Biomedical subjects

K Morohashi

Publications and source records attributed to K Morohashi.

At least 73 records · Page 4Linked to original sources

PubMed1990

Molecular cloning and sequence analysis of cDNA coding for rat liver hemoprotein H-450.

cDNA clones coding for hemoprotein H-450 were isolated from a rat liver cDNA library using anti-H-450 antibody. The molecular weight calculated from the deduced amino acid sequence comprising 547 amino acid residues was 60,085. The N-terminal sequence and a partial internal amino acid sequence of purified H-450, which were determined chemically, were both found in the amino acid sequence of H-450 deduced from the nucleotide sequence. H-450 mRNA is expressed in liver, kidney, and brain. A homology search of amino acid sequences indicated that H-450 shows no homology with cytochrome P-450, but shows significant homology with bacterial O-acetylserine (thiol)-lyases. However, H-450 has no O-acetylserine (thiol)-lyase activity.

Amino Acid Sequence↗

PubMed1989

Molecular cloning of cDNA for cholesterol 7 alpha-hydroxylase from rat liver microsomes. Nucleotide sequence and expression.

A complete cDNA clone encoding cholesterol 7 alpha-hydroxylase was isolated from a rat liver cDNA library by the use of specific antibodies to the enzyme. The isolated cDNA clone was 3.6 kbp long and contained a 1509-bp open reading frame encoding 503 amino acid residues (Mr = 56,880). The identity of the cDNA was confirmed by expression of cholesterol 7 alpha-hydroxylase activity and the immunoreactive protein in COS cells transfected with pSVL expression vector carrying the cDNA insert. The primary structure of cholesterol 7 alpha-hydroxylase deduced from the nucleotide sequence of the cDNA indicated that the enzyme constitutes a novel P-450 family.

Amino Acid Sequence↗

PubMed1989

Cloning and characterization of bovine cytochrome P-450(11 beta) genes.

We isolated 4 different clones of the P-450(11 beta) gene from a bovine genomic library. These genomic clones were highly homologous with each other. Two of the isolated clones were pseudogenes. Determination of its nucleotide sequences indicated that the bovine P-450(11 beta) gene is divided into 9 exons by 8 introns and that it is about 8.5 kb in total length. The number of exons and the locations of intron insertion into the P-450(11 beta) gene are identical with those in the case of P-450(SCC), but different from those of other microsomal P-450s.

Animals↗

PubMed1988

The 5'-flanking region of the human P-450(SCC) gene shows responsiveness to cAMP-dependent regulation in a transient gene-expression system of Y-1 adrenal tumor cells.

The chronic effect of cAMP-dependent regulation on adrenocortical steroidogenesis is known to be revealed in the stimulation of the biosynthesis of steroidogenic enzymes. P-450(SCC), one of the enzymes, catalyzes the first and the rate-limiting reaction in steroidogenesis from cholesterol and its synthesis is regulated by cAMP. In order to investigate cis-acting DNA elements of this gene in response to cAMP-dependent regulation, we have constructed a fusion gene (pSCC5.4k) by ligating the 5'-flanking and the upstream untranslated region (5.4 kb) of the human P-450(SCC) gene to the structural gene for chloramphenicol acetyltransferase (CAT) and transfected it into various culture cells including Y-1 (mouse adrenal tumor), L929 (mouse fibroblast), HTC (rat hepatoma) and Hepa-1 (mouse hepatoma). Only Y-1 cells transfected with pSCC5.4k were found to express transiently the enhanced CAT activity in response to the cAMP analogue, cyclic dibutyryl-AMP (Bt2cAMP). Primer-extension analysis of RNA prepared from the cells treated with or without Bt2cAMP showed that the enhanced CAT activity was due to an increase in the CAT mRNA and that the transcription start site, determined here with the human P-450 gene in the adrenal cortex, was correctly utilized with the fusion gene in the transient expression system. Forskolin and cholera toxin, activators of adenylate cyclase, also increased the expression of the CAT activity in the Y-1 cells. It has been demonstrated, therefore, that the cAMP-dependent regulation of the P-450(SCC) gene in adrenal cortex is faithfully reflected in the transient expression system using Y-1 cells and the fusion gene and that a cis-acting DNA element(s) in response to cAMP is present within the 5'-flanking sequence (5.4 kb) of the P-450(SCC) gene.

Adenylyl Cyclases↗

PubMed1988

A mutant rat strain deficient in induction of a phenobarbital-inducible form of cytochrome P-450 in liver microsomes.

Two phenobarbital-inducible forms of cytochrome P-450, P-450(PB-1), and P-450(PB-4), were purified from the liver microsomes of phenobarbital-treated rats and identified with P-450b and P-450e, respectively. It was found, however, that the content of P-450(PB-4) in the liver microsomes of a strain of SD rat, Qdj:SD, was very low even after phenobarbital-induction. The levels of the mRNAs for P-450(PB-1) and P-450(PB-4) were separately determined using Northern blot hybridization with specific oligonucleotide probes. It was found that the level of P-450(PB-4) mRNA in the livers of phenobarbital-treated Qdj:SD rats was much lower than that of phenobarbital-treated Slc:SD rats. Slc:SD rats are widely used in Japanese laboratories. Genetic analysis using the crossbred animals between Qdj:SD and Slc:SD rats showed that the low expression of P-450(PB-4) in Qdj:SD rats is a recessive trait and is caused by a single gene mutation. However, no difference in the 5' flanking region in P-450(PB-4) gene was found between Qdj:SD rats and Slc:SD rats.

Animals↗

PubMed1988

Expression of a rat liver microsomal cytochrome P-450 catalyzing testosterone 16 alpha-hydroxylation in Saccharomyces cerevisiae: vitamin D3 25-hydroxylase and testosterone 16 alpha-hydroxylase are distinct forms of cytochrome P-450.

Rat cytochrome P-450(M-1) cDNA was expressed in Saccharomyces cerevisiae TD1 cells by using a yeast-Escherichia coli shuttle vector consisting of P-450(M-1) cDNA, yeast alcohol dehydrogenase promoter and yeast cytochrome c terminator. The yeast cells synthesized up to 2 X 10(5) molecules of P-450(M-1) per cell. The microsomal fraction prepared from the transformed cells contained 0.1 nmol of cytochrome P-450 per mg of protein. The expressed cytochrome P-450 catalyzed 16 alpha- and 2 alpha-hydroxylations of testosterone in accordance with the catalytic activity of P-450(M-1), but did not hydroxylate vitamin D3 or 1 alpha-hydroxycholecalciferol at the 25 position. The expressed cytochrome P-450 also catalyzed the oxidation of several drugs and did not show 25-hydroxylation activity toward 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol. However, it cross-reacted with the polyclonal and monoclonal antibodies elicited against purified P-450cc25 which catalyzed the 25-hydroxylation of vitamin D3. These results indicated that P-450(M-1) cDNA coded the 2 alpha- and 16 alpha-hydroxylase of testosterone, and that these two positions of testosterone are hydroxylated by a single form of cytochrome P-450. Vitamin D3 25-hydroxylase and testosterone 16 alpha- and 2 alpha-hydroxylase are different gene products, although these two hydroxylase activities are immunochemically indistinguishable.

Animals↗

PubMed1988

Expression of two kinds of cytochrome P-450(11 beta) mRNA in bovine adrenal cortex.

Using pcP-450(11 beta)-2 cDNA (Morohashi et al. (1987) J. Biochem. 102, 559-568) as the probe, a different cDNA clone, pcP-450(11 beta)-3, was isolated from a cDNA library of bovine adrenal cortex. The restriction enzyme map of pcP-450(11 beta)-3 was highly homologous but not identical with that of pcP-450(11 beta)-2. Nucleotide sequence determination revealed the substitutions of 14 nucleotides and 3 amino acids between pcP-450(11 beta)-2 and -3. Blotting analysis involving two different oligonucleotide probes specific to these two cDNAs indicated that at least two kinds of P-450(11 beta) mRNA were expressed in individual animals and that at least two kinds of P-450(11 beta) genes exist in the bovine genome.

Adrenal Cortex↗

PubMed1988

Molecular cloning and nucleotide sequence of cDNA of microsomal carboxyesterase E1 of rat liver.

cDNA clones of the mRNA for rat liver carboxyesterase E1, one of the carboxyesterases exclusively located on the luminal side of microsomal vesicles, were isolated. Sequence analysis of 2 kbp long cDNA revealed the primary structure of carboxyesterase E1, which consisted of 549 amino acids (Mr 60, 171.71) and contained an extra peptide of 18 amino acids at the NH2-terminus of the mature enzyme. Comparison of the deduced primary structure and sequences of some proteolytic fragments of the purified enzyme indicated the multiplicity of the enzyme. The extra peptide at the NH2-terminal had features in common with the signal peptides of most secretory proteins. However, no polar amino acid residues existed before the hydrophobic core of the signal peptide. A new interpretation is proposed to explain how the signal peptide without the NH2-terminal polar residues works. A tetrapeptide (KDEL) which was shown to keep a few microsomal proteins in the lumen of the endoplasmic reticulum was not found in the primary structure of carboxyesterase E1, which suggested the existence of another mechanism for retention of proteins in the lumen of endoplasmic reticulum. Carboxyesterase E1 showed significant homology with the COOH-terminal portion of thyroglobulin.

Amino Acid Sequence↗

PubMed1987

Structural analysis and specific expression of microsomal cytochrome P-450(M-1) mRNA in male rat livers.

cDNA clones for the P-450(M-1) mRNA, which exhibits a male-specific expression in rat livers, were isolated by using synthetic oligonucleotides as the probes. Sequence analysis of the cDNAs showed that P-450(M-1) mRNA contains 1,853 nucleotides in addition to a poly(A) chain, and a single open reading frame of 1,500 nucleotides encodes a polypeptide of 500 amino acids with a Mr = 57,187. The predicted NH2-terminal sequence of 30 amino acids agrees well with that of the purified protein determined by Edman degradation, and the predicted primary structure included all the partial sequences of six internal peptides of P-450(M-1) obtained by the proteolytic digestion and a conserved amino acid sequence containing a putative heme-binding cysteine, proximate to the COOH terminus of the molecules. P-450(M-1) showed relatively high sequence similarity with P-450b (Fujii-Kuriyama, Y., Mizukami, Y., Kawajiri, K., Sogawa, K., and Muramatsu, M. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2793-2797) (52% similarity), P-450-3b (Ozols, J., Heinemann, F. S., and Johnson, E. F. (1985) J. Biol. Chem. 260, 5427-5434) (64%), P-450-1 (Tukey, R. H., Okino, S., Barnes, H., Griffin, K. J., and Johnson, E. F. (1985) J. Biol. Chem. 260, 13347-13354) (74%), P-450PBc1 (Leighton, J. K., DeBrunner-Vossbrinck, B. A., and Kemper, B. (1984) Biochemistry 23, 4598-4603) (71%), while its sequence similarity with 3-methylcholanthrene-inducible P-450c and P-450d is rather low. Consequently, P-450(M-1) could be structurally classified into the phenobarbital-inducible type of P-450 gene family. RNA blot analysis using a synthetic oligonucleotide specific for P-450(M-1) revealed that P-450(M-1) mRNA was expressed exclusively in the livers of mature male rats in a sex-specific manner, but not in other tissues so far examined.

Amino Acid Sequence↗

PubMed1987

Gene structure of human cytochrome P-450(SCC), cholesterol desmolase.

Four independent clones containing a part of the P-450(SCC), cholesterol desmolase, gene were isolated from human genomic libraries using bovine P-450(SCC) cDNA as a probe. These clones covered the entire P-450(SCC) gene except for a part of the 1st intron. The gene is at least 20 kb long and is split into 9 exons by 8 introns. The sequence analysis revealed that the nine separated exons code for a primary structure consisting of 521 amino acids which shows 72% homology with that of bovine P-450(SCC). A CATT sequence and a TATAAT sequence, which are possibly a "CAT" box, and a "TATA" box, respectively, are present 129 and 91 bp upstream from the initiation codon. An unusual exon/intron junctional sequence that begins with GC was found in the 6th intron of the gene. A putative extension peptide consisting of 39 amino acids was found in the sequence of human P-450(SCC) by comparison with that of the bovine counterpart. Two conserved regions were found in the extension peptide of these two forms of P-450(SCC), suggesting a functional role of the portions in the mitochondrial localization and processing of P-450(SCC) precursor. The mature form of human P-450(SCC) has only one cysteine residue, which was located in the center of the HR2 region (Gotoh et al. (1983) J. Biochem. 97, 807-817). This observation established beyond doubt that the sole cysteine residue in the HR2 region is the 5th ligand to the heme.

Base Sequence↗

PubMed1987

Molecular cloning and nucleotide sequence of DNA of mitochondrial cytochrome P-450(11 beta) of bovine adrenal cortex.

cDNA clones of the mRNA for bovine adrenal cytochrome P-450(11 beta) were isolated. Sequence analysis of a 4 kb long cDNA revealed the primary structure of P-450(11 beta), which consisted of 503 amino acids (Mr: 57,924) and contained an extension peptide of 24 amino acids at the NH2-terminus of the mature P-450(11 beta). molecule. A bovine genomic DNA containing the 1st exon and its leader sequence of P-450(11 beta) gene was also isolated from a bovine gene library. Determination of the transcription initiation site by S1 nuclease analysis using the cloned genomic DNA confirmed that the methionine codon near the 5' side of the 4 kb long cDNA was the initiation codon. Comparisons of the primary structures among P-450(11 beta) and other forms of cytochrome P-450 including P-450(SCC) indicated that the two mitochondrial P-450s, P-450(11 beta) and P-450(SCC), were significantly different from microsomal forms of cytochrome P-450. The homology between P-450(11 beta) and P-450(SCC) was 36%, which is higher than the values between P-450(11 beta) and various microsomal P-450s. An alignment of P-450(11 beta) and P-450(SCC) to give maximum matching showed four highly conserved regions (C-1, C-2, C-3, and C-4). The homology values of these regions were 58-70%, considerably higher than the overall homology between these two mitochondrial P-450s. A putative heme binding site and a steroid binding site were located in the conserved regions. Hydropathy profiles of P-450(11 beta) and P-450(SCC) were very similar. A definite difference was noticed at the NH2-terminal portion between mitochondrial and microsomal types of P-450. Microsomal type of cytochrome P-450 had a hydrophobic sequence consisting of about 20 amino acids, whereas mitochondrial type had an extension peptide containing many positively changed amino acids.

Adrenal Cortex↗

PubMed1987

Site-directed mutagenesis of basic amino acid residues in the extension peptide of P-450(SCC) precursor: effects on the import of the precursor into mitochondria.

The precursor of cytochrome P-450(SCC) (preP-450(SCC], an inner membrane protein of adrenal cortex mitochondria, has an extension peptide consisting of 39 amino acids which is thought to play an essential role in the import of the precursor into mitochondria. The amino terminal portion of the extension peptide contains three positively charged amino acid residues, Arg(4), Arg(9), and Lys(14). To investigate their role in the import of preP-450(SCC) into mitochondria, they were replaced by other amino acids, Ser or Thr, by site-directed mutagenesis. The import of mutated preP-450(SCC)s with single amino acid substitution was much less efficient than with the original precursor. The mutated preP-450(SCC)s with two or three substitutions were not imported. These results suggest that the positively charged amino acid residues in the amino terminal portion of the extension peptide are essential for the import of preP-450(SCC) into mitochondria.

Amino Acids↗

PubMed1987

Structural gene of cytochrome b-562 from the cytochrome b-c1 complex of Rhodobacter sphaeroides.

The structural gene coding for cytochrome b-562 isolated from the cytochrome b-c1 complex of Rhodobacter (Rhodopseudomonas) sphaeroides has been cloned. Its nucleotide sequence has been determined and the amino acid sequence was deduced therefrom. It consists of 157 amino acids (Mr 17,237) and contains four hydrophobic segments. The first 30 residues in the predicted amino acid sequence are the same as those determined for the NH2-terminal portion of purified cytochrome b-562. The amino acid composition is in accord with that determined for the pure protein. From the hydropathy profile and molar ratio of protoheme to cytochrome b-562, it is suggested that the structural and functional unit of the cytochrome is a two-heme cross-linked homodimer.

Amino Acid Sequence↗

PubMed1986

Nucleotide sequence of a nearly full-length cDNA coding for pepsinogen of rat gastric mucosa.

A nearly full-length rat pepsinogen cDNA was isolated from a rat gastric mucosa cDNA library and its nucleotide sequence was determined. The cDNA comprises 1370 base pairs (bp), including the 5'-non-coding region (60 bp), the coding nucleotide sequence (1176 bp) and the 3'-non-coding region (131 bp). The predicted amino acid sequence of rat prepepsinogen (392 residues) contains a 16-residue signal sequence followed by the pepsionogen moiety of 376 residues. Rat pepsinogen has an amino acid composition characteristic of C-type pepsinogens and is much more homologous in amino acid sequence with C-type pepsinogens than A-type pepsinogens. These results indicate that the major form of rat pepsinogen can be classified as a C type pepsinogen.

Amino Acid Sequence↗

PubMed1986

Structural analysis of cloned cDNA for mRNA of microsomal cytochrome P-450(C21) which catalyzes steroid 21-hydroxylation in bovine adrenal cortex.

We have isolated cDNA clones of the mRNA for cytochrome P-450 that catalyzes the steroid C-21 hydroxylation (P-450(C21)), which specifically catalyzes 21-hydroxylation of steroids in the microsomes of bovine adrenal cortex by using synthetic oligonucleotides as probes. Sequence determination of the cloned cDNA showed that it contains 2157 nucleotides and a poly(A) chain and that a single open reading frame of 1488 nucleotides codes for a polypeptide of 496 amino acids with a molecular weight of 56,113. The deduced amino acid composition is in agreement with that determined by direct amino acid analysis of purified P-450(C21) and the predicted primary structure contained amino acid sequences of N-terminal region and two internal tryptic fragments of the protein so far analyzed. Comparing the amino acid sequence with those of other forms of P-450 reveals that a conserved amino acid sequence containing a putative heme-binding cysteine is present in the equivalent position, proximate to the COOH terminus of the molecules and that P-450(C21) is phylogenically situated in an intermediate position between steroidogenic mitochondrial cytochrome P-450 which catalyzes the side-chain cleavage of cholesterol (P-450(SCC)) and drug-metabolizing microsomal P-450s. However, the amino acid sequence of P-450(C21) is much closer to that of drug-metabolizing P-450s than to that of P-450(SCC).

Adrenal Cortex↗

PubMed1986

Processing-independent in vitro translocation of cytochrome P-450(SCC) precursor across mitochondrial membranes.

In the presence of a membrane-permeable metal chelator, bovine adrenal cortex mitochondria imported P-450(SCC) precursor without processing of the amino-terminal extension peptide. The imported precursor was bound to the matrix side surface of the inner membrane. When the inhibition due to the metal chelator was removed, the imported precursor was processed to the mature form. Unprocessed precursor was also detected in mitochondria when the import reaction was carried out at relatively low temperature. These results suggest that the translocation of P-450(SCC) precursor across mitochondrial membranes is independent of its processing to the mature form. Both membrane-bound and solubilized P-450(SCC) could be cleaved by trypsin into two fragments with molecular weights of 29 kDa and 26 kDa, respectively, suggesting a two-domain structure of the molecule. The in vitro-imported and processed P-450(SCC) was also cleaved by trypsin in the same way. This finding indicated that the in vitro-imported and processed P-450(SCC) has the same conformation as the native form.

Adrenal Cortex↗

PubMed1985

Possible steroid binding site common to adrenal cytochrome P-450scc and prostatic steroid binding protein.

By searching the entire PIR-protein-sequence data base, we have found that a dodecapeptide sequence in bovine adrenal cytochrome P-450scc is closely related to that in rat prostatic steroid binding protein. The two proteins belong to unrelated protein families, but both have steroids as substrates or ligands. Thus, the dodecapeptides may be important for substrate/ligand recognition in the individual proteins.

Adrenal Glands↗

PubMed1984

Molecular cloning and nucleotide sequence of cDNA for mRNA of mitochondrial cytochrome P-450(SCC) of bovine adrenal cortex.

We have isolated cDNA clones of the mRNA for cytochrome P-450(SCC), which catalyzes the side-chain cleavage reaction of cholesterol in bovine adrenal cortex mitochondria, by using synthetic oligonucleotides as probes. Sequence analysis of the cloned cDNAs enabled us to deduce the primary structure of the precursor form of P-450(SCC), which consisted of 520 amino acids and contained an extrapeptide of 39 amino acids at the NH2 terminus. The amino acid sequence from the 40th to 55th amino acid residue in the predicted structure completely coincided with the sequence of the NH2-terminal portion of purified P-450(SCC). The amino acid composition calculated from the predicted structure showed an excellent agreement with that determined with the purified protein. The extrapeptide of the precursor molecule resembles those of a few nuclear-encoded yeast mitochondrial proteins reported so far. Although P-450(SCC) is a component of mitochondria, comparison of its primary structure with those of other forms of cytochrome P-450 shows that P-450(SCC) is structurally more related to microsomal cytochrome P-450s than to a bacterial cytochrome P-450, P-450cam. A homologous sequence observed with various forms of cytochrome P-450 is also highly conserved in the P-450(SCC) molecule. Only two cysteinyl residues are present in the mature form of P-450(SCC), one of which is located in the middle of the conserved sequence, confirming the function of this cysteinyl residue as the fifth ligand of the heme.

Adrenal Cortex↗