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S Iwanaga

Publications and source records attributed to S Iwanaga.

At least 289 records · Page 16Linked to original sources
PubMed1986

Structure-function relationships of phospholipases. II: Charge density distribution and the myotoxicity of presynaptically neurotoxic phospholipases.

The charge density distribution of 24 phospholipases A2 has been examined to identify the involvement of charged amino acid residues in the determination of the pharmacological properties of these proteins. There is no characteristic difference between the presynaptically neurotoxic and non-neurotoxic phospholipases, however, presynaptically neurotoxic phospholipases which are also myotoxic have a distinct charge distribution pattern. There is a characteristic cationic site around residues 79-87. This site has a relatively fixed position with respect to the hydrophobic 'neurotoxic' region, on the NH2 terminal side. This cationic region is located on the outer surface in the three-dimensional structure of phospholipase, just before hydrophobic helix E, and is available for interaction with membranes. Such a characteristic region is absent in non-myotoxic phospholipases which are either presynaptically active or inactive. On the other hand, myotoxins, a group of non-enzymatic proteins inducing myotoxicity, also possess such characteristic regions of cationic and hydrophobic sites.

Animals↗
PubMed1986

Comparison of amino terminal region of three isoenzymes of phospholipases A2 (TFV PL-Ia, TFV PL-Ib, TFV PL-X) from Trimeresurus flavoviridis (habu snake) venom and the complete amino acid sequence of the basic phospholipase, TFV PL-X.

Three phospholipases (PLA2s) isolated from Japanese habu snake (Trimeresurus flavoviridis) venom differ from each other in the physiological symptoms induced in animals. The amino acid compositions and amino terminal sequences of these isoenzymes have been compared. TFV PL-Ia and TFV PL-Ib resemble each other very closely, but TFV PL-X has a distinctly different amino acid composition and amino terminal region. The complete amino acid sequence of the basic PLA2, TFV PL-X, was determined by sequencing the four peptides obtained by the cleavage of Asp-Pro bonds. The overlapping peptide was obtained by cleaving tryptophanyl bond and the carboxy terminal region was determined by sequencing the chymotryptic peptide. TFV PL-X consisted of a single chain of 122 amino acid residues, including 14 half-cystine residues. It is a group II A PLA2 and is homologous with the phospholipase super family. However, it has a distinctly different sequence compared with the other PLA2s from the venom of Trimeresurus species.

Amino Acid Sequence↗
PubMed1986

Structure-function relationships of phospholipases. I: Prediction of presynaptic neurotoxicity.

The hydropathic indexes of 24 phospholipases have been calculated from their amino acid sequences. The presynaptic neurotoxic potential of venom phospholipases can be predicted by the use of hydropathy profiles. The presynaptically acting phospholipases have a distinct hydrophobic region around the residues 80-110 which is probably involved in the interaction with the presynaptic membranes. This region is present as a separate helix in the tertiary structure of phospholipases. Such a hydrophobic region is absent in non-neurotoxic phospholipases.

Amino Acid Sequence↗
PubMed1986

Two types of coagulogen mRNAs found in horseshoe crab (Tachypleus tridentatus) hemocytes: molecular cloning and nucleotide sequences.

The complete cDNA sequence coding for the coagulogen present in the horseshoe crab (Tachypleus tridentatus) hemocytes was determined. Clones carrying cDNA fragments for coagulogen were isolated from a cDNA library of the hemocyte mRNA using synthetic oligodeoxyribonucleotides as probes. The nucleotide sequence analyses of the cloned cDNAs revealed that the hemocyte coagulogen consists of 175 amino acids with 20 amino acids in a presegment, and that there are two types of mRNAs for coagulogen. The two mRNAs exhibited three nucleotide substitutions, two of which were in their protein-coding regions, resulting in two amino acid replacements. Subsequently, two molecular species of coagulogen, named coagulogens type I and type II, were identified by tryptic peptide mapping of the mature proteins isolated from the hemocyte lysate. These results suggest that the two types of coagulogens are first synthesized as preproteins and are incorporated into the granules that are abundantly present in the hemocytes with liberation of the signal peptides.

Amino Acid Sequence↗
PubMed1986

Complete amino acid sequence of NADH-cytochrome b5 reductase purified from human erythrocytes.

The complete amino acid sequence of soluble NADH-cytochrome b5 reductase purified from human erythrocytes was determined. The enzyme, which contained 8 methionine residues, was cleaved by cyanogen bromide. The resulting nine peptides were separated by gel filtration and purified further by high-performance liquid chromatography. The purified peptides were sequenced by automated Edman degradation. Three large CNBr peptides, residues 1-101, 109-151, and 169-231, were further fragmented with trypsin, Staphylococcus aureus V8 protease or a lysyl endopeptidase of Achromobacter lyticus. The peptides obtained from the tryptic digest of citraconylated FAD-depleted apoprotein completed the alignments of the other peptides. The enzyme was composed of 275 amino acid residues. The 4 functionally important cysteine residues were located in the COOH-terminal portion. The molecular weight of the protein was calculated to be 31,260 without FAD. A prediction of the secondary structure was made by the method of Chou and Fasman. The protein was hydrophilic as a whole (43% polarity), but some regions were rich in hydrophobic residues. From the sequence homology of this enzyme with the pyridine nucleotide-binding sites of other flavoproteins, three candidates for the FAD and NADH-binding domains were suggested.

Amino Acid Sequence↗
PubMed1986

Proteolytic cleavage of vitamin K-dependent bovine plasma protein S by alpha-thrombin and plasma serine protease.

It is known that protein S, a vitamin K-dependent plasma protein, isolated from a human source, gives a closely spaced doublet on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reduction and that this heterogeneity in molecular size results from a limited proteolysis of protein S mediated by alpha-thrombin in human species. We found here that alpha-thrombin also rapidly converted single-chain bovine protein S to a nicked form, which consisted of the NH2-terminal segment containing gamma-carboxyglutamic acid and the COOH-terminal large segment bridged by a disulfide linkage(s). These two segments were isolated and chemically characterized after S-alkylation of the nicked protein S. The results suggest that the alpha-thrombin-catalyzed hydrolysis of protein S probably occurs at a peptide linkage (Arg-Ser) located in the NH2-terminal portion. The conversion of single-chain protein S to the nicked form was also mediated by plasma kallikrein and plasmin, in addition to alpha-chymotrypsin and trypsin. However, the alpha-thrombin-catalyzed conversion did not occur when calcium ions were added to the reaction mixture.

Animals↗
PubMed1986

Purification and properties of intracellular clotting factor, factor B, from horseshoe crab (Tachypleus tridentatus) hemocytes.

An intracellular clotting factor, factor B, which is closely associated with the hemolymph coagulation system of horseshoe crab (Tachypleus tridentatus), was purified and characterized. The purified preparation gave a single band (Mr = 64,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the absence of 2-mercaptoethanol, while three bands (Mr = 64,000, 40,000, and 25,000) were detected on SDS-PAGE after reduction. This preparation was converted by limulus clotting factor C to an activated form, factor B, with Mr = 56,000 consisting of a heavy chain (Mr = 32,000) and a light chain (Mr = 25,000) bridged by disulfide linkage(s). The factor B, which was produced separately by treating the partially purified factor B with factor C, was also purified. It gave a single band on unreduced SDS-PAGE and two bands on reduced SDS-PAGE. The purified factor B had Mr of 56,000 consisting of a heavy chain (Mr = 32,000) and a light chain (Mr = 25,000). These results indicated that the purified factor B zymogen is a mixture of single-chain and two-chain forms, both of which have the same molecular weight of 64,000, and that these two forms are converted to factor B by factor C. The diisopropyl phosphorofluoridate-sensitive site of factor B was found in the heavy chain. The reconstitution studies using purified factor C, factor B, proclotting enzyme and coagulogen in the presence of lipopolysaccharide indicated that factor B is an essential component to complete sequential activation of the limulus clotting system, and that it specifically activates proclotting enzyme to the active clotting enzyme.

Amino Acids↗
PubMed1986

Isolation and amino acid sequence of a peptide containing an epoxide-reactive residue from the thermolysin-digest of Scytalidium lignicolum acid protease B.

Scytalidium lignicolum acid protease B, a pepstatin-insensitive acid protease, was modified by 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) with the concomitant loss of its enzyme activity, and an EPNP-labeled peptide was isolated from the thermolysin-digest of the modified enzyme by HPLC. The amino acid sequence of the peptide was determined to be Ile-Leu-Glu-Thr-Gly, which corresponds to the sequence of residue Nos. 51-55 of the enzyme. The results of treatment of the labeled peptide with hydroxylamine suggested that the EPNP moiety is ester-linked to Glu53 of the enzyme. The amino acid sequence around Glu53 of the acid protease B showed high homology with those around the active site Asp residues of calf chymosin and porcine pepsin. These results show that it is highly possible that Glu53 of the acid protease B is one of the amino acid residues involved in its catalytic activity.

Amino Acid Sequence↗
PubMed1986

Identification of the plasminogen-binding site of human alpha 2-plasmin inhibitor.

A plasminogen-binding site of human alpha 2-plasmin inhibitor was studied. The chromatogram of digest from the amidinated alpha 2-plasmin inhibitor (67K-daltons, plasminogen-binding form) with trypsin was almost identical with that obtained from the 65K-daltons derivative (non-plasminogen-binding form) treated with the same procedure, except for the three tryptic peptides. One of the three peptides, the deamidinated peptide T-11, was found to have a strong ability to inhibit the interaction of alpha 2-plasmin inhibitor with human plasmin. Moreover, the dissociation constant Kd for interaction between the peptide T-11 and plasmin was estimated to be 5.5 microM, indicating that Kd is about 10-fold lower than that of epsilon-aminocaproic acid. The sequence of the peptide T-11 was determined by the Edman method as follows: NH2-G-D-K-L-F-G-P-D-L-K-L-V-P-P-M-E-E-D-Y-P-Q-F-G-S-P-K-COOH. alpha 2-Plasmin inhibitor and its 65K-daltons derivative were found to have the same NH2-terminal sequence of Asn(Asp)-Gln-Glu-Gln-. These results indicated that the plasminogen-binding site(s) of alpha 2-plasmin inhibitor could be located in the COOH-terminal region of its molecule and that some of epsilon-NH2-groups in the deamidinated peptide T-11 may be involved in the lysine-binding site(s) of plasmin(ogen).

Amino Acid Sequence↗
PubMed1986

Difference in enzymatic properties between "staphylothrombin" and free alpha-thrombin.

Bacterial protein, staphylocoagulase, binds stoichiometrically to human prothrombin resulting in a coagulant complex, staphylothrombin. The enzymatic properties of staphylothrombin differ from those of alpha-thrombin in their substrate specificities toward natural and synthetic substrates, in addition to their interaction with protease inhibitors. In order to obtain information about the region of staphylocoagulase that interacts with human prothrombin, staphylocoagulase was cleaved by alpha-chymotrypsin. This limited alpha-chymotryptic cleavage of staphylocoagulase yielded three large fragments, fragments of 43, 30, and 20 kDa. The 43-kDa fragment exhibited a high affinity for human prothrombin (Kd = 1.7 nM), which is comparable to the affinity observed using intact staphylocoagulase (Kd = 0.46 nM). A complex of the 43-kDa fragment and prothrombin possessed both clotting and amidase activities essentially identical to those observed in a complex of intact staphylocoagulase and prothrombin. The 30-kDa fragment exhibited weaker affinity for prothrombin (Kd = 120 nM). While a complex of this fragment and prothrombin did not exhibit clotting activity, it nonetheless possessed a weak amidase activity. The 20-kDa fragment was found only to bind to prothrombin. The NH2-terminal sequence analyses of these fragments revealed that the 43-kDa fragment constitutes the NH2-terminal portion of staphylocoagulase, and contains the 30-kDa and 20-kDa fragments. It is therefore concluded that the functional region of staphylocoagulase for binding and activation of prothrombin is localized in the NH2-terminal region of the intact protein. The 43-kDa fragment contains 324 amino acids with a molecular weight of 38,098. The 43-kDa fragment has an unusual amino acid composition based on the sequence, in which the sum of Asp (28 residues), Asn (22), Glu (35), Gln (9), and Lys (52) residues accounts for more than 45% of the total residues. A comparison of the amino acid sequence of the 43-kDa fragment with that of streptokinase did not reveal any obvious sequence homology. There was also no sequence homology with those of trypsin, alpha-chymotrypsin, and elastase.

Binding Sites↗
PubMed1985

Tryptase from rat mast cells converts bovine prothrombin to thrombin.

The effect of tryptase purified from rat peritoneal mast cells on bovine prothrombin was examined. Tryptase activated prothrombin, as evidenced by the increase in thrombin activity with a synthetic substrate, t-butyloxy-carbonyl-Val-Pro-Arg-4-methylcoumaryl-7-amide. The apparent Km value toward bovine prothrombin and the kcat value were 2.3 microM and 46.3 s-1, respectively. Studies on the time course of prothrombin activation by tryptase and by activated factor X (Xa), and analysis of the activation products on sodium dodecyl sulfate gel electrophoresis showed that the process of activation of prothrombin by tryptase was similar to that by Xa except that an intermediate of 67,000 daltons was formed.

Alpha-Globulins↗
PubMed1985

Identification of T-kininogen in high and low molecular weight kininogens deficient rat (brown Norway Katholiek strain).

Kinin release in Brown Norway Katholiek (B/N-Ka) rat plasma was compared with those of Brown Norway Kitasato and Sprague-Dawley rats by treating with rat plasma kallikrein, rat urinary kallikrein, snake venom kininogenase and trypsin. B/N-Ka rat plasma yielded no detectable amount of kinin by either plasma kallikrein, urinary kallikrein or snake venom kininogenase, but yielded variable amount of kinin by trypsin. The released kinin was proved to be isoleucylseryl-bradykinin by high performance liquid chromatography and bioassay profiles. B/N-Ka rat plasma formed a precipitation line against antiserum to T-kininogen, but no line against antiserum to HMW kininogen-light chain.

Animals↗
PubMed1985

Purification and structure of the intact Ada regulatory protein of Escherichia coli K12, O6-methylguanine-DNA methyltransferase.

The ada gene of Escherichia coli K12, the regulatory gene for the adaptive response of bacteria to alkylating agents, was cloned and placed under the control of the lac regulatory region on a multicopy runaway plasmid, thereby yielding a hybrid plasmid pYN3059. Ada protein with a molecular weight of about 38,000 was overproduced when cells harboring pYN3059 were incubated at 42 degrees C in the presence of a lac inducer, isopropyl-beta-D-thiogalactoside. Taking advantage of overproduction of Ada protein, we purified the protein to apparent physical homogeneity. The purified 38,000-dalton Ada protein transferred the methyl group from the O6-methylguanine residue of alkylated DNA to the Ada protein, per se. Although the Ada protein was degraded to smaller polypeptides when crude extracts or partially purified preparations were incubated in a high ionic-strength buffer at neutral pH, the purified Ada protein remained stable under the same conditions, indicating that the Ada protein may not undergo autodegradation. An amino-terminal sequence and total amino acid composition of the purified Ada protein were in accord with nucleotide sequence of the ada gene, determined by the dideoxy method using M13 phage. It was deduced that Ada protein comprises 354 amino acids and its molecular weight is 39,385. The promoter for the ada gene was determined by S1 nuclease mapping.

Amino Acid Sequence↗