments revealed, that the destructive effect of active oxygen species decreased in the following order: NH2OH.HCl in alkaline conditions in the presence of copper ions-NH2.HCl in alkaline conditions--high pressure of pure oxygen. The stability of nucleic acids decreased in the following order: covalently closed circular DNA-linear DNA-RNA.
Mol Biol (Mosk)
PMID:[Degradation of nucleic acids during generation of superoxide-anion in the presence of copper ions]. 245 81

Temperature-sensitive mutants have been isolated following hydroxylamine mutagenesis of a plasmid containing Escherichia coli rRNA genes carrying selectable markers for spectinomycin resistance (U1192 in 16 S rRNA) and erythromycin resistance (G2058 in 23 S rRNA). These antibiotic resistance alleles, originally identified by Morgan and co-workers, enable us to follow expression of cloned rRNA genes in vivo. Recessive mutations causing the loss of expression of the cloned 16 S rRNA gene were identified by the loss of the ability of cells to survive on media containing spectinomycin. The mutations were localized by in vitro restriction fragment replacement followed by in vivo marker rescue and were identified by DNA sequence analysis. We report here seven single-base alterations in 16 S rRNA (A146, U153, A350, A359, A538, A1292 and U1293), five of which produce temperature-sensitive spectinomycin resistance and two that produce unconditional loss of resistance. In each case, loss of ribosomal function can be accounted for by disruption of base-pairing in the secondary structure of 16 S rRNA. For the temperature-sensitive mutants, there is a lag period of about two generations between a shift to the restrictive temperature and cessation of growth, implying that the structural defects cause impairment of ribosome assembly.
J Mol Biol 1989 Oct 20
PMID:Isolation of temperature-sensitive mutants of 16 S rRNA in Escherichia coli. 253 Dec 27

A water soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), has been used to crosslink horse heart cytochrome c and trypsin-solubilized bovine liver microsomal cytochrome b5. The reaction was conducted under a variety of solution conditions, and the products were purified by a combination of gel filtration and ion-exchange chromatography. Under all conditions of pH, ionic strength, EDC/protein ratio and reaction time that were studied, multiple 1:1 crosslinked complexes were observed with no evidence of a single, dominant species. Acetate, which is often used as a quencher of such reactions, was found to increase the complexity of the reaction products, presumably through EDC-promoted coupling to cytochrome c. Hydroxylamine treatment of the crosslinked complexes, a procedure frequently used to reverse EDC modification of tyrosyl residues, did not reduce the number of crosslinked components observed. The cytochrome b5 heme group was readily extracted from each of the 1:1 crosslinked complexes by standard techniques, so the crosslinking of heme propionate 7 with Lys79 of cytochrome c that might have been anticipated on the basis of molecular graphics modeling [Salemme, F.R. (1976) J. Mol. Biol. 102, 563-568] was not evident from this analysis. Analysis of HPLC tryptic peptide maps produced from crosslinked complexes revealed reduced specificity of trypsin in hydrolysis of EDC-crosslinked protein-protein complexes and unsatisfactory resolution of crosslinked or branched peptides. Nevertheless, it was possible to demonstrate that residues 52-72 of cytochrome b5, a region predicted to be critical to interaction with cytochrome b5 [Salemme, F.R. (1976) J. Mol. Biol. 102, 563-568] was absent from all peptide maps of 1:1 cytochrome c.cytochrome b5 complexes. Based on these results and a review of the literature involving EDC crosslinking of electron transfer proteins, we conclude that the techniques available for specific protein hydrolysis and separation of crosslinked peptides are not adequate to permit routine unambiguous identification of crosslinking sites in carbodiimide-crosslinked complexes.
 ...
PMID:Crosslinking of cytochrome c and cytochrome b5 with a water-soluble carbodiimide. Reaction conditions, product analysis and critique of the technique. 255 10

We have devised a procedure to generate any single base mismatch in a constant sequence context, and have studied these from two points of view. (1) We have examined electrophoretic mobility of 458 base-pair fragments containing approximately centrally located single mismatches, in polyacrylamide gels, compared to fully matched DNA fragments. We found that no single mismatch caused a significant perturbation of gel mobility, and we conclude that all the mismatches may be accommodated within a helical geometry such that there is no alteration of the path of the helix axis in a straight DNA molecule. (2) We have studied all the single mismatches with respect to reactivity to a number of chemical probes. We found that: (a) No mispaired adenine bases are reactive to diethyl pyrocarbonate and are therefore not simply unpaired such that N-7 is exposed. (b) A number of mispaired thymine bases are reactive to osmium tetroxide, and cytosine bases to hydroxylamine. (c) Where crystal or nuclear magnetic resonance structures are available, the reactivity correlates with exposure of the pyrimidine 5,6 double bonds to attack in the major groove as a result of wobble base-pair formation. This is particularly clear for G.T and I.T base-pairs. (d) Reactivity of bases in mismatched pairs can be dependent on sequence context. (e) Reactivity of the C.C mismatch to hydroxylamine is suppressed at low pH, suggesting that a rearrangement of base-pairing occurs on protonation. The results overall are consistent with the formation of stacked intrahelical base-pairs wherever possible, resulting in no global distortion of the DNA structure, but specific enhancement of chemical reactivity in some cases.
J Mol Biol 1989 Oct 20
PMID:Single base mismatches in DNA. Long- and short-range structure probed by analysis of axis trajectory and local chemical reactivity. 258 2

In vitro mutagenesis with hydroxylamine of a ParD- miniderivative of R1, pAB174, yielded mutants that were less stable in the cell than pAB174. Some of these mutants had a thermosensitive phenotype. The replication of pAB2623, one of the thermosensitive mutants, was inhibited in the cell at the restrictive temperature of 42 degrees C. The efficiency of the RepA protein of pAB2623 to promote replication of R1 in an in vitro assay was greatly reduced. Sequence analysis indicated that the repA gene of pAB2623 contains, close to its 3' end, two GC-AT transitions, separated by a single base, that change two consecutive codons of the gene. These results indicate that the phenotype of the mutant is the consequence of a mutated RepA protein and is consistent with the requirement of RepA for the in vivo replication of this plasmid.
Mol Gen Genet 1989 May
PMID:Isolation and characterization of a conditional replication mutant of the antibiotic resistance factor R1 affected in the gene of the replication protein repA. 267 46

Incubation of rat ovarian plasma membranes with [gamma-32P]guanosine 5'-triphosphate (GTP) in the presence of an adenosine triphosphate (ATP)-trapping system results in the labeling of a single protein, Mr 33,000 +/- 3000 designated 'a' (Amir-Zaltsman, Y., Ezra, E., Walker, M., Lindner, H. R. and Salomon, Y. (1980) FEBS Lett. 122, 166-170). Based on competition with other nucleotides it is concluded that protein 'a' is preferentially phosphorylated by [gamma-32P]GTP (Km = 0.28 microM). Phosphorylation of protein 'a' does not occur at pH less than 5 and progressively increases to plateau levels at pH 7-9. Phosphorylation of protein 'a' is absolutely dependent on the presence of divalent cations 1 mM Mg2+, Ca2+, or Cd2+. At higher concentrations, 5-20 mM, Mg2+ or in the presence of 1 mM Mn2+ ions other proteins are also phosphorylated. While vanadate ions selectively prevent the labeling of protein 'a', molybdate ions were found to inhibit phosphorylation of all the membrane proteins including protein 'a'. In contrast to molybdate ions, vanadate ions were found to accelerate the dephosphorylation of phosphoprotein 'a'. We suggest that phosphoprotein 'a' is a high energy protein intermediate in which the phosphate is present as a phosphoramidate for the following reasons: (i) Guanosine diphosphate (GDP) but not guanosine 5'-O-(2-thiodiphosphate) selectively accelerated the dephosphorylation of phosphoprotein 'a' but only in the presence of Mg2+ ions. (ii) The phosphoprotein intermediate is hydrolyzed in the presence of hydroxylamine. (iii) Phosphoprotein 'a' is labile in the presence of 1 N HCl but stable in 1 N NaOH at 37 degrees C. (iv) Phosphoprotein 'a' is heat labile. Phosphoprotein 'a' is readily digested by several proteolytic enzymes and a single cleavage peptide is generated upon treatment with Staphylococcus aureus V8 protease. The properties of protein 'a' were compared and found different from another phosphoprotein Mr 90,000 +/- 1000, designated 'b' that was selected arbitrarily. We propose that protein 'a' is a GTP requiring enzyme intermediate, of yet unidentified function.
Mol Cell Endocrinol 1989 May
PMID:Phosphorylation of proteins in rat ovarian plasma membranes by [gamma-32P]GTP: evidence for the formation of a high energy phosphoprotein. 275 26

The chemical and enzymatic N-oxygenation of verapamil was investigated. Verapamil N-oxide is readily synthesized by chemical means. It is not indefinitely stable, however, and undergoes Cope-type elimination to produce 3,4-dimethoxystyrene and a hydroxylamine. The major stable metabolite observed during the metabolism of verapamil with rat and hog liver microsomes and purified flavin-containing monooxygenase is 3,4-dimethoxystyrene. 3,4-Dimethoxystyrene is formed at a rate 4 times that of nor-verapamil. Studies suggest that N-oxygenation is catalyzed largely by the flavin-containing monooxygenase and N-demethylation is catalyzed by cytochrome P-450. This conclusion is based on the effects of cytochrome P-450 inhibitors and positive effectors for the flavin-containing monooxygenase as well as on studies with the purified enzyme. In the presence of rat and hog liver microsomes, significant stereoselectivity in N-oxygenation of verapamil is observed (S/R ratio of 3.1 and 4.1, respectively). With purified hog and rat hepatic flavin-containing monooxygenase, the stereoselectivity for verapamil N-oxygenation (S/R ratio of 10.1 and 6.6, respectively) suggests a role for this enzyme in the stereoselective first-pass metabolism of verapamil.
Mol Pharmacol 1989 Sep
PMID:Enantioselective N-oxygenation of verapamil by the hepatic flavin-containing monooxygenase. 277 29

In vitro expression of cDNA encoding bovine opsin is accomplished using the baculovirus expression vector system. Full-length opsin was synthesized which was recognized by poly- and monoclonal antisera raised against bovine rhodopsin. Upon infection with a recombinant virus, 1 x 10(6) insect cells produced up to 3 micrograms opsin. Incubation of the in vitro synthesized opsin with 11-cis retinal produced a hydroxylamine-stable, photosensitive pigment.
Mol Biol Rep 1988
PMID:Synthesis of functional bovine opsin in insect cells under control of the baculovirus polyhedrin promoter. 297 52

Reaction thermodynamics have been calculated for an oxene model for cytochrome P-450 oxidations of four related arylamines: aniline, p-hydroxyaniline, acetanilide, and acetaminophen, by both radical and nonradical mechanisms, using a semiempirical molecular orbital method (modified neglect of differential overlap). The results indicate that for both p-hydroxyaniline and acetaminophen, a recently proposed peroxidase-like mechanism leading directly to p-benzoquinoneimines via radical intermediates is thermodynamically favored over N-hydroxylamine formation by H abstraction or addition rearrangement. These studies also provide a detailed characterization of three candidate species for the toxic reactive intermediate of acetaminophen: 1) p-benzoquinoneimines, 2) the radical intermediate formed by H abstraction from the nitrogen, and 3) the radical intermediate formed by H abstraction from the phenol. Calculated electron and spin densities indicate that the radical formed by H abstraction from the phenol oxygen does not remain localized on the oxygen, but is primarily a semiquinone aryl radical with significant unpaired spin density on the ring carbon atoms, particularly on C-3 and C-5. This result is consistent with the hyperfine splitting pattern observed for a transient radical species in a hydroxyl radical-mediated chemical oxidation of acetaminophen. The radical formed by H abstraction from the nitrogen also delocalizes on the ring carbons, but to a lesser extent and at the 2- and 4-positions. A closed shell mechanism of N oxidation of arylamines appears to lead directly to the hydroxylamines with less likelihood of precursor reactive intermediates. Toxic species could then be formed by loss of H2O from the hydroxylamines.
Mol Pharmacol 1985 Mar
PMID:Metabolic activation and toxicity of acetaminophen and related analogs. A theoretical study. 298 85

The RAD3 gene of Saccharomyces cerevisiae, which is involved in excision repair of DNA and is essential for cell viability, was mutagenized by site-specific and random mutagenesis. Site-specific mutagenesis was targeted to two regions near the 5' and 3' ends of the coding region, selected on the basis of amino acid sequence homology with known nucleotide binding and with known specific DNA-binding proteins, respectively. Two mutations in the putative nucleotide-binding region and one in the putative DNA-binding region inactivate the excision repair function of the gene, but not the essential function. A gene encoding two tandem mutations in the putative DNA-binding region is defective in both excision repair and essential functions of RAD3. Seven plasmids were isolated following random mutagenesis with hydroxylamine. Mutations in six of these plasmids were identified by gap repair of mutant plasmids from the chromosome of strains with previously mapped rad3 mutations, followed by DNA sequencing. Three of these contain missense mutations which inactivate only the excision repair function. The other three carry nonsense mutations which inactivate both the excision repair and essential functions. Collectively our results indicate that the RAD3 excision repair function is more sensitive to inactivation than is the essential function. Overexpression of wild-type Rad3 protein and a number of rad3 mutant proteins did not affect the UV resistance of wild-type yeast cells. However, overexpression of Rad3-2 protein rendered wild-type cells partially UV sensitive, indicating that excess Rad3-2 protein is dominant to the wild-type form. These and other results suggest that Rad3-2 protein retains its affinity for damaged DNA or other substrates, but is not catalytically active in excision repair.
Mol Cell Biol 1986 Apr
PMID:Analysis of the essential and excision repair functions of the RAD3 gene of Saccharomyces cerevisiae by mutagenesis. 302 77


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