Identification, purification, and characterization of a calcium-dependent endonuclease (NUC18) from apoptotic rat thymocytes. NUC18 is not histone H2B

Glucocorticoids stimulate apoptosis in rat thymocytes that is characterized by internucleosomal DNA degradation. We have previously identified an 18-kDa calcium-dependent nuclease whose activity is associated with this DNA degradation. The existence of this nuclease has been challenged by Alnemri and Litwack (1989) J. Biol. Chem. 264, 4104-4111, who suggest that the nuclease we observed was histone H2B. We report here a modified nuclease assay which uses [32P] DNA as a substrate that has enabled the purification and characterization of the 18-kDa nuclease (NUC18). Using Bio-Rex 70 chromatography in conjunction with this assay, we show that NUC18 can be separated from histone H2B. Enzymatically active NUC18, purified to apparent homogeneity, failed to react with two different anti-histone H2B antibodies. NUC18 was inactive in the absence of calcium and known inhibitors of apoptosis, i.e. zinc and aurintricarboxylic acid inhibit its activity. Although NUC18 activity was detected in nuclear extracts of thymocytes of both control and glucocorticoid-treated thymocytes, these activities were distinct. Gel filtration analysis revealed that NUC18 was present as a high molecular weight complex (greater than 100 kDa) in both groups of cells, whereas it also existed as a low molecular weight form in glucocorticoid-treated cells. Thus, NUC18 remains a candidate for the endonuclease responsible for the DNA degradation component of the apoptotic process.