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Biomedical subjects

H Takeya

Publications and source records attributed to H Takeya.

At least 55 records · Page 3Linked to original sources
PubMed1993

Factor IX Fukuoka. Substitution of ASN92 by His in the second epidermal growth factor-like domain results in defective interaction with factors VIIa/X.

Hemophilia B Fukuoka, a moderately severe bleeding disorder, is a naturally occurring mutant of factor IX. Plasma from our patient had 3% clotting activity even though 64% of factor IX antigen was present. The purified mutant protein was cleaved normally by factor Xla, factor VIIa-tissue factor complex, or RVV-X (factor X-activating enzyme from Russell's viper venom), yielding a two-chain factor IXa. Amino acid composition and sequence analyses of one of the lysyl endopeptidase peptides derived from factor IX Fukuoka revealed that Asn92 in the second epidermal growth factor (EGF)-like domain had been replaced by His. The active site of the factor IXa Fukuoka was normally competent for the incorporation of p-aminobenzamidine and for the hydrolysis of a synthetic substrate, N alpha-benzyloxycarbonyl-L-arginine p-nitrobenzyl ester. Factor Xa formation by factor IXa Fukuoka was only 8% of the normal factor IXa, even in the presence of polylysine, and only 0.2% of the normal in the system containing phospholipids, Ca2+, and factor VIIIa, thereby indicating a functional defect in interaction of the mutant with factors VIIIa/X. Furthermore, catalytic efficiency (kcat/Km) of factor IXa Fukuoka toward factor X in the presence of Ca2+, phospholipids, and factor VIIIa was only 2.3% of the normal factor IXa. These results suggest that an Asn-to-His substitution at position 92 in the second EGF-like domain of factor IX Fukuoka would have an untoward effect on the specific conformational state of factor IX for binding with factors VIIIa/X.

Adult↗
PubMed1993

Ad4BP regulating steroidogenic P-450 gene is a member of steroid hormone receptor superfamily.

Bovine cytochrome P-450(11 beta) gene (CYP11B) has six different cis-acting elements, Ad1, Ad2, Ad3, Ad4, Ad5, and Ad6, in the promoter region. The Ad4 site also exists in the promoter regions of other steroidogenic P-450 genes as well as in CYP11B1. An Ad4-binding protein (Ad4BP) which specifically binds to the Ad4 site was purified from bovine adrenal cortex nuclear extract, and its molecular mass was 53 kDa. A complementary DNA encoding Ad4BP was isolated from a bovine adrenal cortex cDNA library. The cDNA clone contained an open reading frame of 1383 base pairs encoding 461 amino acids, whose calculated molecular weight was 51,020. The predicted amino acid sequence of Ad4BP revealed that the protein has a zinc finger domain and a ligand binding/dimerization domain. Ad4BP is a novel member of the steroid hormone receptor superfamily. Comparison of the primary structures of the hormone receptor superfamily showed that Ad4BP was highly homologous to FTZ-F1, which regulates the fushi tarazu gene, and ELP, which is expressed in the murine embryonal carcinoma cells. Transfection of a Ad4BP expression plasmid into CV-1 cells activated the transcription of the CAT reporter gene carrying the Ad4 sequence in the promoter region.

Amino Acid Sequence↗
PubMed1993

The region Ser333-Arg356 of the alpha-chain of human C4b-binding protein is involved in the binding of complement C4b.

Human C4b-binding protein (C4BP) functions as a cofactor to factor I in the degradation of C4b and accelerates the decay rate of the C4b2a complex. In this study we describe a monoclonal antibody directed against the alpha-chain of C4BP that inhibits the binding of C4b to C4BP. In order to identify the structural domain of the alpha-chain of C4BP that interacts with C4b, tryptic fragments of C4BP were generated. Amino acid sequence analysis of the fragments revealed that the residues Ser333-Arg356 of the alpha-chain of C4BP contain the epitope of this antibody, and as a consequence, that this part of the alpha-chain of C4BP is likely to be involved in the interaction with C4b.

Amino Acid Sequence↗
PubMed1993

Primary structures of platelet aggregation inhibitors (disintegrins) autoproteolytically released from snake venom hemorrhagic metalloproteinases and new fluorogenic peptide substrates for these enzymes.

A hemorrhagic protein (60 kDa), HR1B, present in the venom of Trimeresurus flavoviridis is a mosaic protein consisting of an NH2-terminal metalloproteinase-domain, a disintegrin (platelet aggregation inhibitor)-like domain, and a unique COOH-terminal Cys-rich domain. Since the gross structures of HR1B and protein precursors of disintegrins, trigramin, and rhodostomin, all of which contain the metalloproteinase domain, are similar, many disintegrins so far detected in snake venoms are assumed to be autoproteolytic fragments released from precursors. In ongoing related experiments, the newly purified hemorrhagic metalloproteinases, HR1A from T. flavoviridis venom and HT-1 from Crotalus ruber ruber venom, in addition to HR1B, were autoproteolyzed, in the absence of Ca2+, at 37 degrees C for 3-12 h. Under these conditions, HR1A, HR1B, and HT-1 each released a single major fragment of 32, 34, and 31 kDa, respectively. The entire amino acid sequences of the isolated fragments indicated the presence of disintegrin-like and Cys-rich domains in the COOH-terminal regions of HR1A, HR1B, and HT-1, respectively. It seems likely that so-called disintegrins probably originate from various metalloproteinases present in venom. On the bases of peptide sequences close to the autoproteolytic cleavage sites of these metalloproteinases and the sites of fibrinogen cleaved by these enzymes, we synthesized new intramolecularly quenched fluorogenic peptide substrates. Among the 10 peptides tested, 2-aminobenzoyl (Abz)-Ser-Pro-Met-Leu-2,4-dinitroanilinoethylamide (Dna) proved to be the best substrate for venom metalloproteinase, as deduced from kinetic analyses.

Amino Acid Sequence↗
PubMed1993

[Coagulation and fibrinolytic systems and atherogenesis].

Normal blood fluidity and perpetuation of the non-thrombogenic state are primarily maintained by the anticoagulant and fibrinolytic systems of vascular endothelial cells involving heparin-like molecule, thrombomodulin, prostacyclin, and the receptor for tissue plasminogen activator. Atherosclerosis perturbes these activities, resulting in arterial thrombosis. On the other hand, recent experimental evidence suggests that the disordered thromboregulation often promotes atherosclerosis. Several known risk factors for development of atherosclerosis, including homocysteine and lipoprotein (a) perturb anticoagulant and fibrinolytic systems of vascular endothelial cells at an early stage of atherogenesis.

Arteriosclerosis↗
PubMed1992

Coagulation factor X activating enzyme from Russell's viper venom (RVV-X). A novel metalloproteinase with disintegrin (platelet aggregation inhibitor)-like and C-type lectin-like domains.

We determined the complete amino acid sequence of RVV-X, the blood coagulation factor X activating enzyme, isolated from Russell's viper venom and studied structure-function relationships. RVV-X (M(r) 79,000) consists of a disulfide-bonded two-chain glycoprotein with a heavy chain of M(r) 59,000 and a light chain of heterogeneous M(r) 18,000 (LC1) and 21,000 (LC2). These chains were separated after reduction and S-pyridylethylation, and the isolated major component LC1 was used for sequence analysis. The heavy chain consists of 427 residues containing four asparagine-linked oligosaccharides, and its entire sequence was similar to that of the high molecular mass hemorrhagic protein, HR1B, isolated from the venom of Trimere-surus flavoviridis. The heavy chain contains three distinct domains, metalloproteinase, disintegrin (platelet aggregation inhibitor)-like and unknown cysteine-rich domains. On the other hand, light chain LC1 consists of 123 amino acid residues containing one asparagine-linked oligosaccharide and shows sequence homology similar to that found in the so-called C-type (Ca(2+)-dependent) lectins. Therefore, RVV-X is a novel metalloproteinase containing a mosaic structure with distintegrin-like, cysteine-rich, and C-type lectin-like domains. RVV-X potently inhibits collagen- and ADP-stimulated platelet aggregations, probably via its distintegrin-like domain, although this domain does not contain the Arg-Gly-Asp sequence which is conserved in various venom distintegrins and which is thought to be one of the interaction sites for platelet integrins. Our findings also indicate that snake venom factor IX/factor X-binding protein with a C-type lectin structure (Atoda, H., Hyuga, M., and Morita, T. (1991) J. Biol. Chem. 266, 14903-14911) inhibits RVV-X-catalyzed factor X activation; hence, the light chain of RVV-X probably participates in recognizing some portion of the zymogen factor X.

Amino Acid Sequence↗
PubMed1992

Putative inositol 1,4,5-trisphosphate binding proteins in rat brain cytosol.

In previous works, we synthesized a series of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) analogs, with a substituent on the second carbon of the inositol ring. Using these analogs, the Ins(1,4,5)P3 affinity media were also synthesized (Hirata, M., Watanabe, Y., Ishimatsu, T., Yanaga, F., Koga, T., and Ozaki, S. (1990) Biochem. Biophys. Res. Commun. 168, 379-386). When the cytosol fraction from the rat brain was applied to an Ins(1,4,5)P3 affinity column, an eluate with a 2 M NaCl solution was found to have remarkable Ins(1,4,5)P3-binding activity. The active fraction was further fractionated with gel filtration chromatography, and two proteins with an apparent molecular mass of 130 or 85 kDa were found to be Ins(1,4,5)P3-binding proteins but with no Ins(1,4,5)P3 metabolizing activities. Partial amino acid sequences determined after proteolysis and reversed-phase chromatography revealed that the protein with an apparent molecular mass of 85 kDa is the delta-isozyme of phospholipase C and that of 130 kDa has no sequence the same as the Ins(1,4,5)P3-recognizing proteins hitherto examined. Ins(1,4,5)P3 at concentrations greater than 1 microM strongly inhibited 85-kDa phospholipase C delta activity, without changing its dependence on the concentrations of free Ca2+ and H+. Among inositol phosphates examined, Ins(3,4,5,6)P4 inhibited the binding of [3H]Ins(1,4,5)P3 to the 130-kDa protein at much the same concentrations as seen with Ins(1,4,5)P3. This report seems to be the first evidence for the presence of soluble Ins(1,4,5)P3-binding proteins in the rat brain, one of which is the delta isozyme of phospholipase C.

Amino Acid Sequence↗
PubMed1990

Amino acid sequence of human protein Z, a vitamin K-dependent plasma glycoprotein.

Protein Z is a vitamin K-dependent glycoprotein isolated and characterized from human and bovine plasma. A cDNA coding for human protein Z has been obtained by the isolation of phage clones from a liver cDNA library and in vitro amplification of two other liver libraries. Protein Z is synthesized with a prepro-leader sequence of 40 amino acids. The mature protein is composed of 360 residues including a Gla domain of 13 carboxyglutamic acid residues, two epidermal growth factor domains, and a carboxyl terminal region which is highly homologous to the catalytic domain of serine proteases. Human protein Z, however, contains an Asp instead of Ser and a Lys instead of His in the catalytic triad of the active site.

Amino Acid Sequence↗
PubMed1990

Blood clotting factor IX Nagoya 3: the molecular defect of zymogen activation caused by an arginine-145 to histidine substitution.

Factor IX Nagoya 3 (IX Nagoya 3) is a natural mutant of factor IX recognized in a patient with moderately severe hemophilia B. The patient had 0.60 units/ml of factor IX antigen and 2-5% of clotting activity. IX Nagoya 3 was purified from the patient's plasma by immunoaffinity chromatography with an anti-factor IX monoclonal antibody column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the treatment of IX Nagoya 3 with factor XIa/calcium ions resulted in cleavage only at the Arg180-Val181 bond. The amino acid sequence analysis of one of the lysyl endopeptidase peptides derived from IX Nagoya 3 revealed that Arg-145 is replaced by His. This substitution impairs the cleavage between the light chain and the activation peptide by factor XIa/calcium ions.

Amino Acid Sequence↗
PubMed1990

The complete amino acid sequence of the high molecular mass hemorrhagic protein HR1B isolated from the venom of Trimeresurus flavoviridis.

Hemorrhage is a common occurrence in a victim bitten by crotalid and viperid snakes, and hemorrhagic components in these various venoms have been isolated and characterized. Previously, we have shown that a low molecular weight hemorrhagic protein (HR2a, 202 amino acid residues) isolated from the venom of Trimeresurus flavoviridis is a member of a new subfamily of metalloproteinases. We now report the complete amino acid sequence of a high molecular mass hemorrhagic protein isolated from the same venom. This protein, HR1B, is a mosaic protein composed of 416 residues containing four asparagine-linked oligosaccharide chains. The amino-terminal half (residues 1-203) of HR1B contains a metalloproteinase domain, the sequence of which is 62% identical to that of HR2a and 52% identical to that of hemorrhagic toxin d isolated from Crotalus atrox venom. The most interesting finding is that the middle region (residues 204-300) of HR1B shows a striking similarity to disintegrins, Arg-Gly-Asp-containing platelet aggregation inhibitors, recently found in several viper venoms. Interestingly, however, this region of HR1B does not contain the Arg-Gly-Asp sequence which is known to be a putative binding site in the disintegrins for the platelet fibrinogen receptor, the glycoprotein IIb-IIIa complex. We also found that the carboxyl-terminal region (residues 213-336) of the middle part of HR1B shows 30% identity to residues 1543-1656 of von Willebrand factor and that the remaining region at the carboxyl-terminal end is unique and has a cysteine-rich sequence. These results suggest that the middle portion of HR1B, which shows structural similarities to the disintegrins and von Willebrand factor, may be important in synergistically stimulating hemorrhagic activity in the NH2-terminal metalloproteinase domain.

Amino Acid Sequence↗
PubMed1990

Purification, structure, and biochemical properties of human O6-methylguanine-DNA methyltransferase.

The level of O6-methylguanine-DNA methyltransferase activity in a human cell line carrying a 1.1-kilobase cDNA fragment was about 50 times higher than that found in ordinary methyltransferase-proficient (Mer+) cell lines (Hayakawa, H., Koike, G., and Sekiguchi, M. (1990) J. Mol. Biol. 213, 739-747). Taking advantage of this overproduction, the enzyme was purified to apparent physical homogeneity and the physical and biochemical properties investigated. A single polypeptide with a molecular weight of approximately 25,000 was detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the most highly purified preparation. The Stokes radius of 22.5 A and the sedimentation coefficient of 2.0 S were obtained, from which the molecular weight of the native form of the enzyme was calculated to be 19,000. After digestion with lysyl endopeptidase, peptide fragments of the protein were isolated and sequenced. The amino acid sequences of these peptides and the amino acid composition of the protein were in good agreement with those deduced from the nucleotide sequence of the cloned cDNA. The purified enzyme catalyzed transfer of methyl groups from O6-methylguanine and O4-methylthymine, but not from methylphosphotriesters, of methylated DNA to the enzyme molecule.

Amino Acid Sequence↗
PubMed1990

Primary structure of a hemorrhagic metalloproteinase, HT-2, isolated from the venom of Crotalus ruber ruber.

Crotalidae and Viperidae snake venoms contains several kinds of metalloproteinases which cause localized hemorrhage by direct action on blood vessel walls. We report here the entire amino acid sequence and the disulfide bridge locations of HT-2, one of the hemorrhagic toxins isolated from the venom of Crotalus ruber ruber (red rattlesnake). The non-reduced protein was first cleaved at methionine residues to provide a set of 8 fragments, which covered the entire sequence of HT-2. The disulfide bridge locations of HT-2 were also determined by using these primary fragments. The unambiguous sequence for the whole protein was then established by conventional methods using lysyl endopeptidase and thermolysin digests. HT-2 consisted of 202 amino acid residues with two disulfide bridges, which were assigned to Cys-117-Cys-197 and Cys-157-Cys-164. HT-2 had a typical zinc-chelating sequence His-Glu-X-X-His (residues 142-146) found in thermolysin, and its overall sequence showed, respectively, 50, 52, and 53% identities to those of HR2a, H2-proteinase, and the metalloproteinase domain of HR1B. However, the disulfide bridge locations of HT-2 were different from those in the other metalloproteinases. The primary structure of HT-2 was more closely related to that of Ht-d from Crotalus atrox recently determined (81% identity). From the structural comparison with five metalloproteinases so far elucidated, six conservative amino acid residues, which may possibly be related to the induction of the hemorrhagic activity, were suggested to be present in these toxins.

Amino Acid Sequence↗
PubMed1989

Blood clotting factor IX BM Nagoya. Substitution of arginine 180 by tryptophan and its activation by alpha-chymotrypsin and rat mast cell chymase.

Factor IX BM Nagoya (IX Nagoya) is a natural mutant of factor IX responsible for severe hemophilia B. A patient with this mutant is characterized by a markedly prolonged ox brain prothrombin time. IX Nagoya was purified from the patient's plasma by immunoaffinity chromatography with an anti-factor IX monoclonal antibody column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that treatment of IX Nagoya with factor XIa/Ca2+ resulted in cleavage only at the Arg145-Ala146 bond. Reversed-phase high performance liquid chromatography of a trypsin digest of IX Nagoya showed an aberrant peptide, which was further digested with proteinase Asp-N. Primary structure analysis of one of the Asp-N peptides revealed that Arg180 is replaced by Trp. An essentially complete (99%) amino acid sequence of IX Nagoya was obtained by sequencing fragments derived from a lysyl endopeptidase digest in which no other substitutions in the catalytic triad or substrate binding site were found. We also found that IX Nagoya is activated by alpha-chymotrypsin or rat mast cell chymase by monitoring the rate of factor X activation using a fluorogenic peptide substrate in the presence of factor VIII, phospholipids, and Ca2+. These results indicate that the substitution of Arg180 by Trp impairs the cleavage by factor XIa required for activation of this zymogen and that the substitution causes hemophilia BM.

Amino Acid Sequence↗