Identification of an endonuclease secreted by human B lymphoblastic IM9 cells.
We have identified a Mg(2+)-dependent endonuclease from IM9 cell lysates and culture medium using DNA-native-polyacrylamide gel electrophoresis (DNA-native-PAGE) nuclease assay system. This particular endonuclease activity was not detectable in conventional DNA-SDS-polyacrylamide gel electrophoresis assay system which is similar to the method originally described by Rosenthal and Lacks (A.L. Rosenthal and S.A. Lacks, Anal. Biochem. 80 (1977) 76-90). Experimental results clearly demonstrated that the endonuclease activity was not derived from the fetal calf serum in which the cells were grown, but synthesized in the cell and secreted into the culture medium by IM9 cells. Biosynthesis and subsequent release of the endonuclease into the culture medium were significantly decreased by pretreatment of the cells with actinomycin D. Using supercoiled plasmid DNA as a substrate, the endonuclease activity was determined with the enzyme isolated from the cell culture medium by native-PAGE electroelution. The endonuclease, with Mg2+ alone, was able to catalyze the conversion of the plasmid into linear DNA followed by further degradation. This is the first report demonstrating that a distinct Mg(2+)-dependent endonuclease is secreted by a human immune cell line.
Kwon
H J
HJ
Department of Biochemistry, College of Science, Yonsei University, Seoul, South Korea.
Kim
D S
DS
D002051:Burkitt Lymphoma / Q000201:enzymology*; D004278:DNA, Superhelical / Q000737:chemistry*; D003609:Dactinomycin; D003850:Deoxyribonuclease I / Q000096:biosynthesis; D004591:Electrophoresis, Polyacrylamide Gel; D004706:Endodeoxyribonucleases / Q000096:biosynthesis*; D006801:Humans; D007951:Leukemia, Myeloid / Q000201:enzymology / Q000473:pathology; D015459:Leukemia-Lymphoma, Adult T-Cell / Q000201:enzymology / Q000473:pathology; D008274:Magnesium / Q000494:pharmacology; D010957:Plasmids; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D004278:DNA, Superhelical; D011500:Protein Synthesis Inhibitors; D003609:Dactinomycin; D004706:Endodeoxyribonucleases; C029367:calcium magnesium dependent endodeoxyribonuclease; D003850:Deoxyribonuclease I; C019329:deoxyribonuclease II; D008274:Magnesium