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K Sekimizu

Publications and source records attributed to K Sekimizu.

At least 19 recordsLinked to original sources
PubMed1992

Reversal by phosphatidylglycerol and cardiolipin of inhibition of transcription and replication by histones in vitro.

We examined the effects of phospholipids on transcription and replication in vitro in the presence of histones. Phosphatidylglycerol and cardiolipin were shown to reverse the inhibitory effects of histones in both random RNA synthesis with purified RNA polymerase II and accurate transcription initiated from the adenovirus 2 major late promoter in a nuclear extract. Phosphatidylserine, phosphatidic acid, and phosphatidylcholine did not activate RNA synthesis although they bound as strongly as phosphatidylglycerol and cardiolipin to histones. Phosphatidylglycerol and cardiolipin also reversed the in vitro inhibition of SV40 DNA replication by histones. Unsaturation of the fatty acid residues was shown to be necessary for the restoration of transcription and replication by phosphatidylglycerol.

Adenoviruses, Human↗
PubMed1992

Inhibition of Escherichia coli DNA topoisomerase I activity by phospholipids.

The DNA relaxation activity of Escherichia coli DNA topoisomerase I in vitro was greatly inhibited by cardiolipin. Inhibition also occurred to some extent with phosphatidylglycerol from egg yolk. Analysis with synthetic phospholipid revealed that phosphatidylglycerol containing unsaturated fatty acids exhibited a strong inhibitory effect, whereas inhibition by phosphatidylglycerol containing saturated fatty acids was weak. Phosphatidylethanolamine showed no inhibitory effect. Chlorpromazine, which interacts with phospholipids, suppressed the inhibitory effect of cardiolipin. Cardiolipin and phosphatidylglycerol with unsaturated fatty acid precipitated topoisomerase I even at low concentrations, whereas phosphatidylglycerol from egg yolk and a synthetic phosphatidylglycerol containing saturated fatty acids precipitated this enzyme only at high concentrations. One-third of the total topoisomerase I in E. coli was found in the membrane fraction. Treatment of E. coli cells with chlorpromazine resulted in relaxation of plasmid DNA. This DNA relaxation was not observed in a topA mutant, suggesting that this relaxation by chlorpromazine in vivo is catalysed by topoisomerase I.

Chlorpromazine↗
PubMed1992

Purification, gene cloning, and gene disruption of the transcription elongation factor S-II in Saccharomyces cerevisiae.

Saccharomyces cerevisiae S-II was purified to near homogeneity as a protein stimulating RNA polymerase II. Four of seven lysyl endopeptidase-digested fragments of S-II were located in the PPR2 sequence reported previously. Analysis of a genomic clone of S-II revealed that S-II and PPR2 are the same protein consisting of 309 amino acid residues, and frame shifts were found in the sequence of PPR2 gene reported previously. Yeast S-II and mouse S-II showed high similarity in their amino acid sequences, especially in their amino-terminal and carboxyl-terminal regions. A gene disruption experiment showed that an S-II null mutant was not lethal under usual growth conditions, indicating that S-II is not essential for the growth of yeast.

Amino Acid Sequence↗
PubMed1992

Induction by psychotropic drugs and local anesthetics of DnaK and GroEL proteins in Escherichia coli.

We examined effects of psychotropic drugs and local anesthetics on the synthesis of heat shock proteins in Escherichia coli. Chlorpromazine, a phenothiazine derivative, was shown to induce DnaK and GroEL proteins, major heat shock proteins in E. coli. The inductions of these proteins were not observed in an rpoH (= htpR) amber mutant strain, indicating that the heat shock sigma factor sigma 32 was required for their inductions. Northern blot hybridization analysis revealed that chlorpromazine induced increases of messenger RNAs for the DnaK and GroEL proteins. Thus, the induction occurred at the level of transcription. Chlorpromazine also induced non-heat shock proteins with molecular masses of 21 kDa, 20 kDa, and 17 kDa, even in the rpoH mutant strain. Other psychotropic drugs and local anesthetics, namely, dibucaine, lidocaine, imipramine, tetracaine and procaine, also induced DnaK and GroEL proteins and the small molecular weight proteins.

Anesthetics, Local↗
PubMed1992

Decrease by psychotropic drugs and local anaesthetics of membrane fluidity measured by fluorescence anisotropy in Escherichia coli.

The effects of psychotropic drugs and local anaesthetics on the fluidity of Escherichia coli cell membranes were examined. Chlorpromazine was shown to increase 1,6-diphenyl-1,3,5-hexatriene fluorescence anisotropy, indicating that it decreased the membrane fluidity. This increase was significant at a temperature of more than 24 degrees C. Dibucaine, lignocaine, imipramine, tetracaine and procaine also increased the fluorescence anisotropy.

Anesthetics, Local↗
PubMed1991

Binding of simian virus 40 large tumor antigen to phospholipid vesicles.

SV40 T antigen is the initiator protein of SV40 DNA replication. We examined the interaction of purified SV40 T antigen with phospholipids by (i) centrifugation analysis with phospholipid vesicles, (ii) filter binding assay and footprint analysis of T antigen binding to the replication origin of SV40 DNA and (iii) analysis of the initiation of SV40 DNA replication in vitro. In all cases, cardiolipin showed affinity for T antigen and inhibited its DNA binding capacity. Phosphatidylglycerol with unsaturated fatty acids also inhibited the binding of T antigen to the replication origin of SV40 DNA, whereas phosphatidylglycerol with saturated fatty acids did not. This finding suggested the importance of unsaturated fatty acids for the interaction of T antigen with phospholipids. Other phospholipids including phosphatidylserine, phosphatidylinositol and phosphatidylethanolamine showed little or no affinity for T antigen.

Antigens, Polyomavirus Transforming↗
PubMed1991

Inhibitory action of phospholipid-interacting drugs on transcription initiation in a nuclear extract of Ehrlich ascites tumor cells.

Drugs with affinity for phospholipids, such as chlorpromazine, verapamil, tetracaine and imipramine, were found to inhibit accurate transcription from adenovirus 2 major late promoter in a nuclear extract of Ehrlich ascites tumor cells. The transcription activity of the nuclear extract inhibited by chlorpromazine was restored by addition of acidic phospholipids. The nuclear extract was also shown to lose transcription activity when treated with phospholipase A2. Chlorpromazine was found to inhibit transcription at the step of initiation, not elongation. Moreover, it did not affect the activity of purified RNA polymerase II, suggesting the interaction of phospholipids with transcription factors in the nuclear extract. Some transcription factors in the nuclear extract were found to have affinity for cardiolipin, and were precipitated with excess cardiolipin. The transcription factors precipitated with cardiolipin could be solubilized with guanidine hydrochloride, and restored the transcription activity of the cardiolipin-treated nuclear extract.

Adenoviruses, Human↗
PubMed1990

Site-directed mutagenesis of arginine 246, glutamic acid 247, and histidine 248 in the eukaryotic transcription factor S-II.

When His248 of the transcription factor S-II was replaced by alanine or tyrosine, the activity of the resulting mutants was less than 30% of that of the wild-type S-II. When Arg246, Glu247, and His248 were all replaced by leucine, the resulting mutant showed complete loss of activity. These results indicate that the amino acid sequence Arg-Glu-His at positions 246-248 of S-II is important for its stimulatory activity. The mutant S-II with no activity could not form a complex with RNA polymerase II, unlike wild-type S-II, but retained ability to interact with DNA.

Amino Acid Sequence↗
PubMed1990

Acidic phospholipids directly inhibit DNA binding of mammalian DNA topoisomerase I.

Inhibition of mammalian DNA topoisomerase I by phospholipids was investigated using purified enzyme. Acidic phospholipids inhibited the DNA relaxation activity of topoisomerase I whereas neutral phospholipid, phosphatidylethanolamine, did not. Accumulation of a protein-DNA cleavable complex, an intermediate which is known to accumulate upon inhibition by a specific inhibitor camptothecin, did not occur. The filter binding assay revealed that the DNA binding activity of the enzyme was inhibited by acidic phospholipids. Moreover, direct binding of phosphatidylglycerol to topoisomerase I was demonstrated. These results indicated that the inhibitory effect of acidic phospholipids on topoisomerase I was due to the loss of the DNA binding of the enzyme as a result of direct interaction between phospholipids and the enzyme.

Animals↗
PubMed1989

Production of functional S-II in Bombyx mori cells.

A cDNA for murine transcription factor S-II (Hirashima et al., J. Biol. Chem (1988) 263, 3858-3863) was inserted into a silkworm baculovirus vector and expressed in Bm-N cells, a cell line of Bombyx mori maintained in the laboratory. Recombinant S-II was purified from virus-infected cell extracts to near homogeneity by ammonium sulfate fractionation, and chromatographies on DEAE-cellulose and phosphocellulose. About 60 micrograms of recombinant S-II was obtained from 1 g of virus-infected cells. The molecular mass and specific activity of recombinant S-II were exactly the same as those of authentic S-II purified from Ehrlich ascites tumor cells.

Animals↗
PubMed1989

Nuclear GTP-binding proteins of Swiss 3T3 cells.

The GTP-binding proteins of Swiss 3T3 cell nuclei were analyzed by filter binding assay and UV cross-linking analysis. The results showed the presence of multiple GTP-binding proteins in the nuclei. Scatchard analysis revealed that the Kd value for GTP binding to high-affinity components was 69 nM, that to low-affinity components being 2.7 microM. The GTP-binding activities of some nuclear proteins were found to change significantly in response to the growth conditions of the cells. During culture of cells in medium without serum, the GTP-binding activity of a 140 kDa protein clearly decreased, whereas that of a 40 kDa protein increased.

Animals↗
PubMed1989

Restricted degradation of U6 RNA in a nuclear extract of Ehrlich ascites tumor cells.

A new nucleolytic activity that causes restricted digestion of U6 RNA was found in a nuclear extract of Ehrlich ascites tumor cells. This nucleolytic activity specifically degrades U6 RNA in the vicinity of its 3'-end with accumulation of a discrete sized degradation product of RNA of 90-95 nucleotides. Since this degradation product was not digested further by the nuclease under these conditions, this trimming of U6 RNA is supposed to be a biologically meaningful reaction. This nucleolytic activity required Mg2+, and was inhibited by Zn2+ or Ca2+.

Animals↗
PubMed1988

Stimulation of transcription from accurate initiation sites by purified S-II.

The effects of transcription factors S-II and S-II', a phosphorylated form of S-II, on accurate transcription were compared in a reconstituted transcription system greatly depleted of S-II. S-II, but not S-II', stimulated the syntheses of run-off products of various truncated class II genes in this system, suggesting that the activity of this factor is regulated by its phosphorylation and dephosphorylation.

Adenoviruses, Human↗
PubMed1988

Sequential early stages in the in vitro initiation of replication at the origin of the Escherichia coli chromosome.

Complexes previously identified in the reconstitution of stages in the initiation of replication of plasmids (oriC) bearing the origin of the Escherichia coli chromosome have been examined further. These are: (i) an ATP complex of dnaA protein, (ii) an initial complex of ATP.dnaA protein with oriC DNA, (iii) an open complex in which a portion of the oriC duplex has been opened by dnaA protein action, (iv) a prepriming complex of the open complex with dnaB, dnaC, and HU proteins, and (v) a complex with a small bubble opened at oriC by dnaB helicase action and by coating with single strand-binding protein (SSB). Helicase and gyrase actions can enlarge the bubble; coupling to priming and replication propagates bidirectional fork movement. Formation and stability of these complexes are profoundly affected by ATP, Mg2+, and temperature, as well as the levels of the participating proteins, including HU and SSB. As examples, the open complex is stable to isolation at a temperature near 38 degrees C but not at 24 degrees C; the prepriming complex requires an elevated temperature and high ATP levels for its formation, but is maintained at a low temperature and is destabilized by Mg2+. These successive steps, subject to a variety of controls, are designed to open the supercoiled duplex for priming and bidirectional replication.

Adenosine Triphosphate↗
PubMed1988

Cardiolipin activation of dnaA protein, the initiation protein of replication in Escherichia coli.

ATP binding to dnaA protein is essential for its action in initiating the replication of plasmids that bear the unique origin of the Escherichia coli chromosome (oriC). ADP bound to that site renders dnaA protein inactive for replication. Diphosphatidylglycerol (cardiolipin), a diacidic membrane phospholipid, displaces the bound nucleotide, and in the presence of components that reconstitute replication, fully reactivates the inert ADP form of dnaA protein. The monacidic phosphatidylglycerol is one-tenth as active as cardiolipin, whereas the neutral phosphatidylethanolamine, the principal E. coli phospholipid, is inactive. Fluphenazine, a tranquilizer drug, blocks cardiolipin activation of dnaA protein, in keeping with the inhibitory action of such agents on phospholipid-dependent enzymes. With the use of this drug to terminate cardiolipin action, dependence of the activation on time, elevated temperature, and high levels of ATP was demonstrated. Cardiolipin binding of nucleotide-free dnaA protein prevents binding of ATP and initiation of oriC replication. Removal of a fatty acid from cardiolipin by phospholipase A reverses this inhibitory effect. The strong and specific interaction of cardiolipin, a cell membrane component, with an essential nucleotide-binding site of dnaA protein, the protein essential for the initiation of chromosome replication, may be an important element in regulating the cell cycle.

Adenosine Diphosphate↗
PubMed1988

The dnaA protein of Escherichia coli. Abundance, improved purification, and membrane binding.

Immunoassays of dnaA protein in extracts from five strains showed a rather constant abundance relative to cell mass, with a variation of 800-2100 molecules/cell; overproducing cells contained 100-fold that number. About half of the dnaA protein in wild type cells was solubilized by a lysis procedure. Within the insoluble fractions, dnaA protein was identified by its characteristic high-affinity binding of ATP. An improved, rapid procedure for purifying dnaA protein from overproducing cells appears to depend on its coprecipitation with phospholipids and depends on solubilization by guanidine HCl. The procedure, with a 5-fold increased yield, also eliminates a potent ATPase contaminant. Purified dnaA protein, unlike dnaB and dnaC proteins, binds to phospholipid vesicles as judged by analysis on sucrose gradient centrifugation.

Adenosine Triphosphate↗