Deoxyribonuclease IIalpha (DNase IIalpha) is an acid endonuclease found in lysosomes, nuclei, and various secretions. Murine DNase IIa is required for digesting the DNA of apoptotic cells after phagocytosis and for correct development and viability. DNase IIa purified from porcine spleen was previously shown to contain three peptides, two of which were thiol crosslinked, all derived by processing of a single polypeptide. Commercial bovine protein is consistent with this structure. However, screening of 18 human cell lines failed to demonstrate this processing, rather a 45 kDa protein was consistently observed. Incubation of cells with the N-glycosylation inhibitor tunicamycin resulted in a 37 kDa protein, which is close to the predicted formula weight. The protein also contains at least one thiol crosslink. Similar results were obtained with overexpressed DNase IIa. These results suggest that active DNase IIa consists of one contiguous polypeptide. We suggest the previous structure reflects proteolysis during protein purification. (C) 2002 Eisevier Science (USA).