Abstract
Cloned mouse mastocytoma P-815.2-E-6 cells are barely able to synthesize prostaglandins because of a lack of prostaglandin endoperoxide synthase activity. However, the addition of sodium n-butyrate at 1 mM induces synthesis de novo of prostaglandins in this cell line. Employing this system, we could isolate an mRNA for prostaglandin endoperoxide synthase by a combination of cell-free translation and immunoprecipitation. The antibody, prepared in rabbit by injecting purified prostaglandin endoperoxide synthase from bovine vesicular gland, was shown to cross-react with the corresponding enzyme from 2-E-6 cells. The poly(A)-containing mRNA has a sedimentation coefficient of 17S and codes for a single polypeptide chain of Mr 62 000 as estimated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The Mr of the mouse polypeptide chain appears very similar to that of the purified carbohydrate-free prostaglandin endoperoxide synthase from sheep vesicular gland. These findings are a contribution to the isolation of the gene for prostaglandin endoperoxide synthase.
Full text
PDF
Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Kawamura M., Koshihara Y., Senshu T., Murota S. Enhancement of prostaglandin synthesizing activity by the treatment with sodium n-butyrate on cloned mastocytoma cells and various other tissue cell lines. Prostaglandins. 1980 May;19(5):659–669. doi: 10.1016/0090-6980(80)90165-3. [DOI] [PubMed] [Google Scholar]
- Koshihara Y., Inagaki A., Murota S. I. Induction of prostacyclin formation by sodium n-butyrate in a cloned epithelial liver cell line. Biochim Biophys Acta. 1981 May 22;664(2):278–290. [PubMed] [Google Scholar]
- Koshihara Y., Kawamura M., Senshu T., Murota S. Effect of sodium n-butyrate on induction of prostaglandin synthase activity in cloned mastocytoma P-815 2-E-6 cells. Biochem J. 1981 Jan 15;194(1):111–117. doi: 10.1042/bj1940111. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Krystosek A., Cawthon M. L., Kabat D. Improved methods for purification and assay of eukaryotic messenger ribonucleic acids and ribosomes. Quantitative analysis of their interaction in a fractionated reticulocyte cell-free system. J Biol Chem. 1975 Aug 10;250(15):6077–6084. [PubMed] [Google Scholar]
- 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]
- Miyamoto T., Ogino N., Yamamoto S., Hayaishi O. Purification of prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes. J Biol Chem. 1976 May 10;251(9):2629–2636. [PubMed] [Google Scholar]
- Pelham H. R., Jackson R. J. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem. 1976 Aug 1;67(1):247–256. doi: 10.1111/j.1432-1033.1976.tb10656.x. [DOI] [PubMed] [Google Scholar]
- Roth G. J., Siok C. J., Ozols J. Structural characteristics of prostaglandin synthetase from sheep vesicular gland. J Biol Chem. 1980 Feb 25;255(4):1301–1304. [PubMed] [Google Scholar]
- Smith W. L., Wilkin G. P. Immunochemistry of prostaglandin endoperoxide-forming cyclooxygenases: the detection of the cyclooxygenases in rat, rabbit, and guinea pig kidneys by immunofluorescence. Prostaglandins. 1977 May;13(5):873–892. doi: 10.1016/0090-6980(77)90217-9. [DOI] [PubMed] [Google Scholar]
- Van der Ouderaa F. J., Buytenhek M., Nugteren D. H., Van Dorp D. A. Purification and characterisation of prostaglandin endoperoxide synthetase from sheep vesicular glands. Biochim Biophys Acta. 1977 May 25;487(2):315–331. doi: 10.1016/0005-2760(77)90008-x. [DOI] [PubMed] [Google Scholar]