A chromatin-bound neutral protease and its inhibitor in rat peritoneal macrophages.

Rat peritoneal macrophages are known to contain a chymotrypsin-like neutral protease associated with a specific inhibitor. By homogenizing the cells in 0.25 M sucrose (pH 8.0) containing 0.5% Triton X-100, both the protease and the inhibitor were found to be localized in the nuclei, particularly in chromatin. The inhibitory factor in chromatin was then separated from the protease by hydroxylapatite gel chromatography in the presence of 2 M NaCl and 5 M urea. The inhibitor fraction obtained was deproteinized by digestion with Pronase and subsequent extraction with phenol; these treatments did not alter the inhibitory potency. The deproteinized inhibitor fraction had a UV absorption ratio, A280/A260, of 0.61, but it was resistant to digestion with various nucleases, including DNase 1, nuclease P1, and snake venom phosphodiesterase. However, when it was incubated with poly(ADP-ribose) glycohydrolase from calf thymus, the inhibitory potency was markedly decreased. An authentic poly(ADP-ribose), with a mean chain length of approximately 30 ADP-ribose units, produced significant inhibition of the neutral protease isolated from macrophage chromatin. No such inhibition was produced by DNA, single-stranded DNA, RNA, polyadenylate, polyuridylate, polycytidylate, or monomeric ADP-ribose.