Biomedical subjects

R Masaki

Publications and source records attributed to R Masaki.

6 recordsLinked to original sources

PubMed1992

Quantitative immunocytochemical analysis of the induction of cytochrome P450IIB in rat hepatocytes.

We examined whether induction of the phenobarbital (PB)-inducible form of cytochrome P450 (P450IIB) in rat hepatocytes could be analyzed quantitatively by immunogold electron microscopy. Rats received intraperitoneal injections of PB every 24 hr and livers at the various stages of PB induction were fixed by perfusion with a mixture of paraformaldehyde (4%) and glutaraldehyde (0.1%) and embedded in LR White. Ultra-thin sections were cut and labeled by the protein A-gold procedure using affinity-purified anti-P450IIB antibody which was previously immunoabsorbed with liver microsomes from a control rat (not treated with PB). We counted the number of gold particles per micron of the rough ER membranes (particle density). Before PB treatment, the particle density of the rough ER in rat hepatocytes was practically zero and increased markedly at 48 and 72 hr after PB treatment. The rough microsomes were prepared from these PB-treated rat livers. The amount of P450IIB was estimated by immunoblot analysis and the number of gold particles bound to the rough microsomal membrane was determined by the same post-embedding immunogold procedure. The particle density of the rough microsomes increased in parallel with the increase in the amount of P450IIB, indicating good correlation of the two variables. Thus, the induction of cytochrome P450IIB can be quantitatively and reliably investigated by immunogold electron microscopy.

Acrylic Resins↗

PubMed1991

Molecular cloning, sequencing, and expression of cDNA for rat liver microsomal aldehyde dehydrogenase.

The cDNA clone for rat liver microsomal aldehyde dehydrogenase (msALDH) was isolated and sequenced. The deduced amino acid sequence consisting of 484 amino acid residues revealed that the carboxyl-terminal region of msALDH has a hydrophobic segment, which is probably important for the insertion of this enzyme into the endoplasmic reticulum membrane. COS-1 cells transfected with the expression vector pcD containing the full-length cDNA showed that the active enzyme was expressed and localized mainly on the cytoplasmic surface of the endoplasmic reticulum membranes. It has been proposed that ALDH isozymes form a superfamily consisting of class 1, 2, and 3 ALDHs (Hempel, J., Harper, K., and Lindahl, R., (1989) Biochemistry 28, 1160-1167). Comparison of the amino acid sequence of rat liver msALDH with those of rat other class ALDHs showed that msALDH was 24.2, 24.0, and 65.5% identical to phenobarbital-inducible ALDH (variant class 1), mitochondrial ALDH (class 2), and tumor-associated ALDH (class 3), respectively. Several amino acid residues common to the other known ALDHs, however, were found to be conserved in msALDH. Based on these results, we proposed to classify msALDH as a new type, class 4 ALDH.

Aldehyde Dehydrogenase↗

PubMed1991

Structures and characterization of sex-specific mouse cytochrome P-450 genes as members within a large family. Duplication boundary and evolution.

We characterized two female-specific testosterone 16 alpha-hydroxylase mouse cytochrome P-450 genes, 16 alpha oh-a and 16 alpha oh-b. Gene 16 alpha oh-a, consisting of nine exons, is approximately 38 kbp in size. The exon sequence of this P-450 gene is identical to cDNA pf26 nucleotide sequence [Noshiro, M., Lakso, M., Kawajiri, K. & Negishi, M. (1988) Biochemistry 27, 6434-6443], which encodes female-specific testosterone 16 alpha-hydroxylase regulated by the murine Rip locus. Gene 16 alpha oh-b, containing nine exons with the same junctions as the 16 alpha oh-a, spans at least 20 kbp, and encodes a cytochrome P-450 whose deduced amino acid sequence is 90% similar to the hydroxylase. Nucleotide sequences revealed that duplication of the two genes occurred 4-22 million years ago, and that the 5' duplication boundary is located 1336 bp upstream from the putative transcription-start site. In the flanking regions of both genes, there is a long stretch (100 bp) of CA repeats in addition to other motifs, including TATA box, glucocorticoid-response-element-core and Simian-virus-40-enhancer sequences and IgG light-chain gene promoter. We isolated many genomic DNA clones which contain exon 1 sequences, and compared their restriction maps, cross-hybridization and nucleotide sequences. The results indicate that these genomic clones represent closely related genes in the 16 alpha oh family with a minimum of 16 members, which is further divided into classes a, b and c. 16 alpha oh-a and 16 alpha oh-b belong to the first and second classes, respectively. Moreover, extensive segmental gene conversion and nonreciprocal recombination were noted among the genes, particularly among those in class b. All genes in that class contain the long ATTT repeat sequences in intron 1, which may have triggered a rapid gene conversion and/or stabilize the duplicated genes.

Animals↗

PubMed1979

Isolation and characterization of two ferredoxin-NADP+ reductases from Spirulina platensis.

Two ferredoxin-NADP+ reductases (FNRs I and II) [EC 1.6.7.1] were purified from a blue-green alga, Spirulina platensis, by (NH4)2SO4 fractionation, gel filtration on Sephadex G-100 and DEAE-Sephadex A-50 chromatography. FNRs I and II were both FAD-containing enzymes with molecular weights of 33,000, and could photochemically reduce NADP+ to the same extent in the presence of S. platensis ferredoxin, using FNR-depleted membrane fragments of S. platensis. They had similar physical and enzymatic properties, except for chemical properties such as the amino (N)-terminal sequences and the patterns of their peptide maps. The significance of the presence of two FNRs in S. platensis as as of the multiple forms found in other organisms is discussed.

Amino Acid Sequence↗

PubMed1977

Chemical modification of spinach ferredoxin. Properties of acetylated spinach ferredoxin.

The acetylation of spinach ferredoxin by acetic anhydride modified about four moles of amino groups. The absorption spectra, CD spectra, the fluorescence of sole tryptophan residue and the biological activity of acetylated ferredoxin were investigated. An equilibrium existed between two different states, D- and N-form, of the acetylated ferredoxin and was dependent on the cation concentration. D-form completely reverted to N-form upon the binding of one mole of cation, Na+ or Mg2+. Although the N-form was indistinguishable from native ferredoxin in every property tested, the D-form was significantly different from the N-form or native ferredoxin and was very unstable, especially at low salt concentrations. It is suggested that the amino groups might be important in maintaining the protein conformation by forming salt linkages, but may not be essential for the activity. Furthermore, since the D-form, unlike the N-form and native ferredoxin, was inactive in the ferredoxin-NADP+ reductase [EC 1.6.7.1] assay system and had no inhibitory effect in this system, it was considered to be incapable of forming a complex with ferredoxin-NADP+ reductase. On the other hand, the N-form of the modified ferredoxin was as active as native ferredoxin. It is suggested that amino groups of spinach ferredoxin are not essential for the redox reaction of ferredoxin or for complex formation with the reductase.

Acetylation↗

PubMed1976

Structure and function of chloroplast-type ferredoxins.

Comparison of various chloroplast-type ferredoxin sequences, chemical and enzymic modifications, reconstitution experiments, and fluorescence measurement of chloroplast-type ferredoxins have led to the following conclusions. 1. Tyrosine, histidine, and tryptophan residues are not directly involved in the oxidation-reduction mechanism of ferredoxins. The four indispensible cysteine residues in spinach ferredoxin which constitutes a part of the iron-sulfur cluster are located at residues 39, 44. 47 and 77. Two out of six cysteine residues in Spirulina ferredoxin could be easily modified with vinylpyridine without the loss of reconstitutive ability i.e. the apoferredoxin could be converted to the holoform by the addition of iron and sulfide. 2. Spinach ferredoxin was digested with carboxypeptidase A and the terminal alanine could be removed without loss of the spectral properties of native ferredoxin. However, the removal of the terminal three residues gave rise to the loss of reconstitutive ability. 3. The amino groups of spinach ferredoxin were modified by acetic anhydride and four residues were acetylated. The acetylated preparation of ferredoxin had an unique spectrum. Upon the addition of high concentration of ions the spectrum of this derivative resembled the spectrum of native ferredoxin. Acetylferredoxin did not combine with ferredoxin-NADP reductase, but upon the addition of moderate concentrations of cations, it did bind to this enzyme.

Amino Acid Sequence↗