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Biomedical subjects

Y Sukenaga

Publications and source records attributed to Y Sukenaga.

13 recordsLinked to original sources
PubMed1995

Bronchodilator and cardiovascular effects of NKH477, a novel water-soluble forskolin derivative, in guinea pigs.

The bronchodilator and cardiovascular effects of NKH477 (6-(3-dimethylaminopropionyl)forskolin hydrochloride) were evaluated. In anesthetized guinea pigs, i.v. bolus injections of NKH477 (1-100 micrograms/kg) inhibited the bronchoconstriction induced by inhaled leukotriene D4, increased the heart rate (HR) and decreased the diastolic arterial blood pressure (DBP) in a dose-dependent manner. The bronchodilator effect of NKH477 was 1500 times more potent than that of aminophylline and 17 times less potent than that of isoproterenol. The selectivity of NKH477 for bronchodilation vs an increase in HR was 15 times higher than that of isoproterenol and similar to that of aminophylline; and vs a decrease in DBP, the selectivity was 4 times higher than that of aminophylline and similar to that of isoproterenol. I.v. infusion of NKH477 (0.1-3 micrograms/kg/min) for 2 hr dose-dependently inhibited the bronchoconstriction induced by i.v. histamine. Isoproterenol (0.1 microgram/kg/min, i.v.) enhanced the bronchoconstriction after termination of the infusion, whereas NKH477 did not. In conscious guinea pigs, inhalation of NKH477 (0.1-5 mg/ml) concentration-dependently inhibited the bronchoconstriction induced by inhaled histamine, and a high concentration of NKH477 (35.4 mg/ml) increased the HR. The bronchodilator effect of inhaled NKH477 was 15 times less potent than that of isoproterenol. The selectivity of inhaled NKH477 was similar to that of isoproterenol. These results indicate that NKH477 may be useful as a bronchodilator.

Animals↗
PubMed1994

Purification and characterization of a ubenimex (Bestatin)-sensitive aminopeptidase B-like enzyme from K562 human chronic myeloid leukemia cells.

A ubenimex-sensitive aminopeptidase B-like enzyme was purified from the non-membrane-bound fraction of K562 cells by a series of chromatographic procedures and slab-gel electrophoresis. The apparent molecular mass of the enzyme was estimated to be 73 kDa by SDS-PAGE. The aminopeptidase activity was activated by chloride ions and inhibited by Zn2+, Cu2+, Cd2+, and p-chloromercuribenzoic acid. Ubenimex was a potent inhibitor of this aminopeptidase in the nanomolar range. The sequence of the N-terminus of the protein was not determined. Partial amino acid sequencing revealed that the N-terminus of this aminopeptidase B-like enzyme was blocked by acylation. The partial sequences of the two fragments produced by CNBr cleavage and an acylamino acid-releasing reaction showed this enzyme to be a new aminopeptidase.

Amino Acid Sequence↗
PubMed1993

Purification and molecular cloning of chymase from human tonsils.

A chymotrypsin-like protease was purified to homogeneity from human tonsils by a series of chromatographic procedures. The purified enzyme gave a single protein band with an apparent molecular mass of 30 kDa on SDS-PAGE. The sequence of the first 21 amino acids at the N-terminus of the enzyme was determined. A cDNA for the enzyme was cloned by PCR amplification from extracted tonsillar mRNA using a supposed N-terminal oligonucleotide primer and a conserved C-terminal primer of the chymase family. The deduced amino acid sequence of the isolated clone was identical to that of human chymase in connective tissue-type mast cells from heart except for a Ser instead of a Cys at the N-terminal 7th position.

Amino Acid Sequence↗
PubMed1992

The 5' untranslated region of the human cellular glutathione peroxidase gene is indispensable for its expression in COS-7 cells.

We studied the expression of the human cellular glutathione peroxidase (GPx) gene, from which a key enzyme containing selenocysteine (Scy) at the active site is produced. Expression of some human GPx gene mutants in COS-7 cells revealed that the 5' untranslated region (utr) was necessary for expression of the GPx gene, since mutant genes having 10 base pairs (bps) at the 5'utr (the complete had 311 bps) expressed GPx at very low levels. The genes with 311 or 408 bps at the 5'utr were better expressed than those having 257 bps. The GPx gene having 133 bps at the 3'utr (80 bps shorter than the entire length) was highly expressed. This deletion did not influence expression. We constructed some mutants in which 3 bases were altered at the upstream region of the Scy UGA codon in the frame of the GPx gene, by site-directed mutagenesis. GPx expression decreased but the expression was restored. Therefore, the upstream region of the in-frame Scy codon was not essential in the Scy decoding mechanisms. Finally, the 5'utr was essential for the expression of GPx gene. However, the deletion of a part of the 3'utr and the site-directed mutation upstream of the Scy codon did not show drastic effects on the expression.

Amino Acid Sequence↗
PubMed1991

Changes in Cu,Zn-superoxide dismutase gene during induced erythroid and myeloid differentiation.

We investigated the alteration of Cu,Zn-superoxide dismutase during erythroid and myeloid differentiation in order to elucidate its physiological significance in different types of cells. We measured enzyme activity and mRNA levels of superoxide dismutase in the process of differentiation to erythroid cells or myeloid cells. When human leukemia K562 cells are incubated in the presence of 80 microM hemin, benzidine-positive cells appear on day 1 and 80% of the cells become positive on day 5. During hemin-induced erythroid differentiation, Cu,Zn-superoxide dismutase activity increases 3.5-fold of the initial value and mRNA for Cu,Zn-superoxide dismutase increases prior to the activity to the same extent. On the other hand, when human promyelocytic leukemia HL-60 cells are incubated in the presence of 1.3% dimethyl sulfoxide, nitroblue tetrazolium-positive cells reach approximately 90% on day 5. During dimethyl sulfoxide-induced myeloid differentiation, the activity of Cu,Zn-superoxide dismutase decreases below 15% of the initial value on day 5 and mRNA for Cu,Zn-superoxide dismutase decreases as well. The results indicate that the synthesis of superoxide dismutase is linked with either the erythroid or myeloid differentiation program.

Blotting, Southern↗
PubMed1989

The detection of natural opal suppressor seryl-tRNA in Escherichia coli by the dot blot hybridization and phosphorylation by a tRNA kinase [corrected] .

It was believed that there was no natural suppressor tRNA in Escherichia coli, however, it has been suggested that selC, relating to the synthesis of formate dehydrogenase of a selenoprotein [(1988) Nature 331, 723-725], codes for tRNA, even though the presence of tRNA has not yet been demonstrated. We detected the product of selC in the tRNA preparation of the E. coli MC 4100 strain by the dot blot hybridization method with a DNA probe (ACCGCTGGCGGC) corresponding to the extra arm of selC tRNA. Two hybridization peaks were found in the chromatographic pattern from Sephadex A50. The amount of tRNA was estimated to be about 0.03% of the total tRNA. Some tRNA [corrected] was phosphorylated by a tRNA kinase in E. coli B. These results suggest that the opal suppressor seryl-tRNA in E. coli should be converted to selenocysteyl-tRNA [corrected] and occurs in vertebrates as a general phenomenon.

Chromatography↗
PubMed1988

Physicochemical properties of charge isomers of recombinant human superoxide dismutase.

Recombinant human Cu2Zn2SOD expressed in Escherichia coli consisted of mainly three isomers with isoelectric points of 5.14 (A), 5.06 (B), and 4.99 (C). Each isomer was isolated by DEAE-Toyopearl chromatography and the physiochemical properties were investigated. No significant differences in chemical and spectrophotometric properties, such as specific activity, metal contents, amino acid composition, and UV and ESR spectra, were found. The result of labeling of free cysteine residues with ABD-F showed the disulfide bond to be formed between 57Cys and 146Cys in every isomer. A few differences were found in the CD spectrum around 260 nm and in the elution patterns on reverse-phase HPLC. The isoelectric points of the three isomers became the same after treatment by reduction and carboxymethylation and even after reduction only, pI of isomers tended to be at the value of component (A). These results suggest that the three isomers are identical in primary structure but slightly different in secondary or tertiary structure. These differences are probably derived from structural alterations around 111Cys.

Amino Acids↗
PubMed1980

Ligand bindings of bovine carboxypeptidase B. II. Affinity chromatography and cooperative ligations.

Affinity chromatography was used to characterize the binding properties of carboxypeptidase B with its ligands. The affinity adsorbents employed included arginine directly attached to agarose beads, arginine attached to the same support through hydrophilic and hydrophobic spacers, and immobilized caproylphenylalanine. The enzyme showed marked retardation on all of the arginine columns but only slight retardation on the phenylalanine column. Several hydrophobic ligands in solution enhanced to some extent the enzyme retardation on columns having arginine directly attached to the solid support, while several amino- and guanidino-alkylcarboxylic acids (lysine and arginine analogs) greatly enhanced the enzyme retardation on the phenylalanine column and somewhat enhanced it on the other columns having hydrophobic spacer arms. These observations confirmed that the enzyme has twobinding sites for the soluble and immobilized ligands and that these two sites exhibit cooperative ligations. Binding constants of the enzyme for various soluble ligands were also calculated from their chromatographic effects and the resulting values were interpreted in terms of the cooperative action of the two bindings sites, i.e., one for the primary binding of basic amino acid analogs (SITE I) and the other for hydrophobic compounds (Site II). In this chromatographic study, however, such cooperation of the two sites was obscure when arginine, acylarginine, or alkylarginine was the ligand directing to Site I.

Animals↗
PubMed1980

Ligand bindings of bovine carboxypeptidase B. III. Hydrophobic activators in dipeptide hydrolysis.

Several hydrophobic compounds acted as activators in dipeptide (Bz-Gly-L-Arg-OH, Z-Gly-L-Phe-OH) hydrolysis by bovine carboxypeptidase B. These hydrophobic compounds include Bz-Gly-OH, Z-Gly-OH, Z-L-Phe-OH, and Z-L-Phe-GLy-OH. These compounds were indicated to bind to the secondary substrate binding sites which is proposed to be responsible for substrate activation kinetics in dipeptide hydrolysis. Of the compounds Z-L-Phe-OH alone acted also as a inhibitor at higher concentrations, indicating that it binds to both primary and secondary sites as the dipeptide substrates do. Comparison of the activation effects of the compounds employed indicated that hydrophobic interaction played an important role in binding to the secondary site. Substrate and modifier binding constants were also determined and the results indicated that modifier binding increased both affinity and catalytic rate constant of the primary site. On the other hand, Z-Gly-OH and Z-L-Phe-Gly-OH inhibited the hydrolyses of tri and tetrapeptide substrates. This observation suggests that the secondary site is contained in the extended active center which the enzyme possibly has.

Alcohols↗
PubMed1980

Ligand binding of bovine carboxypeptidase B. IV. Oligopeptide substrates and extended active center.

Catalytic and binding properties of bovine carboxypeptidase B were studied by kinetic and affinity chromatographic methods both using several oligopeptides as substrates or immobilized ligands. These oligopeptides contained either arginines or phenylalanines at carboxy termini as well as phenylalanyl residues in one of the other positions. The chromatographic studies showed that the phenylalanyl residues in endo-positions play a significant role in binding of the immobilized peptides to the enzyme, while the kinetics studies indicated further that the presence of an internal hydrophobic residue in a substrate was advantageous for the catalytic release of the carboxyl terminal residue from the substrate. These observations support the supposition that the enzyme has an extended active center which contains an extended hydrophobic binding site. Several hydrophobic compounds, which have been shown to act as activators in dipeptide substrate hydrolyses, showed inhibitory effect on hydrolyses of oligopeptide substrates. This observation suggests that these hydrophobic compounds bind to a portion of the hydrophobic site in the active center.

Animals↗