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S Iwanaga

Publications and source records attributed to S Iwanaga.

At least 127 records · Page 7Linked to original sources
PubMed1994

cDNA cloning and characterization of mitochondrial import stimulation factor (MSF) purified from rat liver cytosol.

We identified a liver cytosolic protein factor that stimulated the import of wheat germ lysate-synthesized precursor proteins into mitochondria. It was termed mitochondrial import stimulation factor or MSF [Hachiya, N. et al. (1993) EMBO J. 12, 1579-1586]. It consisted of 32-kDa (MSFL) and 30-kDa (MSFS) polypeptides as assessed by SDS-PAGE. MSF recognized the presequence portion of mitochondrial precursor proteins and catalyzed the depolymerization and unfolding of in vitro synthesized mitochondrial precursor proteins in an ATP-dependent manner. We report here the cDNA cloning and characterization of MSF. Microsequencing of MSFL and MSFS showed that they belonged to a highly conserved, widely distributed eukaryotic protein family, collectively designated as 14-3-3 proteins. We cloned the cDNA of MSFL and that of one component of MSFS (MSFS1) from a rat liver cDNA library. The cloned cDNAs were separately expressed in Escherichia coli and the expressed proteins were purified to homogeneity. The purified recombinant MSFL and MSFS1 stimulated mitochondrial import of adrenodoxin precursor (pAd) synthesized in vitro with wheat germ lysate translation system. Recombinant MSFL or MSFS1 had the ability to bind with denatured pAd and they kept the precursor in an import-competent state. Rabbit polyclonal antibodies raised against the recombinant proteins inhibited the import-stimulation activity of rat liver cytosol as well as that of authentic purified MSF. Identification of MSF as 14-3-3 proteins establishes a novel function for this family of proteins and indicates their role as cytosolic chaperones to aid many important cellular events.

14-3-3 Proteins↗
PubMed1994

A carcinoma in situ arising in an endocervical polyp--a case report.

A rare case of a carcinoma in situ arising from an endocervical polyp is described. The covering epithelium of the polyp transformed gradually into a carcinoma in situ via squamous metaplasia and dysplasia, spreading horizontally with glandular involvement, and colliding with the surface columnar cells. The lack of tumor tissue in the pedicle and adjacent cervical region confirms the case was a carcinoma in situ originating from the surface epithelium of the endocervical polyp.

Adenomatous Polyps↗
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

Horseshoe crab coagulation factor B. A unique serine protease zymogen activated by cleavage of an Ile-Ile bond.

Horseshoe crab factor B is an intracellular serine protease zymogen involved in the bacterial endotoxin-responsive hemolymph coagulation cascade. cDNAs for factor B were isolated utilizing a polymerase chain reaction product using two primers derived from the partial amino acid sequence. The cloned cDNA of 1928 base pairs encoded 400 amino acid residues of factor B precursor. The first 23 amino acid residues constitute a presumed prepropeptide that may be processed by both a signal peptidase and a processing protease, similar to mammalian vitamin K-dependent protease precursors. The mature protein consists of 377 amino acids with a calculated molecular mass of 40,570 Da. The overall structure is highly homologous to that of limulus proclotting enzyme (35.9% identity), the substrate for active factor B in the cascade. Like the proclotting enzyme, mature factor B is composed of an amino-terminal "clip"-like domain and a carboxyl-terminal serine protease domain homologous to that of human plasma prekallikrein (36.5%). Internal sequences encode a unique activation peptide. Surprisingly, the cleavage sites of the zymogen factor B for activation by limulus active factor C were found to be an Arg-Ser and an Ile-Ile bond, the latter of which has not been found in any other protease zymogens. These cleavages result in the release of the activation peptide, which consists of 21 residues with a carboxyl-terminal isoleucine. These results indicate that the intracellular clotting system of the limulus hemocyte, like mammalian plasma clotting cascade, proceeds with the sequential activation of three serine protease zymogens: factor C, factor B, and proclotting enzyme.

Amino Acid Sequence↗
PubMed1993

[Clinical evaluation of myocardial perfusion imaging with 99mTc-tetrofosmin].

Myocardial perfusion imaging with Tc-99 m-1,2-bis (bis (2-ethoxyethyl) phosphino) ethane (Tc-99 m-tetrofosmin) was performed in 26 patients with ischemic heart disease (IHD) or suspected to have IHD. The agent was administered both at rest and at peak exercise. Exercise and rest studies were performed on the same day or on different days. The injected dose ranged from 296 MBq-740 MBq. Image quality was adequate for diagnosis, and was superior to that of T1-201 scintigraphy. Results from the tetrofosmin imaging corresponded well with findings of T1 scintigraphy, with a segmental concordance of 87%. Sensitivity and specificity were 70% and 93%, respectively. Our results suggested that tetrofosmin could be a promising Tc-99m labeling agent for the evaluation of myocardial perfusion.

Evaluation Studies as Topic↗
PubMed1993

Development of antitissue factor antibodies in patients after liver surgery.

After liver surgery, two patients developed unexplainable prolonged prothrombin times (PT) that were not associated with bleeding tendency. The substitution of rabbit thromboplastin for either human or monkey thromboplastin in performing PT tests resulted in a normal clotting time. Tissue factor (TF) procoagulant activity assays and an immunoblotting analysis showed that these patients had developed IgG lambda-type immediate anticoagulants directed against both rabbit and bovine TF that did not crossreact with human or monkey TF. In a chromogenic assay, the patient IgG caused a decrease in both the Km and the Vmax of the factor X activation by rabbit TF-factor VIIa complex. The lack of reactivity of the patient IgG with human TF presumably explained why there was no clinical bleeding. Both patients had been treated earlier with a topical hemostatic agent prepared from bovine corium, microfibrillar collagen hemostat, while undergoing previous surgery. In an immunoblotting analysis, the patient IgG stained a 42-Kd band in the Triton extract of the collagen preparation under either reducing or nonreducing conditions. The Triton extract of the collagen preparation blocked the binding of the patient IgG to bovine TF. Thus, it is suggested that the iatrogenic immunization by intraoperative exposure of bovine TF retained in the collagen preparation may be responsible for the development of anti-TF antibodies in these patients. The anti-TF antibodies resulted in a clinical error in the evaluation of coagulation status after the use of rabbit thromboplastin.

Animals↗
PubMed1993

Rabbit liver microsomal endopeptidase with substrate specificity for processing proproteins is structurally related to rat testes metalloendopeptidase 24.15.

The detergent extract of rabbit liver microsomes contains an endopeptidase (MEP) with substrate specificity for peptides containing Arg residues at the P1 and P4 positions in the cleavage site (Kawabata, S., and Davie, E. W. (1992) J. Biol. Chem. 267, 10331-10336). These sequences occur in many proproteins such as the vitamin K-dependent proproteins and prohormones. A cDNA coding for MEP has been obtained from three overlapping clones isolated from two rabbit liver lambda gt10 cDNA libraries. The longest open reading frame of the 3507-base pair cDNA codes for a protein of 704 amino acids, of which 406 residues were confirmed by amino acid sequence analysis. MEP contains a putative active site of -His-Glu-X-X-His-, which is typical of mammalian zinc metallopeptidases. Based on a hydropathy plot, MEP is a hydrophilic protein with no transmembrane domain and no NH2-terminal signal sequence. Amino acid sequence analysis identified Asn at the three potential N-glycosylation sites in the enzyme, indicating that MEP contains no N-linked sugar. MEP is homologous with rat testes metalloendopeptidase 24.15 (60% identity), rat mitochondrial intermediate peptidase (24% identity), Escherichia coli dipeptidyl carboxypeptidase (25% identity), and the open reading frame YCL57w present in yeast chromosome III (35% identity).

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

Limulus hemocyte transglutaminase. Its purification and characterization, and identification of the intracellular substrates.

To investigate further the molecular events of the intracellular coagulation cascade in limulus hemocytes, a transglutaminase (TGase), which may be involved in the formation of a stabilized gel, was purified and characterized. Through the purification procedures consisting of six steps, 1.6 mg of TGase with a specific activity of 940 amine incorporation unit/mg was obtained from 32.4 g of Tachypleus tridentatus hemocytes. The purified TGase gave a single band on SDS-polyacrylamide gel electrophoresis with a molecular mass of 86 kDa, and demonstrated mammalian-type II TGase-like enzymatic properties. The TGase activity was Ca(2+)-dependent and was inhibited by primary amines, EDTA, and SH-reagents. Moreover, two major potential substrates for TGase were identified in the hemocyte lysate by using dansylcadaverine (DCA) incorporation in the presence of 10 mM CaCl2 and 10 mM dithiothreitol. Of these protein substrates, an 80-kDa protein contained a large number of proline residues, amounting to about 22% of the total amino acids. On the other hand, an 8.6-kDa protein abundantly present in the hemocytes was characterized as a Cys-rich protein consisting of 81 amino acid residues and a calculated molecular mass of 8,671. The entire amino acid sequence of this protein was established. Also, the 8.6-kDa protein was readily cross-linked intermolecularly by TGase, forming multimers as large as pentamers. We speculate that like plasma factor XIIIa, limulus TGase and its two protein substrates in the hemocytes may play an important role in the defense of this animal against invading microorganisms.

Amino Acid Sequence↗
PubMed1993

Limulus hemocyte transglutaminase. cDNA cloning, amino acid sequence, and tissue localization.

We have evidence that the limulus (Tachypleus tri-dentatus) hemocyte transglutaminase (TGase) has a molecular mass of 86 kDa and properties of the mammalian type II TGase-like enzyme (Tokunaga, F., Yamada, M., Miyata, T., Ding, Y.-L., Hiranaga-Kawabata, M., Muta, T., Iwanaga, S., Ichinose, A., and Davie, E.W. (1993) J. Biol. Chem. 268, 252-261). We present here the cDNA and amino acid sequences, and localization of the TGase in various tissues of limulus. The cloned cDNA for TGase consists of 2,884 base pairs. An open reading frame of 2,292 base pairs encodes a sequence comprising 764 residues of the mature protein with molecular masses of 87,021 and 87,110 Da, due to two different clones. The discrepancies of nucleotides in these two clones result in 3 amino acid exchanges at positions Gly452(GGT)-Arg(CGT), Ser477(AGT)-Cys(TGT), and Ile486(ATC)-Ser(AGC), respectively. Northern blot analysis on a total RNA extracted from various tissues of limulus revealed that TGase is expressed with 3.0 kilobases of a single type of mRNA, mainly in hemocytes, hepatopancreas, and gastric tissues. Limulus TGase shows significant sequence similarity with the mammalian TGase family, as follows: guinea pig liver TGase (32.7%), human factor XIIIa subunit (34.7%), human keratinocyte TGase (37.6%), and human erythrocyte band 4.2 (23.0%). Limulus TGase has a unique NH2-terminal cationic extension of 60 residues with no homology to the NH2 termini of mammalian TGases. Based on the alignment of the amino acid sequence of limulus TGase with those of the known TGase family, a phylogenetic tree representing an evolutionary relationship among the family members was inferred by the neighbor joining method.

Amino Acid Sequence↗
PubMed1993

Secondary structural features of modules M2 and M3 of barnase in solution by NMR experiment and distance geometry calculation.

Proteins consist of structural units such as globular domains, secondary structures, and modules. Modules were originally defined by partitioning a globular domain into compact regions, each of which is a contiguous polypeptide segment having a compact conformation. Since modules show close correlations with the intron positions of genes, they are regarded as primordial polypeptide pieces encoded by exons and shuffled, leading to yield new combination of them in early biological evolution. Do modules maintain their native conformations in solution when they are excised at their boundaries? In order to find answers to this question, we have synthesized modules of barnase, one of the bacterial RNases, and studied the solution structures of modules M2 (amino acid residues 24-52) and M3 (52-73) by 2D NMR studies. Some local secondary structures, alpha-helix, and beta-turns in M2 and beta-turns in M3, were observed in the modules at the similar positions to those in the intact barnase but the overall state seems to be in a mixture of random and native conformations. The present result shows that the excised modules have propensity to form similar secondary structures to those of the intact barnase.

Amino Acid Sequence↗