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Biomedical subjects

S Iwanaga

Publications and source records attributed to S Iwanaga.

At least 253 records · Page 14Linked to original sources
PubMed1988

Blood clotting factor IX Niigata: substitution of alanine-390 by valine in the catalytic domain.

Factor IX Niigata is a mutant factor IX responsible for the moderately severe hemophilia B in a patient who has a normal level of factor IX antigen with reduced clotting activity (1-4% of normal). We reported previously that the purified mutant protein could be converted to the factor IXa beta form by factor XIa/Ca2+ at a rate similar to that in the case of normal factor IX, but the resulting mutant factor IXa beta could not activate factor X in the presence of factor VIII, Ca2+, and phospholipids (Yoshioka, A. et al. (1986) Thromb. Res. 42, 595-604). In the present study, we analyzed factor IX Niigata at the structural level to elucidate the molecular abnormality responsible for the loss of clotting activity. Amino acid sequence analysis of a peptide obtained on lysyl endopeptidase digestion, coupled with subsequent SP-V8 digestion, demonstrated that the alanine at position 390 was substituted by valine in the catalytic domain of the factor IX Niigata molecule.

Alanine↗
PubMed1988

A novel immunohistochemical method for in vivo detection of endotoxin using horseshoe crab factor C.

We developed a novel immunohistochemical method for in vivo detection of endotoxin (LPS) localization in relation to the biologically active region, by use of factor C, an initiation factor in the Limulus clotting system which is mediated by LPS, as a specific affinoligand to LPS, and using rabbit anti-factor C IgG. The competitive inhibition of various LPS, lipid A, anti-LPS factor, or polymyxin B to factor C binding indicates that the immunohistochemical reaction is specific to LPS. Investigating the time course of LPS distribution during 6 hr after IV injection of 5 mg/kg to rats, the greatest uptake of LPS was evident in the reticuloendothelial system (RES), particularly in Kupffer cells, 5 min after injection, and in adrenocortical cells 3 hr after the injection. Shortly after the injection, LPS also appeared in platelet thrombi, intravascular monocytes, and a few neutrophils, and on the surface of endothelial cells in liver, kidney, spleen, lung, and aorta. Both smooth and rough forms of LPS were detectable and there was no apparent difference in their localization. This approach facilitates immunohistochemical analyses of the mechanisms involved in development of endotoxemia.

Adrenal Glands↗
PubMed1988

[A case of HBs antigen negative hepatocellular carcinoma without liver cirrhosis in a young woman].

A 27-year-old woman, post parturition, was admitted to hospital because of epigastric pain. Ultrasonography and abdominal computerized tomography revealed a giant mass in the left lobe of the liver. Celiac angiography showed a hypervascular mass with irregular vascularity. Serum HBs antigen proved negative. A left hepatic lobectomy was performed in March, 1985. Histology of the tumor revealed the trabecular type of a hepatocellular carcinoma with Edmondson grade 2 atypism, whereas the non-cancerous region of the liver showed chronic inactive hepatitis. This case is presented, because of the rarity of a hepatocellular carcinoma developing in such a young patient. In addition, reported cases of a hepatocellular carcinoma in the young in Japan are reviewed.

Adult↗
PubMed1987

The chymotrypsin-like activity of human prostate-specific antigen, gamma-seminoprotein.

gamma-Seminoprotein (gamma-Sm) is a human prostate-specific antigen and a serine protease judging from the complete amino acid sequence which shows extensive homology with the kallikrein family. The enzymatic activity of gamma-Sm was defined as a chymotrypsin-like activity using reduced and S-3-(trimethylated amino)propylated lysozyme and insulin-oxidized A and B chains as substrates. The -Leu/Ser- peptide bond of lysozyme was rapidly hydrolyzed by gamma-Sm. gamma-Sm also hydrolyzed the -Phe/Glu- of lysozyme and the -Leu/Cys(SO3H)- of insulin B chain. Insulin A chain and arginyl- or lysyl-linkage of these proteins were not hydrolyzed by gamma-Sm at all.

Amino Acids↗
PubMed1987

A new sensitive assay for bovine activated factor XI (factor XIa) using a reconstituted coagulation cascade system.

A sensitive assay for quantitating bovine activated Factor XI (Factor XIa) in vitro was developed by measuring the amidolytic activity of thrombin generated in a mixture of Factors XIa, IX and X and prothrombin prepared from bovine source and washed bovine platelets. In this system, the rate of thrombin generation increased linearly with increasing amounts (fmoles) of Factor XIa. The assay system for Factor XIa was not significantly affected by the presence of plasma kallikrein, Factor XIIa, high-molecular-weight kininogen, amylose sulfate or sulfatide within the range of the amounts used for surface-mediated activation of Factor XII, prekallikrein and Factor XI. Following surface-mediated activation of Factor XI, further generation of Factor XIa was blocked by adding freeze-thawed platelets that contain cationic proteins which bind to negatively-charged surfaces (J. Biochem. 97, 139-151, 1985). The method is useful to the kinetic analysis of the surface-mediated activation of Factors XII and XI, although it is not applicable to the activation in plasma.

Animals↗
PubMed1987

Isolation and characterization of ornitho-kininogen.

Ornitho-kininogen was purified from chicken blood plasma by a two-stage method using chromatography on columns of S-alkylated papain-Cellulofine and DEAE-5PW. The yield was 1.7 mg from 44 ml plasma. The isolated preparation gave a single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) with or without 2-mercaptoethanol and on disc/polyacrylamide gel electrophoresis. The relative molecular mass, Mr, of ornitho-kininogen was estimated as 74,000 on SDS-PAGE using the Ferguson plot method. Ornitho-kininogen was found to have the similar properties to those of mammalian high-Mr kininogen, in terms of the amino acid composition, molecular mass, and susceptibility to plasma kallikrein. No kininogen corresponding to mammalian low-Mr kininogen and rat T-kininogen could be detected in chicken plasma. In fact, ornitho-kininogen was degraded rapidly by bovine plasma kallikrein, liberating a kinin. This kinin was isolated from the digest by reversed-phase HPLC. The primary structure of the isolated kinin was determined as Arg1-Pro2-Pro3-Gly4-Phe5-Thr6-Pro7-Leu8-Arg9. The sequence of this peptide, named ornitho-kinin, was similar to that of bradykinin except for the substitution of Thr6 and Leu8 for Ser6 and Phe8. The isolated ornitho-kinin induced a contraction of chicken smooth muscle and had a strong hypotensive effect in the chicken. However, it did not contract the isolated rat uterus. It is suggested that this specificity difference is due to the replacement of Phe8 by Leu8. The sequence of residues 1-30 of ornitho-kininogen exhibited 43% identity with that of bovine kininogen.

Amino Acid Sequence↗
PubMed1987

Lipopolysaccharide-sensitive serine-protease zymogen (factor C) of horseshoe crab hemocytes. Identification and alignment of proteolytic fragments produced during the activation show that it is a novel type of serine protease.

The horseshoe crab clotting factor, factor C, present in the hemocytes is a serine-protease zymogen activated with lipopolysaccharide. It is a two-chain glycoprotein (Mr = 123,000) composed of a heavy chain (Mr = 80,000) and a light chain (Mr = 43,000) [T. Nakamura et al. (1986) Eur. J. Biochem. 154, 511-521]. In our continued study of this zymogen, we have now also found a single-chain form of factor C (Mr = 123,000) in the hemocyte lysate. The heavy chain had the NH2-terminal sequence of Ser-Gly-Val-Asp-, consistent with that of the single-chain factor C, indicating that the heavy chain is derived from the NH2-terminal part of the molecule. The light chain had an NH2-terminal sequence of Ser-Ser-Gln-Pro-. Incubation of the two-chain zymogen with lipopolysaccharide resulted in the cleavage of a Phe-Ile bond between residues 72 and 73 of the light chain. Concomitant with this cleavage, the A (72 amino acid residues) and B chains derived from the light chain were formed. The complete amino acid sequence of the A chain was determined by automated Edman degradation. The A chain contained a typical segment which is similar in sequence to a family of repeats in human beta 2-glycoprotein I, complement factors B, protein H, C4b-binding protein, and coagulation factor XIII b subunit. The NH2-terminal sequence of the B chain was Ile-Trp-Asn-Gly-. This chain contained the serine-active site sequence-Asp-Ala-Cys-Ser-Gly-Asp-Ser-Gly-Gly-Pro-. These results indicate that horseshoe crab factor C exists in the hemocytes in a single-chain zymogen form and is converted to an active serine protease by hydrolysis of a specific Phe-Ile peptide bond.

Amino Acid Sequence↗
PubMed1987

Complete nucleotide sequence of cDNA and deduced amino acid sequence of rat liver arginase.

Arginase (EC 3.5.3.1) catalyzes the last step of urea synthesis in the liver of ureotelic animals. The nucleotide sequence of rat liver arginase cDNA, which was isolated previously (Kawamoto, S., Amaya, Y., Oda, T., Kuzumi, T., Saheki, T., Kimura, S., and Mori, M. (1986) Biochem. Biophys. Res. Commun. 136, 955-961) was determined. An open reading frame was identified and was found to encode a polypeptide of 323 amino acid residues with a predicted molecular weight of 34,925. The cDNA included 26 base pairs of 5'-untranslated sequence and 403 base pairs of 3'-untranslated sequence, including 12 base pairs of poly(A) tract. The NH2-terminal amino acid sequence, and the sequences of two internal peptide fragments, determined by amino acid sequencing, were identical to the sequences predicted from the cDNA. Comparison of the deduced amino acid sequence of the rat liver arginase with that of the yeast enzyme revealed a 40% homology.

Amino Acid Sequence↗
PubMed1987

Bovine high molecular weight kininogen. The amino acid sequence, positions of carbohydrate chains and disulfide bridges in the heavy chain portion.

The existence of two types of circulating bovine plasma high molecular weight kininogen (HMWK) was predicted from analyses of complementary DNAs coding for this protein (Kitamura, N., Takagaki, Y., Furuto, S., Tanaka, T., Nawa, H., and Nakanishi, S. (1983) Nature 305, 545-549). The present protein-based study provided evidence in support of the proposed amino acid sequence derived from analysis of the cDNA clone, and the results confirm the existence of two types of circulating HMWK. Type I HMWK contains a heavy chain composed of 361 residues, while the heavy chain of type II HMWK contains 359 residues. The amino acid sequences of type I and type II HMWK determined in this study were identical to that inferred from the cDNA sequence with the exception of microheterogeneity observed in the cDNA at position 87 (Glu/Gln) and 168 (Lys/Arg). The heavy chain of type I HMWK contains 4 asparagine-linked carbohydrate chains at Asn-69, -150 (or -151), -179, and -186, while the heavy chain of type II HMWK contains these and an additional carbohydrate chain at Asn-264. In addition, a carbohydrate chain was found to be O-glycosidically linked to Thr-118 in both chains. Among nine disulfide linkages found in HMWK, eight intrachain disulfide pairs were established in the heavy chain. One interchain disulfide bridge occurs between the heavy chain and the light chain. This disulfide pairing, as well as repeating amino acid sequences observed in the heavy chain, provides strong evidence for the existence of three homologous domains in the heavy chain of bovine HMWK.

Amino Acid Sequence↗
PubMed1987

Prothrombin Tokushima, a replacement of arginine-418 by tryptophan that impairs the fibrinogen clotting activity of derived thrombin Tokushima.

Structural studies on a hereditarily abnormal prothrombin, prothrombin Tokushima, have been performed to identify the difference responsible for its reduced fibrinogen clotting activity upon conversion to thrombin. The prothrombin sample used was from a heterozygote but contained exclusively a defective prothrombin molecule, since the patient was heterozygous for both dysprothrombinemia and hypoprothrombinemia. Amino acid sequence analysis of a peptide isolated from a lysyl endopeptidase digest of the abnormal thrombin indicated that Arg-418 (equivalent to Asn-101 in the chymotrypsin numbering system) had been replaced by Trp. This amino acid substitution can result from a single nucleotide change in the codon for Arg-418 (CGG----TGG). The Arg----Trp replacement found in the thrombin portion of prothrombin Tokushima appears to reduce its interaction with various substrates including fibrinogen and platelet receptors and accounts for the recurrent bleeding episode observed in the propositus.

Amino Acid Sequence↗
PubMed1987

Complete amino acid sequence of cytochrome c551 from Erythrobacter species strain OCh 114.

The complete amino acid sequence of cytochrome c551 isolated from an aerobic photosynthetic bacterium, Erythrobacter sp. strain OCh 114, was determined. The cytochrome molecule was composed of a total of 119 amino acid residues and its molecular weight including heme was calculated to be 13,235. The sequence was (Sequence: see text). Its molecular weight indicates that this cytochrome is of the L-type. Sequence alignment with other bacterial cytochromes c shows that this cytochrome is similar to cytochromes c of Rhodobacter capsulatus, Rhodobacter sphaeroides, and Paracoccus denitrificans, which were grouped into the alpha-3 subcluster from the 16S rRNA sequence analysis.

Amino Acid Sequence↗
PubMed1987

Structural comparison of bovine erythrocyte, brain, and liver NADH-cytochrome b5 reductase by HPLC mapping.

NADH-cytochrome b5 reductases purified from bovine erythrocytes and from bovine brain and liver microsomes solubilized with lysosomal protease were subjected to structural analysis by using HPLC mapping, amino acid analysis of the resulting peptides, and NH2-terminal sequence analysis of apoproteins. HPLC maps of the tryptic peptides derived from these enzymes were very similar to each other, and amino acid analysis of the HPLC-separated peptides indicated that the structures of these enzymes are identical except for the NH2-terminal region. The NH2-terminal sequence of the brain enzyme determined by automated Edman degradation was as follows: NH2-Phe-Gln-Arg-Ser-Thr-Pro-Ala-Ile-Thr-Leu-Glu-Asn-Pro-Asp- Ile-Lys-Tyr-Pro-Leu-Arg-Leu-Ile-Asp-Lys-Glu-Val-Ile- This sequence is identical to that of liver enzyme except that the liver enzyme started at the 3rd Arg or 4th Ser. The NH2-terminal amino acid residue of the soluble erythrocyte enzyme was not detected by automated Edman degradation. The sequence analysis of a tryptic peptide from the erythrocyte enzyme indicated that Leu is present before the NH2-terminal Phe of the brain enzyme. The recently reported sequence of the apparently identical protein (Ozols et al. (1985) J. Biol. Chem. 260, 11953-11961) differs in two amino acid assignments from our sequence.

Amino Acid Sequence↗
PubMed1987

Purification and amino acid sequence of Kunitz-type protease inhibitor found in the hemocytes of horseshoe crab (Tachypleus tridentatus).

A low molecular weight protein protease inhibitor was purified from Japanese horseshoe crab (Tachypleus tridentatus) hemocytes. It consisted of a single polypeptide with a total of 61 amino acid residues. This protease inhibitor inhibited stoichiometrically the amidase activity of trypsin (Ki = 4.60 X 10(-10) M), and also had inhibitory effects on alpha-chymotrypsin (Ki = 5.54 X 10(-9) M), elastase (Ki = 7.20 X 10(-8) M), plasmin, and plasma kallikrein. However, it had no effect on T. tridentatus clotting enzyme and factor C, mammalian blood coagulation factors (activated protein C, factor Xa and alpha-thrombin), papain, and thermolysin. The complete amino acid sequence of this inhibitor was determined and its sequence was compared with those of bovine pancreatic trypsin inhibitor (BPTI) and other Kunitz-type inhibitors. It was found that the amino acid sequence of this inhibitor has a high homology of 47 and 43% with those of sea anemone inhibitor 5-II and BPTI, respectively. Thus, this protease inhibitor appeared to be one of the typical Kunitz-type protease inhibitors.

Amino Acid Sequence↗