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F Shishikura

Publications and source records attributed to F Shishikura.

17 recordsLinked to original sources
PubMed1999

Purification, characterization, and amino acid sequence of an embryonic lectin in perivitelline fluid of the horseshoe crab.

Hemagglutinating activity in perivitelline fluid of the horseshoe crab embryo dramatically increases during the third and fourth molt of the embryo. A 27-kDa lectin, which we named tachylectin-P (TL-P), was newly identified in perivitelline fluid of the horseshoe crab Tachypleus tridentatus. TL-P preferentially agglutinated human A-type erythrocytes, and the activity was inhibited by N-acetyl group-containing monosaccharides. The amino acid sequence analysis indicated that TL-P is almost structurally the same as a hemocyte-derived lectin with no hemagglutinating activity, tachylectin-1 (TL-1), and that 218 out of 221 amino acid residues in total were conserved between the two lectins. Despite the high sequence similarity, biological and biochemical characteristics of TL-P differed from those of TL-1: (i) unlike TL-1, TL-P agglutinates several animal-derived erythrocytes; (ii) unlike TL-1, TL-P has no significant affinity for bacterial lipopolysaccharides or antibacterial activity; (iii) Based on apparent molecular masses determined by gel filtration, TL-P forms a dimer in solution, while TL-1 is present as a monomer; (iv) and TL-P interacts with endogenous proteins of 13 and 14 kDa present in the perivitelline fluid; however, neither has any affinity for TL-1. We propose that TL-P may have an important role in completing embryonic development by interacting with endogenous glycoproteins or N-acetylhexosamines.

Amino Acid Sequence↗
PubMed1997

Purification and characterization of a 39,000-Da serine proteinase from the hemolymph of a solitary ascidian, Halocynthia roretzi.

A new endogenous serine proteinase from the cell-free hemolymph of a solitary ascidian, Halocythia roretzi, was purified by a combination of ammonium sulfate fractionation, hydrophobic interaction chromatography on TSKgel Toyopearl HW 65 F, ion exchange chromatography on TSKgel DEAE-Toyopearl 650 M, affinity chromatography on Arginine-Sepharose 4B, gel filtration on TSKgel Toyopearl HW 65F and hydroxyapatite chromatography on Bio-Gel HT. The serine proteinase is a single polypeptide chain whose molecular weight and isoelectric point are 39 kDa and about 7.6 pI, respectively. The most susceptible substrate was Boc-Leu-Gly-Arg-4-methyl-coumaryl-7-amide (MCA), and activity was optimal at pH 8. The enzyme was relatively stable at high temperatures; about 50% activity was retained even at 60 degrees C for 30 min in 50 mM Tris-HCl, pH 8.0, containing 0.5 M NaCl, and 0.05% Brij-35. The enzyme was characterized by the inhibitory effects of synthetic or natural inhibitors, substrate specificity toward 26 peptidyl-MCAs, proteinase activity toward natural proteins and complex formation with a serine proteinase inhibitor (58 kDa) previously found in H. roretzi hemolymph, indicating that the enzyme was a member of serine proteinases and strongly inhibited by the 58 kDa serine proteinase inhibitor as well as human antithrombin III. We also demonstrated the clotting enzyme activity of the purified serine proteinase toward bovine fibrinogen and Limulus coagulogen, a fibrinogen-like clottable protein of horseshoe crabs.

Amino Acid Sequence↗
PubMed1997

Leech extracellular hemoglobin: two globin strains that are akin to vertebrate hemoglobin alpha and beta chains.

Leech (Whitmania edentula, Haemadipsa zeylanica var. japonica and Erpobdella lineata) extracellular hemoglobins are basically composed of three constituent subunits, a dimer (D1 and D2 chains) and two monomers (M1 and M2 chains). We isolated these four chains from respective species by a combination of reversed-phase chromatography on a Resource RPC column and gel-filtration on a Superdex 75 column. The apparent molecular masses of the four globin chains were estimated by SDS-PAGE analysis to be 13 kDa (M1), 16 kDa (M2; 19 kDa in its reduced form) and about 27 kDa for the dimer subunit (13 kDa for D1; 15 kDa for D2), regardless of the source. The amino (N)-terminal segments (21-30 residues) from twelve globin chains of the above three species were determined and aligned. It was found that the twelve sequences could be separated into two distinct globin groups A and B. This finding supports the original idea of "two globin strains in annelid hemoglobin", which was proposed without any evidence for leech hemoglobins. Comparing the sequences in the three classes of Annelida, Hirudinea, Oligochaeta and Polychaeta, we found two invariant amino acids, Cys and Trp, which are interposed by eleven amino acid residues. Furthermore, the globin chains belonging to strain A were readily discernible as they had three more invariants, Ser-13, Asp-16 and Trp-28, while the globin chains of strain B had two more invariants, Lys-12 and Arg-27. Consequently, we propose that each of the three classes of Annelida have two distinct groups of globin chains that are akin to vertebrate hemoglobin alpha and beta chains.

Amino Acid Sequence↗
PubMed1996

Purification and characterization of a 58,000-Da proteinase inhibitor from the hemolymph of a solitary ascidian, Halocynthia roretzi.

A new endogenous proteinase inhibitor from the cell-free hemolymph of a solitary ascidian, Halocynthia roretzi, was purified by a combination of ammonium sulfate fractionation, hydrophobic interaction chromatography on Ether-Toyopearl and affinity chromatography on Heparin-Sepharose. The purity of the inhibitor was examined by SDS-PAGE, gel-permeation chromatography, reversed-phase chromatography, isoelectric focusing, immunological analysis and amino-terminal amino acid sequence analysis. The inhibitor is a single polypeptide chain whose molecular weight, isoelectric point and the first 10 amino-terminal amino acid sequences are 58 kDa, pI 9.2 and NH2-Thr-Lys-Lys-Asp-Gly-Glu-Glu-Lys-Val-Ala, respectively. The purified protein inhibits plasma enzyme(s) of H. roretzi, and the rate of inhibition to the plasma enzyme(s) activity was accelerated by incubation with dextran sulfate, but the effect was neutralized by further incubation with polycation, such as polybrene or protamine sulfate. The inhibitory activity was not affected appreciably by pH 7-10 but ceased completely below pH 5 or by heating at 50 degrees C for 30 min.

Amino Acid Sequence↗
PubMed1996

Amino acid sequence of the monomer subunit of the extracellular hemoglobin of the earthworm, Pheretima hilgendorfi.

The complete amino acid sequence of the monomer subunit of Pheretima hilgendorfi hemoglobin was determined: It consists of 140 amino acid residues, including a disulfide bond but no methionine, and has a molecular weight of 16,107 Da. Using computed analyses (amino acid maximum homology) with known sequences of monomer subunits of earthworm's hemoglobins, 115 (82%) were found to be identical with those in the corresponding positions of chain I (monomer subunit) of Pheretima sieboldi hemoglobin; 81 residues (55%), 71 residues (47%), and 66 residues (43%) were found to be in identical positions of the sequences of chain I of Lumbricus terrestris hemoglobin, chain I of Tubifex tubifex hemoglobin and chain I of Tylorrhynchus heterochaetus hemoglobin. Orthologous sequence data of monomer globins that belong to the strain A of annelid hemoglobins are discussed as useful clues for investigation of the divergence pattern of Pheretima species.

Amino Acid Sequence↗
PubMed1996

A comparative study on earthworm hemoglobins: an amino acid sequence comparison of monomer globin chains of two species, Pontodrilus matsushimensis and Pheretima communissima that belong to the family Megascolecidae.

The monomer subunits of giant extracellular hemoglobins from earthworms Pontodrilus matsushimensis and Pheretima communissima that belong to the family Megascolecidae, Oligochaeta, were purified by a reversed-phase column, Resource RPC, and sequenced. The complete amino acid sequences of the two monomeric globin chains were determined: 141 amino acid residues with a molecular weight of 16,366 Da for Pontodrilus matsushimensis and 140 amino acid residues with a molecular weight of 16,000 for Pheretima communissima, respectively. The Pontodrilus matsushimensis monomer globin has three cysteine residues, and the two located at positions 2 and 131 are conserved as those observed in all annelids and contribute to form a disulfide-bonded interchain. The third cysteine residue at position 73 is the first evidence for the annelid monomer globin subunits. The physiological functions of the third cysteine residue, however, are still unknown. The monomer sequences of the two species were aligned with those of five known sequences from annelids, including a polychaete, Tylorrhynchus heterochaetus, and four oligochaetes, Pheretima hilgendorfi, Pheretima sieboldi, Lumbricus terrestris and Tubifex tubifex. Using computer analysis, a 87.9% identity of the amino acid sequences between two monomeric subunits of Pheretima communissima and Pheretima hilgendorfi hemoglobins showed the highest degree of sequence similarity. A molecular phylogenetic tree of seven species of annelids has constructed, suggesting that the divergence times among the three species of Pheretima and between Pheretima and Pontodrilus were 50 to 100 and about 209 million years ago, respectively.

Amino Acid Sequence↗
PubMed1995

A case of orthologous sequences of hemocyanin subunits for an evolutionary study of horseshoe crabs: amino acid sequence comparison of immunologically identical subunits of Carcinoscorpius rotundicauda and Tachypleus tridentatus.

About 83% of the amino acid sequence of hemocyanin subunit HR6 from the Southeast Asian horseshoe crab, Carcinoscorpius rotundicauda, has been determined. There is a difference of about 43% between HR6 and complete sequences of chelicerate hemocyanin subunits from the American horseshoe crab, Limulus polyphemus, and a tarantula, Eurypelma californicum. However, the immunologically identical subunits HR6 and HT6 from Tachypleus tridentatus (Japanese horseshoe crab) show 2.7% sequence difference. Based on the amino acid sequences of HR6 and HT6, the divergence between C. rotundicauda and T. tridentatus occurred about 9.6 million years ago. In the case of horseshoe crab hemocyanin subunits, it seems that the orthologous homologues in many homologous subunits between species are immunologically detectable.

Amino Acid Sequence↗
PubMed1988

An evolutionary tree for invertebrate globin sequences.

A phylogenetic tree was constructed from 245 globin amino acid sequences. Of the six plant globins, five represented the Leguminosae and one the Ulmaceae. Among the invertebrate sequences, 7 represented the phylum Annelida, 13 represented Insecta and Crustacea of the phylum Arthropoda, and 6 represented the phylum Mollusca. Of the vertebrate globins, 4 represented the Agnatha and 209 represented the Gnathostomata. A common alignment was achieved for the 245 sequences using the parsimony principle, and a matrix of minimum mutational distances was constructed. The most parsimonious phylogenetic tree, i.e., the one having the lowest number of nucleotide substitutions that cause amino acid replacements, was obtained employing clustering and branch-swapping algorithms. Based on the available fossil record, the earliest split in the ancestral metazoan lineage was placed at 680 million years before present (Myr BP), the origin of vertebrates was placed at 510 Myr BP, and the separation of the Chondrichthyes and the Osteichthyes was placed at 425 Myr BP. Local "molecular clock" calculations were used to date the branch points on the descending branches of the various lineages within the plant and invertebrate portions of the tree. The tree divided the 245 sequences into five distinct clades that corresponded exactly to the five groups plants, annelids, arthropods, molluscs, and vertebrates. Furthermore, the maximum parsimony tree, in contrast to the unweighted pair group and distance Wagner trees, was consistent with the available fossil record and supported the hypotheses that the primitive hemoglobin of metazoans was monomeric and that the multisubunit extracellular hemoglobins found among the Annelida and the Arthropoda represent independently derived states.

Amino Acid Sequence↗
PubMed1987

Amino acid sequence of the monomer subunit of the extracellular hemoglobin of Lumbricus terrestris.

The giant extracellular hemoglobin (3,800 kDa) of the oligochaete Lumbricus terrestris consists of four subunits: a monomer (chain I), two subunits each of about 35 kDa (chains V and VI), and a disulfide-bonded trimer (50 kDa) of chains II, III, and IV. The complete amino acid sequence of chain I was determined: it consists of 142 amino acid residues and has a molecular weight of 16,750 including a heme group. Fifty-nine residues (42%) were found to be identical with those in the corresponding positions in Lumbricus chain II (Garlick, R. L., and Riggs, A. F. (1982) J. Biol. Chem. 257, 9005-9015); 45 (32%), 56 (40%), 44 (31%), and 45 (32%) residues were found to be in identical positions in the sequences of chains I, IIA, IIB, and IIC, respectively, of Tylorrhynchus heterochaetus hemoglobin (Suzuki, T., and Gotoh, T. (1986) J. Biol. Chem. 261, 9257-9267). When the sequences of all six annelid chains are compared, 18 invariant residues are found in the first 104 residues of the molecule; very little homology exists among the annelid chains in the carboxyl-terminal 38-residue region. Nine of the 18 invariant residues are also found in the human beta-globin chain.

Amino Acid Sequence↗
PubMed1987

Two globin strains in the giant annelid extracellular haemoglobins.

The constituent polypeptide chains I, II, III and IV of the giant extracellular haemoglobin of the oligochaete Lumbricus terrestris were isolated by mono Q ion-exchange chromatography and C8 reverse-phase chromatography. The N-terminal amino acid sequences of Lumbricus chains I, III and IV were determined and aligned with those of Lumbricus chain II and the four chains of the extracellular haemoglobin of the polychaete Tylorrhynchus heterochaetus. Three invariant amino acid residues, Cys-7, Val-15 and Trp-19, were found to occur in the N-terminal segments (17-22 residues) of the eight chains of Lumbricus and Tylorrhynchus haemoglobins. In addition, it was found that the eight sequences could be separated into two groups: 'A', consisting of Lumbricus chains I and II and Tylorrhynchus chains I and IIA, having invariant Lys-14 and Lys-16, and 'B', consisting of Lumbricus chains III and IV and Tylorrhynchus IIB and IIC, having invariant Cys-6, Ser-8 and Asp-11. This result suggests that there are two strains of globin chain in the annelid extracellular haemoglobins.

Amino Acid Sequence↗
PubMed1986

Preparation and characterization of monoclonal antibodies to the extracellular hemoglobin of Lumbricus terrestris.

Murine monoclonal antibodies to the extracellular hemoglobin of Lumbricus terrestris were prepared by a modification of the method of Kohler and Milstein. 224 hybridomas were found to produce antibodies which bound to the hemoglobin; they were tested for binding to the four subunits of the hemoglobin: M (chain I, 16 kDa), D1 (chain V, 31 kDa), D2 (chain VI, 37 kDa) and T (50 kDa), a disulfide-bonded trimer of chains II, III and IV, each of about 17 kDa. 150 hybridomas bound to all four subunits and 40 hybridomas bound to various combinations of subunits. The remaining 34 hybridomas combined only with the hemoglobin. The twelve hybridomas obtained after subculturing and cloning were tested for their binding to the two fractions II and III, consisting of subunits D1 + D2 + T and M, respectively, obtained by dissociation at pH 9.5 and at pH 4.0 and to the reassociated whole molecules, obtained subsequent to return to neutral pH. Eight hybridomas which combined only with the hemoglobin also combined with all the reassociated molecules but not with any of the fractions: these monoclonal antibodies probably recognize conformation-dependent antigenic sites that are present only in the hexagonal bilayer structure characteristic of the native and reassociated hemoglobin molecules. Of the remaining four hybridomas, two bound to subunit T and two combined with subunits T and D2; they also combined with the reassociated molecules and with the fractions II. In addition, the hybridomas did not bind to the hemoglobins of Tubifex, Limnodrilus, Arenicola, Tylorrhynchus and Macrobdella or to the chlorocruorins of Myxicola and Eudistylia.

Amino Acids↗
PubMed1986

A disulfide-bonded trimer of myoglobin-like chains is the principal subunit of the extracellular hemoglobin of Lumbricus terrestris.

The extracellular hemoglobin of Lumbricus terrestris (3900 kDa) consists of at least six different polypeptide chains: I through IV (16-19 kDa), V (31 kDa) and IV (37 kDa) (Vinogradov, S.N., Shlom, J.M., Hall, B.C., Kapp, O.H. and Mizukami, H. (1977) Biochim. Biophys. Acta 492, 136-155). SDS-polyacrylamide gel electrophoresis of the unreduced hemoglobin shows that chains II, III and IV form a disulfide-bonded 50 kDa subunit. This subunit was isolated by gel filtration of the hemoglobin on Sephacryl S-200 (a) at neutral pH in 0.1% SDS and (b) in 0.1 M sodium acetate buffer (pH 4.0); in the latter case it retains heme. The 50 kDa subunit obtained by method (b) was reduced and subjected to chromatofocusing on PBE 94 column: the elution pattern obtained with Polybuffer 74 (pH 4.5) and monitored at 280 nm, consisted of three peaks A, B and C; peaks A and B but not C, had absorbance at 410 nm. SDS-polyacrylamide gel electrophoresis showed that peaks A, B and C corresponded to chains II, IV and III, respectively. Amino acid analyses and N-terminal sequence determinations identified chain II as the whose primary structure had been determined (Garlick, R. and Riggs, A. (1982) J. Biol. Chem. 257, 9005-9015). Carbohydrate analysis of the native hemoglobin shows it to contain 2.0 +/- 0.5% carbohydrate consisting of mannose and N-acetylglucosamine in a mole ratio of about 9:1. The carbohydrate content of the 50 kDa subunit is 1.8 +/- 0.5%; it consists of mannose and N-acetylglucosamine in the same ratio and it appears to be associated with chain IV. Rabbit polyclonal antisera to 50 kDa subunit, prepared by method (a), and to the native hemoglobin were shown to cross-react with the hemoglobin and the 50 kDa subunit, respectively, by immunodiffusion. One of eight mouse monoclonal antibodies directed against the native hemoglobin reacted strongly with the 50 kDa subunit prepared by methods (a) and (b) in an enzyme-linked immunosorbent assay (ELISA). Another monoclonal antibody reacted strongly with the 50 kDa subunit obtained by method (b). Neither of the two hybridomas exhibited a strong reaction with any of the three constituent chains of the 50 kDa subunit. These results suggest that the unusual disulfide-bonded 50 kDa subunit, consisting of three myoglobin-like polypeptide chains of which only two have heme, is an integral part of the native Lumbricus hemoglobin molecule.

Amino Acid Sequence↗
PubMed1984

Studies on perivitelline fluid of horseshoe crab embryo. I. Purification and properties of agglutinin from the perivitelline fluid of Tachypleus gigas embryo.

Agglutinin in the perivitelline fluid (PVF) of Tachypleus gigas, horseshoe crab, embryo was isolated and purified by a combination of affinity column chromatography on Sepharose 4B coupled with bovine submaxillary gland mucin and gel-filtration of Fractogel TSK (Toyopearl) HW-60 in Tris-NaCl-CaCl2 (0.05 M Tris-HCl, pH 7.5, containing 0.5 M NaCl and 0.1 M CaCl2) buffer, containing 1 M urea. The specific activity of the purified protein was increased about 1,300 times in comparison with that of the starting material. The active protein was present in highly polymerized forms which were multimers of an identical subunit with a molecular weight of approximately 40,000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This agglutinin was shown to have multimeric activity towards different kinds of erythrocytes and its hemagglutinating activity was inhibited by N-acetylamino sugars and bovine submaxillary gland mucin containing sialic acid. Urea and guanidine-HCl inhibited the agglutinating activity but the activity recovered after dilution or dialysis, whereas the effect of HCl, NaOH, or 2-mercaptoethanol was irreversible.

Animals↗
PubMed1984

Studies on perivitelline fluid of horseshoe crab embryo. II. Purification of agglutinin-binding substance from the perivitelline fluid of Tachypleus gigas embryo.

Three glycoproteins with potent agglutinin-binding activity have been isolated from the perivitelline fluid of Tachypleus gigas, horseshoe crab, embryo. In the native form, these agglutinin-binding substances were highly aggregated. After being dissociated in 10 M urea, these proteins were fractionated by gel-filtration on a Fractogel TSK (Toyopearl) HW-60 in Tris-NaCl-CaCl2 (0.05 M Tris-HCl, pH 7.5, containing 0.5 M NaCl and 0.1 M CaCl2) containing 10 M urea. The proteins thus obtained were designated as ABS-I, -II, and -III in the order of elution and have apparent molecular weights of 25,000 (ABS-II) and 10,000 (ABS-III) as judged by both gel-filtration on Fractogel TSK (Toyopearl) HW-60 in 10 M urea and sodium dodecyl sulfate-gel electrophoresis; the molecular weight of ABS-I could not be estimated in the two systems since it was too high. ABS-I, -II, and -III, of which only ABS-I is water-soluble, inhibit one hemagglutination unit of activity with minimum quantities of 0.5 micrograms/ml, 7.8 micrograms/ml, and 1.0 micrograms/ml, respectively. They were found to be glycoproteins in which 6.6% of the dry weight (ABS-I), 4.2% of the dry weight (ABS-II), and 7.5% of the dry weight (ABS-III) were carbohydrate. The dry weight ratio of hexosamines in these substances is 3:1:2 (ABS-I: ABS-II: ABS-III), and that of sialic acid is also 3:1:2. Amino acid analyses of these proteins indicated that they have high contents of aspartic acid, glutamic acid, and glycine in common.

Amino Acids↗
PubMed1983

Agglutinins in the horseshoe crab hemolymph: purification of a potent agglutinin of horse erythrocytes from the hemolymph of Tachypleus tridentatus, the Japanese horseshoe crab.

Agglutinins from Tachypleus (Tachypleus tridentatus, the Japanese horseshoe crab) hemolymph were isolated by affinity chromatography on BSM-coupled Sepharose 4B. The agglutinins showed multiple species and were composed of eight heterogeneous subunits with molecular weights of 45,000, 42,000, 41,000, 39,000, 33,000, 29,000, 27,000, and 22,000 as determined by SDS-polyacrylamide gel electrophoresis. The affinity-isolated agglutinins were fractionated into four groups by gel filtration on a Fractogel TSK (Toyopearl) HW-65 column, and these were designated as Tachypleus tridentatus agglutinin (TTA)-I, -II, -III, and -IV in the order of elution. These agglutinins were demonstrated to be heterogeneous as judged by their specificity towards horse erythrocytes, subunit structures, and immunological properties. TTA-III showed a potent agglutination activity towards horse erythrocytes and was further purified by gel filtration on a Cellulofine GC-700 column. The purified TTA-III is a highly purified (46,000-fold) protein composed of homogeneous subunits (Mr, 42,000) as judged by SDS-polyacrylamide gel electrophoresis and immunological analysis.

Animals↗
PubMed1983

Coagulogens from four living species of horseshoe crabs (Limulidae): comparison of their biochemical and immunochemical properties.

Coagulogens from four kinds of horseshoe crabs (Limulus polyphemus (LP), Tachypleus tridentatus (TT), Tachypleus gigas (TG), and Carcinoscorpius rotundicauda (CR)) were compared in biochemical and immunochemical properties. On SDS-polyacrylamide gel electrophoresis, the reduced coagulogens had a common apparent molecular weight of 20,000, whereas the molecular weights of unreduced samples were 24,000 for the LP protein and 21,000 for the proteins of the three Asian species (TT, TG, and CR). Gel-filtration yielded apparent molecular weights of 19,500 for the LP protein and 15,500 for the other proteins. All coagulogens consisted of 173-175 amino acid residues and their compositions were very similar to one another, e.g., no methionine, and high proportions of basic amino acids, cystine, valine, and phenylalanine. The amino-terminal 14-residue sequence of the LP coagulogen was Gly-Asp-Pro-Asn-Val-Pro-Thr-Cys-Leu-Cys-Glu-Glu-Pro-Thr. Those of the other coagulogens, Ala-Asp-Thr-Asn-Ala-Pro-Ile-Cys-Leu-Cys-Asp-Glu-Pro-Gly, were identical to one another except for the amino-terminal aspartic acid residue of the TG protein and leucine of the CR protein. The carboxyl-terminal residues of the coagulogens were identified as serine (LP), phenylalanine (TT and TG), and tyrosine (CR), respectively. An antiserum to the LP coagulogen produced a single precipitin line against the LP protein but none against the Asian coagulogens. On the other hand, each of the antiserums to TT, TG, and CR coagulogens yielded a precipitin line against the Asian proteins but gave no line against the LP protein.

Amino Acid Sequence↗