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Biomedical subjects

K Fujikawa

Publications and source records attributed to K Fujikawa.

At least 253 records · Page 14Linked to original sources
PubMed1983

Amino acid sequence of human beta-factor XIIa.

Human factor XII was activated by limited proteolysis with trypsin, and the resulting beta-factor XIIa (Mr = 30,000) was isolated by DEAE-Sephacel column chromatography. The complete amino acid sequence of beta-factor XIIa was then determined on peptides produced by enzymatic digestion with either trypsin, chymotrypsin, or Staphylococcus aureus V8 protease and by chemical cleavage at methionyl and tryptophyl bonds. beta-Factor XIIa is a glycoprotein composed of a heavy chain (243 amino acid residues) and a light chain (9 amino acid residues), and these two chains are held together by a disulfide bond. The carbohydrate is attached to asparagine residue 61 in the heavy chain. The amino acid sequence of the heavy chain shows a high degree of homology to the corresponding regions of other plasma serine proteases, such as plasmin, thrombin, factor IXa and factor Xa, as well as the pancreatic digestive enzymes. These results demonstrate that factor XII is the precursor of a typical serine protease that participates in the coagulation cascade.

Amino Acid Sequence↗
PubMed1983

The occurrence of beta-hydroxyaspartic acid in the vitamin K-dependent blood coagulation zymogens.

Previous work has shown that two vitamin K-dependent plasma zymogens, factor X and protein C, each contain one residue of erythro-beta-hydroxyaspartic acid. In the present study, prothrombin, factor VII and factor IX were subjected to amino acid analyses for beta-hydroxyaspartic acid. Factor IX and factor VII each contain one residue of erythro-beta-hydroxyaspartic acid. Edman sequence analyses revealed that this residue occurs at position 64 in human and bovine factor IX. Inasmuch as the nucleotide sequence codes for aspartic acid at this position, it appears highly likely that beta-hydroxyaspartic acid is formed in these proteins by a post-translational hydroxylation of aspartic acid. In contrast, neither human nor bovine prothrombin contain beta-hydroxyaspartic acid.

Amino Acid Sequence↗
PubMed1983

Complete amino acid sequence of the light chain of human blood coagulation factor X: evidence for identification of residue 63 as beta-hydroxyaspartic acid.

The complete amino acid sequence of the light chain of human blood coagulation factor X has been determined by automated Edman degradation of peptides isolated from chemical and enzymatic digests of the carboxymethylated light chain. The protein consists of 139 amino acid residues, which include 11 residues of gamma-carboxyglutamic acid. The first 100 residues of the human factor X light chain exhibit approximately 80% homology when compared to the amino-terminal sequence of bovine factor X light chain. This homology decreases to approximately 50% in the remaining 39 residues of the carboxyl-terminal region of the protein. Proton nuclear magnetic resonance spectroscopy and mass spectrometry analyses of isolated residue 63 identified this residue as L-erythro-beta-hydroxyaspartic acid, a hitherto unrecognized amino acid in proteins. Evidence is also presented for the presence of this residue in the corresponding regions of the light chains of bovine factor X and bovine protein C. The biological function of beta-hydroxyaspartic acid in these proteins is unknown.

Amino Acid Sequence↗
PubMed1983

The structures of the carbohydrate moieties of bovine blood coagulation factor IX (Christmas factor).

Bovine blood coagulation factor IX (Christmas factor) contains four asparagine-linked sugar chains in one molecule. The sugar chains were quantitatively liberated as radioactive oligosaccharides from the polypeptide moiety by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. The structures of these sugar chains were determined by sequential exoglycosidase digestion in combination with methylation analysis. Bovine factor IX contained two unique penta- and tetrasialyl triantennary sugar chains with the structures shown below in addition to tetra-, tri-, and disialyl biantennary sugar chains of Sia alpha 2 leads to 3 Gal beta 1 leads 3(Sia alpha 2 leads to 6)GlcNAc beta 1 leads to 2Man alpha 1 leads to 6[Sia alpha 2 leads to 3Gal beta 1 leads to 3(Sia alpha 2 leads to 6)GlcNac beta 1 leads to 2Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc, Sia alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6[Sia alpha 2 leads to 3Gal beta 1 leads to 3(Sia alpha 2 leads to 6)GlcNAc beta 1 leads to 2Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc, and Sia alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(Sia alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc and their partially desialized forms.

Acetylation↗
PubMed1983

Comparative induction of somatic eye-color mutations and sex-linked recessive lethals in Drosophila melanogaster by tryptophan pyrolysates.

The mutagenicities of the products of pyrolysis of tryptophan, Trp-P-1 and Trp-P-2, on Drosophila melanogaster were examined by measuring the effects of these compounds in inducing recessive lethals and somatic eye-color mutations. Since negative results have already been obtained by the standard procedure in males, Trp-P-1 and Trp-P-2 (0.75 to 6 mg/ml) in sucrose solution were given to females for assay of recessive lethal mutations in X-chromosomes. These compounds caused a marginal increase above the control level in the mutation frequency. For the assay of effects on somatic eye-color mutations, Trp-P-1 (200 and 400 ppm) and Trp-P-2 (400 and 800 ppm) were fed to male larvae of a tester strain carrying a genetically unstable marker set of z and w+ on the X-chromosome. These compounds caused dose-dependent increases above the control level in somatic eye-color mutations in adults. It is concluded that, under the conditions used, the somatic eye-color mutation system was more sensitive than the recessive lethal system to the mutagenic effects of tryptophan pyrolysates.

Animals↗
PubMed1983

Enzymological aspects of blood coagulation.

Recent studies on the mechanism of initiation and regulation of blood coagulation are reviewed. In the intrinsic blood coagulation pathway, factor XII, prekallikrein (or factor XI) and high molecular weight kininogen from a complex on an anionic surface, such as exposed subendothelium at the site of vascular trauma. In complex, zymogen factor XII activates prekallikrein (or factor XI) by limited proteolysis to initiate the coagulation cascade. A similar initiating mechanism may be operative in the extrinsic pathway, where zymogen factor VII, complexed with a lipoprotein (tissue factor) and calcium ions, converts factor X to factor Xa. Factor Xa converts prothrombin to thrombin which converts soluble fibrinogen to an insoluble fibrin network which physically arrests the flow of blood from the damaged vasculature. In addition, thrombin converts protein C to activated protein C. Activated protein C functions as a negative regulator in the coagulation process by degrading factor VIIIa and factor Va.

Blood Coagulation↗
PubMed1981

Mapping the active sites of bovine thrombin, factor IXa, factor Xa, factor XIa, factor XIIa, plasma kallikrein, and trypsin with amino acid and peptide thioesters: development of new sensitive substrates.

The subsite specificities of bovine factor IXa, factor Xa, factor XIa, factor XIIa, thrombin, plasma kallikrein, and trypsin were mapped with amino acid, dipeptide, and longer peptide thioester substrates. Each substrate contained a P1 Arg residue. The P1' residues included thiol residues which are analogues of valine, leucine, and isoleucine, respectively, and the P2 residue included 12 representative amino acid residues. Longer substrates with the sequence at the antithrombin III reactive site and at the zymogen activation site of various coagulation factors were also studied. The enzymatic hydrolysis of the thioesters was measured in the presence of 4,4'-dithiodipyridine which provides a very sensitive assay for the free thiol. The thioesters were excellent substrates for the coagulation factors studied, and the kcat/Km values for the best thioester substrates were higher than those previously reported for most of these enzymes. Thrombin and plasma kallikrein were the most active of the coagulation factors toward the thioester substrates. The best substrate for thrombin was Z-Gly-Arg-SCH2C6H5, although substrates containing proline in the P2 position were also quite effective. Some of the better substrates for plasma kallikrein had a P2 Phe or Trp residue. Factor IXa was the least reactive of the coagulation factors and hydrolyzed only four of the dipeptide thioesters. Substrates with bulky hydrophobic groups such as Phe or Trp in the P2 position were the most reactive with factor IXa. Factor Xa hydrolyzed all the thioester substrates tested, the most reactive being Z-Gly-Arg-SCH2C6H5. This is consistent with the fact that glycine and arginine are present in the P2 and P1 positions, respectively, of the factor Xa sensitive bonds in prothrombin which is the physiological substrate for factor Xa. Bovine factor XIa showed the least amount of specificity of the various coagulation factors and was quite reactive toward all of the thioester substrates. The most sensitive substrate for this enzyme was also Z-Gly-Arg-SCH2C6H5. Factor XIIa preferred the dipeptide with a P2 Phe, although the simpler thioester Z-Arg-SCH2CH(CH3)2 was more reactive. Trypsin hydrolyzed all of the thioester substrates at a high rate and showed little substrate specificity. With all enzymes studied, extension of the thioester substrate beyond P2 or the P1' thiol leaving group did not lead to an improvement in hydrolysis. Due to their high kcat/Km values and the ease of detecting the thiol leaving group, thioester substrates should be extremely useful for future studies of coagulation proteases.

Amino Acids, Sulfur↗