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The Reaction Mechanism Of “Limulus Test”

Morita, T ; Tanaka, S ; Nakamura, T ; Ohki, M ; Iwanaga, S

Thrombosis and Haemostasis, 1981, Vol.46 (1) [Periódico revisado por pares]

Stuttgart: Schattauer GmbH

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  • Título:
    The Reaction Mechanism Of “Limulus Test”
  • Autor: Morita, T ; Tanaka, S ; Nakamura, T ; Ohki, M ; Iwanaga, S
  • Assuntos: Coagulation ; IV: Thrombin, Thrombin-like Enzymes
  • É parte de: Thrombosis and Haemostasis, 1981, Vol.46 (1)
  • Descrição: The principle of the so-called “Limulus Test” for detection of bacterial endotoxins (LPS) is based on the LPS- induced coagulation reaction, using horseshoe crab amebocyte lysate. To establish the overall molecular events in such a reaction system, we have previously studied on the complete amino acid sequence of a clottable protein, coagulogen, and the enzymatic properties of active clotting enzyme. We also developed a new chromogenic substrate method for assay of LPS, using Limulus amebocyte lysate. During these investigations, we found that proclotting enzyme, which has been characterized as a LPS-sensitive protein, is not sensitive to LPS but that another unknown factor sensitive to LPS is involved in the reaction sequence which mediates the activation of the known proclotting enzyme. The evidences were as follows. When amebocyte lysate prepared from Tachypleus tridentatus (Japanese horseshoe crab) was applied to a column of heparin-Sepharose CL-6B, two components beside coagulogen, all of which are associated with the coagulation system, were separated into the breakthrough fraction (fraction A) and the adsorbed fraction (fraction B). Among them a component eluted in fraction A was identified as the known proclotting enzyme, but this proenzyme did not show any clotting activity even after preincubation with LPS. The other component, tentatively named factor B, eluted in fraction B showed a very weak LPS-dependent amidase activity towards Boc-Leu-Gly-Arg-pNA. However, when a catalytic amount of factor B treated with LPS was added to fraction A, a strong clotting activity appeared, indicating that both components are essential to develop the clotting activity in the presence of LPS. The same experiments as made for the lysate from T. tridentatus were performed using the lysate from Limulus polyphemus. The data was the same as those described above. These results indicate that the LPS- mediated coagulation system in the amebocyte consists of a multi-enzyme system and that this cascade system may provide an extremely high sensitivity of the lysate to endotoxins.
  • Editor: Stuttgart: Schattauer GmbH
  • Idioma: Inglês
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  • Realização: ABCD-USP USP   Logos de Redes Sociais: Freepik

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