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I Sagami

Publications and source records attributed to I Sagami.

49 records · Page 3Linked to original sources
PubMed1987

Purification and characterization of chicken ovalbumin upstream promoter transcription factor from HeLa cells.

A transcription factor which binds to the chicken ovalbumin upstream promoter (COUP) sequence spanning between -70 and -90 is required for efficient transcription of the ovalbumin gene. This COUP transcription factor has been purified approximately 200,000-fold by a combination of conventional column and sequence-specific DNA affinity column chromatography. A few polypeptides were identified in the purified preparation on sodium dodecyl sulfate gel. Upon renaturation, all the major polypeptides in the molecular size range between 43 and 53 kDa bound specifically to the COUP sequence. Furthermore, at least one of the renatured polypeptides in the region of 43-45 kDa retained transcriptional activity. The binding of the COUP transcription factor to the ovalbumin promoter cannot be competed by DNA fragments which contain the CCAAT box promoter sequence. Since the COUP and CCAAT binding proteins can be separated on an S300 column, they are distinct molecules. Using band-shifting assays and 3' and 5' deletion mutants and oligonucleotide mutants, the sequence important for binding was mapped.

Animals↗
PubMed1987

Interactions between a DNA-binding transcription factor (COUP) and a non-DNA binding factor (S300-II).

We have identified previously two transcription factors, COUP (chicken ovalbumin upstream promoter) and S300-II, from HeLa cell nuclear extracts. In this paper, the purine base and the phosphate backbone contact sites for the COUP transcription factor were defined. These studies indicate that the COUP box transcription factor interacts with specific base residues in the major groove of the DNA helix. In addition, we have purified the S300-II factor over 100,000-fold. The polypeptide possessing functional transcriptional activity has been identified by SDS-PAGE followed by gel-slice elution and a renaturation assay. It is absolutely required for in vitro function of the ovalbumin promoter. In addition, S300-II stimulates transcription from the MMTV and lysozyme promoters. Kinetic studies probing the interaction of S300-II with COUP factor suggest that it may stabilize COUP-promoter complexes by slowing their rate of dissociation.

Animals↗
PubMed1987

Characterization of hepatic and pulmonary cytochromes P-450 in 3-methylcholanthrene-treated hamsters.

Two major forms of hepatic cytochrome P-450 (hepatic P-450MCI and P-450MCII) were purified approximately 5-fold from liver microsomes in Syrian golden hamsters treated with 3-methylcholanthrene (MC). The purified preparations of hepatic P-450MCI and P-450MCII contained 9.6 and 8.3 nmol cytochrome P-450 (P-450) per mg protein, respectively, and were essentially free from NADPH-cytochrome c (P-450) reductase (fpT), NADH-cytochrome b5 reductase and cytochrome b5. By sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the molecular weights of hepatic P-450MCI and P-450MCII were estimated to be 56,000 and 53,500. Further, a major form of pulmonary P-450 (P-450MC) were purified from lung microsomes of MC-treated hamster, and contained 14.2 nmol P-450 per mg protein, and estimated to be 56,000 in monomeric molecular weight, indicating the similar molecular weight to hepatic P-450MCI in the hamster. From the absorption spectra the oxidized forms of hepatic P-450MCI and P-450MCII were high- and low-spin ferric hemoproteins, respectively, and pulmonary P-450MC was similar to hepatic P-450MCII in their hemoprotein spin state. No difference, however, was observed in the CO-reduced forms among hepatic P-450MCI, P-450MCII and pulmonary P-450MC, all exhibiting 446.5 nm Soret bands. In a reconstituted system containing fpT and dilauroylphosphatidylcholine (DLPC), pulmonary P-450MC efficiently catalyzed benzo[a]pyrene (BP) hydroxylation at a rate of 11.4 mol formed per min per mol P-450, but hepatic P-450MCI and P-450MCII both exhibited lower levels, e.g., 0.49 and 0.54, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Benzo[a]pyrene metabolism by purified cytochrome P-450 from 3-methylcholanthrene-treated rats.

The metabolism of benzo[a]pyrene in reconstituted pulmonary mono-oxygenase systems has been studied. Metabolites formed by pulmonary cytochrome P450MC, a major form of pulmonary cytochrome P-450 isolated from 3-methylcholanthrene-treated rats, were analysed by h.p.l.c. The profiles of benzo[a]pyrene metabolites formed by the reconstituted P-450MC systems were different from that obtained with rat-lung microsomes, indicating the presence of several unknown metabolites in the reconstituted systems containing NADPH-cytochrome P-450 reductase and epoxide hydrolase. 3-Hydroxybenzo[a]pyrene was a major product formed by pulmonary cytochrome P-450MC, in the absence or presence of epoxide hydrolase. The addition of purified epoxide hydrolase to the reconstituted systems increased the formation of dihydrodihydroxy-benzo[a]pyrenes, particularly 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene. The 9,10-dihydro-9,10-dihydroxybenzo[a]pyrene was the major dihydrodiol formed by pulmonary cytochrome P-450MC. By the addition of epoxide hydrolase the total amount of phenols decreased in parallel with an increased production of dihydrodiol, but the amount of quinones was not changed. Similar results concerning the related production of phenols and dihydrodiols, in the absence and presence of epoxide hydrolase, were obtained in reconstituted systems of hepatic cytochrome P-450MC, the major form of hepatic cytochrome P-450 from 3-methylcholanthrene-treated rats.

Animals↗
PubMed1986

Pulmonary microsomal cytochrome P-450 from 3-methylcholanthrene-treated hamsters: purification, characterization, and metabolism of benzo(a)pyrene.

A major form of pulmonary cytochrome P-450 (pulmonary P-450MC) was purified approximately 165-fold from lung microsomes of 3-methylcholanthrene (MC)-treated hamsters. The purified preparation contained 14.2 nmol of cytochrome P-450 (P-450) per mg protein and was essentially free from NADPH-cytochrome P-450 (cytochrome c)-reductase (NADPH-reductase) and epoxide hydrolase. Pulmonary P-450MC exhibits an absorption maximum at 446.5 nm in the difference spectrum of reduced hemoprotein-CO complex, and a low-spin state of ferric iron in the heme. By sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, the molecular weight of pulmonary P-450MC was estimated to be 56,000. In a reconstituted system, pulmonary P-450MC efficiently catalyzed benzo(a)pyrene (BP) hydroxylation, but showed low activities for 7-ethoxycoumarin O-deethylation and benzphetamine N-demethylation. In Ouchterlony double diffusion analysis, hamster pulmonary P-450MC reacted to the antibody prepared against rat hepatic P-450MC to form a faint precipitation line with a spur, indicating that the two P-450MCs have a common antigenic site but are not immunologically identical. When incubated with [14C]BP in a reconstituted system containing NADPH-reductase and epoxide hydrolase, hamster pulmonary P-450MC formed much higher amounts of BP diols, especially 7,8-diol, than were formed by rat pulmonary P-450MC.

Animals↗
PubMed1986

Identification of two factors required for transcription of the ovalbumin gene.

Two transcription factors, COUP and S300-II, were isolated and partially purified from HeLa cell nuclear extracts. Both factors are required for the efficient transcription in vitro of the ovalbumin gene but not the simian virus 40 early genes. COUP factor binds to the chicken ovalbumin upstream promoter (COUP) sequence which lies between -70 to -90 base pairs upstream from the cap site. A series of competition experiments with a band-shifting assay was carried out to determine the relative affinity of COUP box transcription factor for various promoters. We found that a promoter DNA fragment isolated from the ovalbumin gene competes better than those isolated from the ovomucoid, Y, and alpha-genes. In contrast, the the simian virus 40 early genes, the beta-globin gene, and the adenosine deaminase gene promoters do not compete well in this assay. The molecular weight of the COUP factor was estimated by S-300 column chromatography, glycerol gradient centrifugation to be 90,000. However, two bands were observed in sodium dodecyl sulfate gel electrophoresis of cross-linked COUP factor to a 32P-labeled oligonucleotide containing the COUP sequence. The protein moieties of the major and minor bands were estimated to be 85,000 to 90,000 and 40,000 to 45,000, respectively. The S300-II factor with an apparent molecular weight of 45,000 in an S-300 column is required for function in an in vitro reconstituted transcription system. In contrast to the COUP factor, the S300-II factor does not have apparent specificity for binding to the ovalbumin gene promoter. The S300-II factor may function by interacting with RNA polymerase or other DNA-binding transcription factors.

Base Sequence↗
PubMed1985

Liver microsomal drug metabolism in ethanol-treated hamsters.

Administration of ethanol in drinking water to Syrian golden hamsters for 1-3 weeks caused alterations of microsomal cytochrome P-450-dependent monooxygenase activities in the liver accompanied by a slight elevation in cytochrome P-450 content. Ethanol treatment resulted in an increase in the activities for ethanol oxidation, aniline p-hydroxylation and dimethylnitrosamine N-demethylation. In particular, when dimethylnitrosamine was used as a substrate, the rate of formaldehyde formation was enhanced by 2- to 2.7-fold, while ethanol oxidation and aniline p-hydroxylation were increased by 1.5- to 2- and 1.2- to 1.3-fold, respectively. On the other hand, the activities of 7-ethoxycoumarin O-deethylase, benzphetamine N-demethylase and benzo[a]pyrene 3-hydroxylase were apparently decreased after ethanol treatment. These results for hamsters were significantly different from those reported for rats.

Animals↗
PubMed1985

Microsomal monooxygenase system in Morris hepatoma: purification and characterization of cytochromes P-450 from Morris hepatoma 5123D of 3-methylcholanthrene-treated rats.

Two forms of cytochrome P-450 (hepatoma P-450MCI and P-450MCII) were purified from hepatoma 5123D microsomes of tumor-bearing rats treated with 3-methylcholanthrene. Hepatoma P-450MCI had a specific content of 18.4 nmol/mg protein and showed a main protein band with a minimum molecular weight of 56,000 on sodium dodecyl sulfate-polyacrylamide gel. Hepatoma P-450MCII had a specific content of 7.38 nmol/mg protein and a minimum molecular weight of 50,000. The carbon monoxide-reduced difference spectral peak of hepatoma P-450MCI was at 446.5 nm, whereas the peak of hepatoma P-450MCII was at 451 nm. In the reconstituted system, hepatoma P-450MCI catalyzed 3-hydroxylation of benzo[a]pyrene and O-deethylation of 7-ethoxycoumarin, but showed low activities for N-demethylation of benzphetamine and aminopyrine, O-demethylation of p-nitroanisole, and p-hydroxylation of aniline. On the other hand, hepatoma P-450MCII did not catalyze hydroxylation of any of the substrates tested. By Ouchterlony double-diffusion analysis, hepatoma P-450MCI was immunologically indistinguishable from rat liver cytochrome P-450c, but hepatoma P-450MCII was distinct from hepatoma P-450MCI and rat liver cytochrome P-450c. Peptide maps of hepatoma P-450MCI and rat liver cytochrome P-450c after proteolysis with Staphylococcus aureus V8 protease demonstrated the similarity of the two cytochromes P-450.

Animals↗
PubMed1983

Purification and characterization of pulmonary cytochrome P-450 from 3-methylcholanthrene-treated rats.

A major form of pulmonary cytochrome P-450 (pulmonary P-450MC) was purified approximately 313 fold from lung microsomes of 3-methylcholanthrene (MC)-treated rats. The purified preparation contained 12.5 nmol of cytochrome P-450 per mg protein and was essentially free from NADPH-cytochrome c-reductase, NADH-cytochrome b5-reductase, and cytochrome b5. By SDS-polyacrylamide gel electrophoresis, the molecular weight of pulmonary P-450MC was estimated to be 54,000, which is smaller than that of a major form of hepatic cytochrome P-450 (hepatic P-450MC) purified from MC-treated rats. The peptide patterns of the purified pulmonary P-450MC were apparently different from those of hepatic P-450MC on partial proteolysis with either S. aureus V8 protease or papain, indicating that the primary structure of pulmonary P-450MC is different from that of hepatic P-450MC. In reconstituted systems, pulmonary P-450MC efficiently catalyzed benzo(a)pyrene hydroxylation and ethoxycoumarin O-deethylation, but showed a low activity for benzphetamine N-demethylation. Pulmonary P-450MC formed a single precipitation line with the antibody prepared against hepatic P-450MC in Ouchterlony double diffusion analysis. The pulmonary and hepatic P-450MC activities of benzo(a)pyrene hydroxylation and ethoxycoumarin O-deethylation were inhibited in the same manner by the antibody against hepatic P-450MC. These results suggest that pulmonary P-450MC is immunologically very similar to hepatic P-450MC.

Cytochrome P-450 Enzyme System↗