Skip to main content
Biochemical Journal logoLink to Biochemical Journal
. 1987 Nov 15;248(1):217–222. doi: 10.1042/bj2480217

Purification of Sarcophaga (fleshfly) lectin and detection of sarcotoxins in the culture medium of NIH-Sape-4, an embryonic cell line of Sarcophaga peregrina.

H Komano 1, E Kasama 1, Y Nagasawa 1, Y Nakanishi 1, K Matsuyama 1, K Ando 1, S Natori 1
PMCID: PMC1148521  PMID: 3435439

Abstract

An established cell line originating from a Sarcophaga peregrina (fleshfly) embryo, NIH-Sape-4, was found to synthesize mRNAs for Sarcophaga lectin and sarcotoxin IA, but not those for storage protein or 25 kDa protein. These four proteins are known to be synthesized in the fat-body of third-instar larvae, and the two former in particular are known to participate in the defence mechanism of this insect and to be induced in response to injury of the body wall. Thus the embryonic cell line NIH-Sape-4 synthesizes certain defence proteins constitutively. This cell line will be useful for large-scale purification of Sarcophaga lectin, since 50 micrograms of purified Sarcophaga lectin could be obtained from about 400 ml of culture medium.

Full text

PDF
217

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ando K., Okada M., Natori S. Purification of sarcotoxin II, antibacterial proteins of Sarcophaga peregrina (flesh fly) larvae. Biochemistry. 1987 Jan 13;26(1):226–230. doi: 10.1021/bi00375a030. [DOI] [PubMed] [Google Scholar]
  2. Fairbanks G., Steck T. L., Wallach D. F. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry. 1971 Jun 22;10(13):2606–2617. doi: 10.1021/bi00789a030. [DOI] [PubMed] [Google Scholar]
  3. Hultmark D., Steiner H., Rasmuson T., Boman H. G. Insect immunity. Purification and properties of three inducible bactericidal proteins from hemolymph of immunized pupae of Hyalophora cecropia. Eur J Biochem. 1980 May;106(1):7–16. doi: 10.1111/j.1432-1033.1980.tb05991.x. [DOI] [PubMed] [Google Scholar]
  4. Itoh A., Amano F., Natori S. Activation of murine macrophage-like cell line J774.1 by Sarcophaga peregrina lectin. Gan. 1984 Aug;75(8):714–723. [PubMed] [Google Scholar]
  5. Itoh A., Iizuka K., Natori S. Induction of TNF-like factor by murine macrophage-like cell line J774.1 on treatment with Sarcophaga lectin. FEBS Lett. 1984 Sep 17;175(1):59–62. doi: 10.1016/0014-5793(84)80569-4. [DOI] [PubMed] [Google Scholar]
  6. Kessler S. W. Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A. J Immunol. 1975 Dec;115(6):1617–1624. [PubMed] [Google Scholar]
  7. Komano H., Mizuno D., Natori S. Purification of lectin induced in the hemolymph of Sarcophaga peregrina larvae on injury. J Biol Chem. 1980 Apr 10;255(7):2919–2924. [PubMed] [Google Scholar]
  8. Komano H., Natori S. Participation of Sarcophaga peregrina humoral lectin in the lysis of sheep red blood cells injected into the abdominal cavity of larvae. Dev Comp Immunol. 1985 Winter;9(1):31–40. doi: 10.1016/0145-305x(85)90057-6. [DOI] [PubMed] [Google Scholar]
  9. Komano H., Nozawa R., Mizuno D., Natori S. Measurement of Sarcophaga peregrina lectin under various physiological conditions by radioimmunoassay. J Biol Chem. 1983 Feb 25;258(4):2143–2147. [PubMed] [Google Scholar]
  10. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  11. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  12. Matsumoto N., Nakanishi Y., Natori S. Homologies of nucleotide sequences in the 5'-end regions of two developmentally regulated genes of Sarcophaga peregrina. Nucleic Acids Res. 1986 Mar 25;14(6):2685–2698. doi: 10.1093/nar/14.6.2685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Matsumoto N., Okada M., Takahashi H., Ming Q. X., Nakajima Y., Nakanishi Y., Komano H., Natori S. Molecular cloning of a cDNA and assignment of the C-terminal of sarcotoxin IA, a potent antibacterial protein of Sarcophaga peregrina. Biochem J. 1986 Nov 1;239(3):717–722. doi: 10.1042/bj2390717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Matsumoto N., Sekimizu K., Soma G., Ohmura Y., Andoh T., Nakanishi Y., Obinata M., Natori S. Structural analysis of a developmentally regulated 25-kDa protein gene of Sarcophaga peregrina. J Biochem. 1985 May;97(5):1501–1508. doi: 10.1093/oxfordjournals.jbchem.a135205. [DOI] [PubMed] [Google Scholar]
  15. Okada M., Natori S. Primary structure of sarcotoxin I, an antibacterial protein induced in the hemolymph of Sarcophaga peregrina (flesh fly) larvae. J Biol Chem. 1985 Jun 25;260(12):7174–7177. [PubMed] [Google Scholar]
  16. Okada M., Natori S. Purification and characterization of an antibacterial protein from haemolymph of Sarcophaga peregrina (flesh-fly) larvae. Biochem J. 1983 Jun 1;211(3):727–734. doi: 10.1042/bj2110727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Tahara T., Maeda Y., Kuroiwa A., Ueno K., Obinata M., Natori S. Identification of storage-protein messenger RNA of the fleshfly Sarcophaga peregrina. Biochem J. 1982 Jun 1;203(3):571–575. doi: 10.1042/bj2030571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Takahashi H., Komano H., Kawaguchi N., Kitamura N., Nakanishi S., Natori S. Cloning and sequencing of cDNA of Sarcophaga peregrina humoral lectin induced on injury of the body wall. J Biol Chem. 1985 Oct 5;260(22):12228–12233. [PubMed] [Google Scholar]
  19. Tamura H., Tahara T., Kuroiwa A., Obinata M., Natori S. Differential expression of two abundant messenger RNAs during development of Sarcophaga peregrina. Dev Biol. 1983 Sep;99(1):145–151. doi: 10.1016/0012-1606(83)90261-0. [DOI] [PubMed] [Google Scholar]
  20. Tamura M., Natori S. Induction of human gamma interferon by Sarcophaga peregrina humoral lectin. FEBS Lett. 1984 Oct 1;175(2):325–328. doi: 10.1016/0014-5793(84)80761-9. [DOI] [PubMed] [Google Scholar]
  21. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Weinstock R., Sweet R., Weiss M., Cedar H., Axel R. Intragenic DNA spacers interrupt the ovalbumin gene. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1299–1303. doi: 10.1073/pnas.75.3.1299. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES