Skip Navigation

This Article
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Request Permissions
Google Scholar
Right arrow Articles by HARADA-SUZUKI, T.
Right arrow Articles by NIWA, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by HARADA-SUZUKI, T.
Right arrow Articles by NIWA, M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

J. Biochem, 1982, Vol. 92, No. 3 793-800
© 1982 Japanese Biochemical Society

research-article

Further Studies on the Chromogenic Substrate Assay Method for Bacterial Endotoxins Using Horseshoe Crab (Tachypleus tridentatus) Hemocyte Lysate1

Toshie HARADA-SUZUKI*, Takashi MORITA*,2, Sadaaki IWANAGA*,2, Shin NAKAMURA** and Makoto NIWA***

*Institute for Protein Research, Osaka University Suita, Osaka 565
**Department of Biochemistry, Primate Research Institute, Kyoto University Inuyama, Aichi 484
***Department of Bacteriology, Osaka City University Medical School Abeno-ku, Osaka, Osaka 565

Basic studies on the chromogenic substrate method for the assay of bacterial endotoxins were performed using Tachypleus hemocyte lysate. From the substrate specificity studies, Tachypleus clotting enzyme was found to prefer tripeptidyl-p-nitroanilide (pNA) having glycine and leucine (valine) in the P2 and P3 positions, and the apparent Km values for these peptidyl-pNA's were in the order of 10–5 M. Coagulogen, a natural substrate of the clotting enzyme, competitively inhibited the amidase-catalyzed hydrolysis of Tos-Ile-Glu-Gly-Arg-pNA with a K1 of 3.8 × 10–6 M, indicating that the amidase activity is due to catalytic action of the clotting enzyme.

Using the chromogenic substrate, the optimal conditions for the endotoxin-induced activation of the latent amidase in Tachypleus hemocyte lysate was examined. As a result, it was shown that the following points should be taken into consideration for the assaying of endotoxins; (1) the endotoxin-induced amidase activity in the lysate occurs optimally at pH 8.0 and at 40°C, (2) the induction requires Mg2+, (3) the endotoxin-induced amidase activity is unstable on prolonged incubation, (4) the sensitivity of the lysate to endotoxin is highly dependent upon its protein concentration, and (5) the latent amidase activity in the lysate is induced by a variety of endotoxin preparations with considerable differences in reactivity. In conclusion, the chromogenic substrate method using Tachypleus hemocyte lysate is valuable for the detection and quantitation of bacterial endotoxins, and under certain conditions the method is fifty-times more sensitive than the Limulus gelation test.

2Present address: Department of Biology, Faculty of Science, Kyushu University 33, Higashi-ku, Fukuoka 812.

1This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?




Disclaimer: Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.