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31.
Sadaaki Iwanaga Takashi Morita Toshiyuki Miyata Takanori Nakamura Jun Aketagawa 《Journal of Protein Chemistry》1986,5(4):255-268
A hemocyte lysate from horseshoe crab produced a gel, when exposed to Gram-negative bacterial endotoxins. This gelation reaction of the lysate, so-called Limulus test, has been widely employed as a simple and very sensitive assay method for endotoxins. Recent biochemical studies on the principle of Limulus test indicate that the hemocytes contain several serine protease zymogens, which constitute a coagulation cascade triggered by endotoxins, and that there is a (1 3)--d-glucan-mediated coagulation pathway which also results in the formation of gel. Up to now, six protein components, designated coagulogen, proclotting enzyme, factor B, factor C, factor G and anti-LPS factor, all of which are closely associated with the endotoxin-mediated coagulation pathway, have been purified and biochemically characterized. Among these components, the complete amino acid sequences of coagulogens isolated from one American and three Asian species of horseshoe crabs have been established. Moreover, the reconstitution experiment using the isolated clotting factors, C, B, proclotting enzyme and coagulogen in the presence of endotoxin, leads to the formation of coagulin get. Based on these results, we propose here a mechanism for the Limulus coagulation cascade. 相似文献
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S Lal M Mitsuyama N Ogata M Miyata S Miake K Nomoto 《Journal of general microbiology》1983,129(1):93-98
Mice infected with Mycobacterium bovis, BCG, were shown to be highly susceptible to subsequent challenge with Pseudomonas aeruginosa. The susceptibility was characterized by the enhanced mortality and shortened survival after challenge with P. aeruginosa. BCG-treated mice did not show any enhanced susceptibility to challenge with Gram-positive bacteria such as Staphylococcus aureus or Listeria monocytogenes. BCG-treated mice eliminated P. aeruginosa from their organs in a pattern similar to that in untreated mice. There was no significant difference in the bactericidal activities of polymorphonuclear cells and macrophages between BCG-treated and untreated mice. An equal amount of endotoxin was detected by the Limulus lysate assay in the blood of both BCG-treated and untreated mice after challenge with P. aeruginosa. The enhanced susceptibility induced by BCG pretreatment could be decreased when the mice were rendered LPS-tolerant by injections of small amounts of LPS. These results suggest that BCG-induced susceptibility to P. aeruginosa can be ascribed to an enhanced susceptibility to the lethal effect of LPS produced by challenge bacteria, and not to the impairment of the ability to eliminate infected bacteria. 相似文献
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What causes the aphid 28S rRNA to lack the hidden break? 总被引:1,自引:0,他引:1
38.
Primary structure of hemorrhagic protein, HR2a, isolated from the venom of Trimeresurus flavoviridis 总被引:5,自引:0,他引:5
T Miyata H Takeya Y Ozeki M Arakawa F Tokunaga S Iwanaga T Omori-Satoh 《Journal of biochemistry》1989,105(5):847-853
The complete amino acid sequence and disulfide bridge location of HR2a, one of the hemorrhagic proteins isolated from the snake venom of Trimeresurus flavoviridis, have been determined by analysis of peptides derived from digests with cyanogen bromide, lysyl endopeptidase, trypsin, and Staphylococcus aureus V8 protease. Peptides were purified by gel filtration followed by reversed-phase HPLC. HR2a has the amino-terminal sequence of less than Glu-Gln-Arg- and consists of a total of 202 residues with a calculated molecular weight of 23,015. Sequence analysis indicates the presence of another isoform which lacks the amino-terminal residue, making 201 amino acid residues with a molecular weight of 22,887. Three disulfide bridges of HR2a link Cys-118 to Cys-197, Cys-159 to Cys-181, and Cys-161 to Cys-164. HR2a contains a segment which is similar to the zinc-chelating sequences found in thermolysin and several mammalian metalloproteinases, suggesting that HR2a is a metalloproteinase with limited substrate specificity. However, there is no other significant sequence homology with thermolysin except for the zinc-ligand region. 相似文献
39.
Overexpression and purification of the recombinant Ca2+-binding protein, apoaequorin 总被引:3,自引:0,他引:3
The small, monomeric Ca2+-binding photoprotein, aequorin, emits blue light by an intramolecular reaction when mixed with Ca2+. The photoprotein is made up of coelenterazine and molecular oxygen, bound noncovalently to apoaequorin (apoprotein). The chemical steps leading to light emission, involving the oxidative degradation of coelenterazine, have been studied extensively, but little is known about the active site and how the molecule catalyzes the oxidation of coelenterazine. The three-dimensional structure of the protein has not been determined and therefore answers to these questions have remained unavailable. The present paper describes a procedure for preparing fairly large amounts of apoaequorin and aequorin for X-ray crystallographic studies. It consists of fusing the apoaequorin cDNA to the signal peptide coding sequence of the outer membrane protein A of Escherichia coli, which is under the control of the lipoprotein promoter. When the cDNA was expressed in E. coli, a large excess of the recombinant protein was produced and released into the culture medium. Purification of the protein was accomplished by acid precipitation and DEAE-cellulose chromatography. The procedure yielded 7.4 mg of recombinant apoaequorin with a purity greater than 95% from 200 ml of culture medium. On regeneration with coelenterazine, the recombinant aequorin was fully active with Ca2+. 相似文献
40.
K Suehiro S Kawabata T Miyata H Takeya J Takamatsu K Ogata T Kamiya H Saito Y Niho S Iwanaga 《The Journal of biological chemistry》1989,264(35):21257-21265
Factor IX BM Nagoya (IX Nagoya) is a natural mutant of factor IX responsible for severe hemophilia B. A patient with this mutant is characterized by a markedly prolonged ox brain prothrombin time. IX Nagoya was purified from the patient's plasma by immunoaffinity chromatography with an anti-factor IX monoclonal antibody column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that treatment of IX Nagoya with factor XIa/Ca2+ resulted in cleavage only at the Arg145-Ala146 bond. Reversed-phase high performance liquid chromatography of a trypsin digest of IX Nagoya showed an aberrant peptide, which was further digested with proteinase Asp-N. Primary structure analysis of one of the Asp-N peptides revealed that Arg180 is replaced by Trp. An essentially complete (99%) amino acid sequence of IX Nagoya was obtained by sequencing fragments derived from a lysyl endopeptidase digest in which no other substitutions in the catalytic triad or substrate binding site were found. We also found that IX Nagoya is activated by alpha-chymotrypsin or rat mast cell chymase by monitoring the rate of factor X activation using a fluorogenic peptide substrate in the presence of factor VIII, phospholipids, and Ca2+. These results indicate that the substitution of Arg180 by Trp impairs the cleavage by factor XIa required for activation of this zymogen and that the substitution causes hemophilia BM. 相似文献