Genetic transfer of endothelin converting enzyme activity to CHO-K1 cells: detection of positive cells by reverse hemolytic plaque assay.
We have established a novel method of molecular cloning of endothelin converting enzyme, a key enzyme in the production of a potent vasoconstrictor endothelin-1, by modification of the reverse hemolytic plaque assay. Also, we demonstrated that a cell line, CHO-K1, showed no detectable activity of endothelin converting enzyme. This cell line was transfected with a cDNA library of bovine endothelial cells. The modified reverse hemolytic plaque assay was shown to detect even a single CHO-K1 cell that was changed to produce mature ET-1 by transfection. Thus, this novel method is suggested to be useful for the molecular cloning of other secreted antigens and their processing enzyme.
Shiraki
T
T
Department of Pharmacology, Faculty of Medicine, Kyoto University, Japan.
Sawamura
T
T
Ikura
T
T
Kobayashi
S
S
Miwa
S
S
Masaki
T
T
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000235:genetics*; D001483:Base Sequence; D016466:CHO Cells; D002417:Cattle; D003001:Cloning, Molecular / Q000379:methods*; D006224:Cricetinae; D000072396:Endothelin-Converting Enzymes; D004730:Endothelium, Vascular / Q000502:physiology; D006462:Hemolytic Plaque Technique; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D014162:Transfection
D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; D000072396:Endothelin-Converting Enzymes