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Biochemical principle of Limulus test for detecting bacterial endotoxins.

Iwanaga S - Proc. Jpn. Acad., Ser. B, Phys. Biol. Sci. (2007)

Bottom Line: This gelation reaction of the lysate, so-called Limulus test, has been widely employed as a simple and very sensitive assay method for endotoxins.The molecular structures of these proteins have also been elucidated.Moreover, the reconstitution experiments using the isolated clotting factors, factor C, factor B, proclotting enzyme and coagulogen in the presence of endotoxin, leads to the formation of coagulin gel.

View Article: PubMed Central - PubMed

Affiliation: The Chemo-Sero-Therapeutic Research Institute, 1-6-1 Okubo, Kumamoto 812-8581, Japan . ; Research adviser.

ABSTRACT
A hemocyte lysate from horseshoe crab (Limulus) produced a gel, when exposed to Gram-negative bacterial endotoxins, lipopolysaccharides (LPS). 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)-β-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, and factor G, all of which are closely associated with the endotoxin-mediated coagulation pathway, have been purified and biochemically characterized. The molecular structures of these proteins have also been elucidated. Moreover, the reconstitution experiments using the isolated clotting factors, factor C, factor B, proclotting enzyme and coagulogen in the presence of endotoxin, leads to the formation of coagulin gel. Here, I will focus on the biochemical principle of Limulus test for detecting bacterial endotoxins, and its activation and regulation mechanism on the LPS-mediated coagulation cascade.

No MeSH data available.


Related in: MedlinePlus

Hypothetical mechanism of coagulogen gel formation.10) Upon gelation of coagulogen (pink) by a horseshoe crab clotting enzyme, peptide C (green) is released from the inner portion of the parent molecules. The resulting coagulin (yellow) monomer may selfassemble to form the dimer, trimer, and multimers. The background of this figure shows a fiber-like coagulin gel.
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f5-83_110: Hypothetical mechanism of coagulogen gel formation.10) Upon gelation of coagulogen (pink) by a horseshoe crab clotting enzyme, peptide C (green) is released from the inner portion of the parent molecules. The resulting coagulin (yellow) monomer may selfassemble to form the dimer, trimer, and multimers. The background of this figure shows a fiber-like coagulin gel.

Mentions: A stereo view of the coagulogen monomer, showing the A chain, peptide C, and B chain, and the secondary structure is presented in Fig. 4. The coagulogen monomer is an elongated molecule of approximate dimensions, 60×30×20Å. The structure is mainly dominated by the β-strands in blue, labeled sequentially B1 to B6. The multiple coils and turns of the B chain are colored green. Short helical segments in orange, in the B chain are in the background. The mainly α-helical peptide C, in red, covers a reasonable part of the top surface. The NH2-terminal A chain, in violet, is connected to the B chain via two disulfide bridges colored yellow. The whole cysteines comprise 8 disulfide bridges. The peptide C helix covers an extended hydrophobic core, which becomes accessible upon the cleavage and release of peptide C. Thus, the uncovered cove newly exposed in one molecule after the release of peptide C might interact with a hydrophobic edge of a second molecule, like a head to tail, to form a multimer, as shown in Fig. 5.38)


Biochemical principle of Limulus test for detecting bacterial endotoxins.

Iwanaga S - Proc. Jpn. Acad., Ser. B, Phys. Biol. Sci. (2007)

Hypothetical mechanism of coagulogen gel formation.10) Upon gelation of coagulogen (pink) by a horseshoe crab clotting enzyme, peptide C (green) is released from the inner portion of the parent molecules. The resulting coagulin (yellow) monomer may selfassemble to form the dimer, trimer, and multimers. The background of this figure shows a fiber-like coagulin gel.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3756735&req=5

f5-83_110: Hypothetical mechanism of coagulogen gel formation.10) Upon gelation of coagulogen (pink) by a horseshoe crab clotting enzyme, peptide C (green) is released from the inner portion of the parent molecules. The resulting coagulin (yellow) monomer may selfassemble to form the dimer, trimer, and multimers. The background of this figure shows a fiber-like coagulin gel.
Mentions: A stereo view of the coagulogen monomer, showing the A chain, peptide C, and B chain, and the secondary structure is presented in Fig. 4. The coagulogen monomer is an elongated molecule of approximate dimensions, 60×30×20Å. The structure is mainly dominated by the β-strands in blue, labeled sequentially B1 to B6. The multiple coils and turns of the B chain are colored green. Short helical segments in orange, in the B chain are in the background. The mainly α-helical peptide C, in red, covers a reasonable part of the top surface. The NH2-terminal A chain, in violet, is connected to the B chain via two disulfide bridges colored yellow. The whole cysteines comprise 8 disulfide bridges. The peptide C helix covers an extended hydrophobic core, which becomes accessible upon the cleavage and release of peptide C. Thus, the uncovered cove newly exposed in one molecule after the release of peptide C might interact with a hydrophobic edge of a second molecule, like a head to tail, to form a multimer, as shown in Fig. 5.38)

Bottom Line: This gelation reaction of the lysate, so-called Limulus test, has been widely employed as a simple and very sensitive assay method for endotoxins.The molecular structures of these proteins have also been elucidated.Moreover, the reconstitution experiments using the isolated clotting factors, factor C, factor B, proclotting enzyme and coagulogen in the presence of endotoxin, leads to the formation of coagulin gel.

View Article: PubMed Central - PubMed

Affiliation: The Chemo-Sero-Therapeutic Research Institute, 1-6-1 Okubo, Kumamoto 812-8581, Japan . ; Research adviser.

ABSTRACT
A hemocyte lysate from horseshoe crab (Limulus) produced a gel, when exposed to Gram-negative bacterial endotoxins, lipopolysaccharides (LPS). 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)-β-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, and factor G, all of which are closely associated with the endotoxin-mediated coagulation pathway, have been purified and biochemically characterized. The molecular structures of these proteins have also been elucidated. Moreover, the reconstitution experiments using the isolated clotting factors, factor C, factor B, proclotting enzyme and coagulogen in the presence of endotoxin, leads to the formation of coagulin gel. Here, I will focus on the biochemical principle of Limulus test for detecting bacterial endotoxins, and its activation and regulation mechanism on the LPS-mediated coagulation cascade.

No MeSH data available.


Related in: MedlinePlus