Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

DNA polymerase alpha from germinated wheat embryos was purified by ammonium sulphate fractionation, chromatography on DEAE-Toyopearl, followed by phosphocellulose and heparin Sepharose columns. The specific activity of the purified enzyme was more than 60,000 units/mg. It belongs to the alpha-type according to the large molecular mass, high sensitivity to NEM, aphidicoline, 200 mM KCl, low sensitivity to ethidium bromide and the absence of inhibition by ddTTP. DNA polymerase alpha consists of four subunits as shown by SDS-PAGE and seems to be homogeneous under non-denaturing conditions.
Mol Biol Rep 1992 Feb
PMID:Isolation of DNA polymerase alpha from germinated wheat embryos. 154 80

Eight respiratory-deficient mutants of Chlamydomonas reinhardtii have been isolated after mutagenic treatment with acriflavine or ethidium bromide. They are characterized by their inability to grow or their very reduced growth under heterotrophic conditions. One mutation (Class III) is of nuclear origin whereas the seven remaining mutants (Classes I and II) display a predominantly paternal mt- inheritance, typical of mutations residing in the mitochondrial DNA. Biochemical analysis has shown that all mutants are deficient in the cyanide-sensitive cytochrome pathway of the respiration whereas the alternative pathway is still functional. Measurements of complexes II + III (antimycin-sensitive succinate-cytochrome c oxido-reductase) and complex IV (cytochrome c oxidase) activities allowed to conclude that six mutations have to be localized in the mitochondrial apocytochrome b (COB) gene, one in the mitochondrial cytochrome oxidase subunit I (COI) gene and one in a nuclear gene encoding a component of the cytochrome oxidase complex. By using specific probes, we have moreover demonstrated that five mutants (Class II mutants) contain mitochondrial DNA molecules deleted in the terminal end containing the COB gene and the telomeric region; they also possess dimeric molecules resulting from end-to-end junctions of deleted monomers. The two other mitochondrial mutants (Class I) have no detectable gross alteration. Class I and Class II mutants can also be distinguished by the pattern of transmission of the mutation in crosses. An in vivo staining test has been developed to identify rapidly the mutants impaired in cyanide-sensitive respiration.
Plant Mol Biol 1992 Feb
PMID:Biochemical, genetic and molecular characterization of new respiratory-deficient mutants in Chlamydomonas reinhardtii. 155 49

A method is described for producing fragments of a protein suitable for studies of protein folding. The codon for a single methionine residue is introduced into the cloned gene of barnase, and the gene product cleaved with cyanogen bromide. The site of mutation was chosen to be at the surface of the protein in a region connecting segments of secondary structure in the native enzyme. The alpha + beta protein was mutated from Val36----Met, and split into two fragments, B(1-36) containing the alpha-helical regions and B(37-110), the beta-sheet. The fragments were purified by ion exchange chromatography. Neither retains catalytic activity. Fluorescence, circular dichroism, and 1H nuclear magnetic resonance data indicate that their structures are each close to that of random-coil peptides. The two fragments associate to form a tight complex (Kd = 0.2 to 0.6 microM), which displays spectroscopic properties similar to those of the uncleaved protein. The catalytic activity is restored in the complex with a value for Km similar to that for native enzyme but with kcat reduced about three- to fourfold. The second-order rate constant for association on mixing fragments in the concentration range 2.5 to 7.5 microM is 1 x 10(5) s-1 M-1.
J Mol Biol 1992 Apr 05
PMID:Dissection of an enzyme by protein engineering. The N and C-terminal fragments of barnase form a native-like complex with restored enzymic activity. 156 53

A fragment of barnase comprising amino acids 1 to 36 (B(1-36)) that encompasses the region containing the two large helices (residues 6-18 and 26-34) of the native protein has been obtained by cleavage of the barnase mutant Val36----Met with cyanogen bromide. The circular dichroism (c.d.) spectrum of B(1-36) in the far ultraviolet indicates that the fragment is only weakly structured in water at neutral pH. The two-dimensional 1H nuclear magnetic resonance spectrum of B(1-36) shows, however, that a fraction of the population does have helical structure, spanning amino acid residues 8 to 18. B(1-36) becomes more helical in 35% trifluoroethanol. This is indicated by the c.d. spectrum and the increase from 6.6 to 7.0 in the pKa of His18, which is known to interact with the dipole of helix 6-18 in native barnase. The helical region of B(1-36) in 35% trifluoroethanol extends to residue 6. It is calculated from extrapolation of a trifluoroethanol titration of the ellipticity at 222 nm that B(1-36) exhibits in water approximately 6% of helical structure, calculated for a 36 residue alpha-helical peptide. This corresponds to approximately 20% of that expected for an 11-residue alpha-helical region. In trifluoroethanol, c.d. measurements indicate that approximately 30% of the 36-residue peptide is helical. It has been shown from extensive studies of the refolding of barnase that there is a folding intermediate that contains residues 8 to 18 in a helical conformation and that residue 6 is mainly unfolded. The experiments on the conformation of B(1-36) show that a small, but significant fraction, of its population in water adopts the conformation of the major alpha-helix during the barnase folding pathway, in the absence of tertiary interactions. Thus, in the folding of native barnase, secondary structure formation can precede the docking of the major alpha-helix onto the beta-sheet.
J Mol Biol 1992 Apr 05
PMID:An N-terminal fragment of barnase has residual helical structure similar to that in a refolding intermediate. 156 54

Cloned parasites of the FCR3 strain of Plasmodium falciparum that survived treatment with either mitomycin C or ethidium bromide were grown and subcloned. The chromosomes of 10 cloned isolates from each population were analysed by contour-clamped homogenous electric field gel electrophoresis. Eight of 10 isolates from the mitomycin C-treated population contained a detectable polymorphic chromosome change while none of the isolates from the ethidium bromide-treated population did. One of the polymorphic changes involved chromosome #4. A pyrimethamine resistant derivative of FCR3, FCR3-D5, that had previously been shown to contain a single nucleotide change in the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene but no detectable chromosome polymorphisms, was sequentially treated with mitomycin C and a higher concentration of pyrimethamine than the strain had previously been shown to be resistant to in order to determine if there was a correlation between the selection of chromosome #4 polymorphisms and the applied selective pressure. Most of the cloned survivors of this treatment were found to contain chromosome polymorphisms that involved chromosome #4, the chromosome on which the DHFR-TS gene is located. The polymorphic changes were usually different in the different isolates. The selection of mitomycin C-treated, pyrimethamine resistant strains with chromosome #4 polymorphisms will be discussed.
Mol Biochem Parasitol 1992 Apr
PMID:An examination of the mitomycin C induction of chromosome polymorphisms in cultures of Plasmodium falciparum. 157 78

In the present paper we report the characterization of a repetitive DNA sequence from the genome of the Maracay strain of Trypanosoma cruzi. The sequence is 1025 nucleotides long and represent about 7% of the total nuclear DNA of the parasite with a copy number of 1-2 x 10(4) copies per genome. It is also present in a high copy number in several strains of T. cruzi but absent from other trypanosomatids and from the human genome. The DNA sequence is interspersed and present on many chromosomes. We show that the different copies of this repeat present extensive endonuclease restriction polymorphisms. Using 2 conserved oligonucleotide primers derived from the repetitive sequence, together with the polymerase chain reaction technique, the presence of less than 1/30 of a single parasite can be readily detected by ethidium bromide staining.
Mol Biochem Parasitol 1992 Apr
PMID:Characterization of a highly repeated interspersed DNA sequence of Trypanosoma cruzi: its potential use in diagnosis and strain classification. 157 85

Exposure of mammalian cells to a variety of agents leads to the activation of pre-existing proteins and the induction of specific genes. We have recently described the appearance of a specific DNA-binding protein in nuclei from cells exposed to ionizing radiation (Singh, S. P., and Lavin, M. F. (1990) Mol. Cell. Biol. 10, 5279-5285). This protein is present in the cytoplasm of unperturbed cells and is apparently translocated to the nucleus in response to radiation damage. We describe here the purification and characterization of this specific DNA-binding protein. Purification involved the use of affinity chromatography employing a multimeric form of the DNA-binding motif conjugated to cyanogen bromide-activated Sepharose. Three DNA-binding species were recognized by UV-cross-linking and South-Western analysis. The major species or that with the highest affinity was approximately 70 kDa in size. DNase-1 footprint analysis revealed a single binding site in the kappa immunoglobulin gene enhancer and in a putative control sequence upstream from the c-myc gene. At salt concentrations as high as 1 M, up to 40% of the DNA-binding activity was maintained and the Kd was calculated to be 1.205 x 10(-6) M-1. Binding activity was found to be modulated by phosphorylation. Removal of phosphate groups from the protein resulted in a major loss of binding activity. It is not clear at this stage whether the factor(s) described here plays a role in transcription control or a more general DNA-processing role in response to radiation damage.
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PMID:Purification and characterization of a DNA-binding protein activated by ionizing radiation. 158 18

5-Lipoxygenase-activating protein (FLAP) is specifically labeled by [125I]L-669,083 and [125I]L-691,678, photoaffinity analogues of two classes of potent leukotriene biosynthesis inhibitors. Because human FLAP contains only a single tryptophan residue at position 72 and two internal methionine residues at positions 89 and 125, we have used reagents that specifically cleave at these residues, in conjunction with antipeptide antisera, to localize the site of attachment of the photoaffinity ligands. Immunoprecipitation of specifically labeled peptide fragments after digestion of photoaffinity-labeled FLAP by iodosobenzoic acid at 72Trp demonstrates that the inhibitors bind to FLAP amino-terminal to this residue. This finding is consistent with similar immunoprecipitation studies after digestion at methionine residues using cyanogen bromide. These findings localize the site of attachment of the inhibitors to a region of FLAP that includes the hydrophilic loop between the proposed first and second transmembrane regions. Based on these findings, site-directed mutagenesis of human FLAP was performed to define key amino acids involved in inhibitor binding. Using a radioligand binding assay, analysis of mutants of human FLAP expressed in COS-7 cells demonstrates that a number of residues in the amino-terminal half of the first hydrophilic loop of the protein can be deleted without significantly affecting inhibitor binding. In contrast, no inhibitor binding was detectable with mutants in which amino acid residues in the carboxyl-terminal half of this loop were deleted. Furthermore, a point mutation of 62Asp to asparagine results in a mutant with dramatically reduced affinity for inhibitors. This loss of affinity was not displayed by a mutant in which 62Asp was mutated to a glutamate residue, suggesting that a negative charge associated with residue 62 may be critical for inhibitor binding. The roles that amino acid residues in the carboxyl-terminal half of the first hydrophilic loop of FLAP may play in the binding of leukotriene biosynthesis inhibitors are currently under investigation.
Mol Pharmacol 1992 Jul
PMID:Identification of amino acid residues of 5-lipoxygenase-activating protein essential for the binding of leukotriene biosynthesis inhibitors. 163 56

Oleosins (oil body membrane proteins) of 20.5 and 18 kDa have been purified from sunflower (Helianthus annuus) seeds and polyclonal antibodies raised against them. The precipitated rabbit immunoglobulin fraction was purified by affinity chromatography on cyanogen bromide-activated Sepharose and specifically recognised polypeptides of 18 and 20.5 kDa in sunflower homogenate and oil body fractions assayed by western blotting. A near-full-length cDNA clone was isolated for the 20.5 kDa oleosin. The 694 bp cDNA contained an open reading frame of 534 bp, followed by an untranslated region of 81 bp and a poly(A) region of 70 bp. The open reading frame encoded a polypeptide of 19.8 kDa. Study of transcript localisation revealed message to be abundant in the embryo during the later stage of development and still present in the dry seed. No signal was observed in RNA prepared from expanding leaves.
Plant Mol Biol 1992 Aug
PMID:cDNA sequence of a sunflower oleosin and transcript tissue specificity. 164 88

Incubation of rocker-cultured neonatal rat heart cells with 3 mM L(+)-lactate led to a sharp increase in the sensitivity of cardiomyocytes to the beta-adrenergic agonist isoprenaline, as measured by their chronotropic response. This effect was accompanied by a reduction in the arachidonic acid content of the total phospholipids. The phospholipase A2-activator melittin as well as free arachidonic acid induced this supersensitivity to the same degree. On the other hand, the L(+)-lactate-evoked supersensitivity could be blocked by the phospholipase A2 inhibitors mepacrine and n-bromophenacyl-bromide, suggesting an involvement of phospholipase A2 in the process of beta-adrenergic sensitization. The sensitizing action of arachidonic acid was blocked by the lipoxygenase inhibitors esculetin and nordihydroguaiaretic acid, but not by the cyclo-oxygenase inhibitor indomethacin. Supersensitivity was likewise evoked by 15-S-hydroxyeicosatetraenoic acid (15-S-HETE), but not by 5-S-HPETE or 5-S-HETE. These findings suggest that the phospholipase A2-15-lipoxygenase pathway plays a role in the induction of beta-adrenergic supersensitivity in the cultured cardiomyocytes and point to a new physiological role of the lipoxygenase product 15-S-HETE.
Mol Cell Biochem 1991 Mar 27
PMID:Modulation of the beta-adrenergic response in cultured rat heart cells. I. Beta-adrenergic supersensitivity is induced by lactate via a phospholipase A2 and 15-lipoxygenase involving pathway. 164 55


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