Purification and characterization of new anti‐adrenocorticotropin rabbit neutrophil peptides (defensins)

Neutrophil peptides (NPs, defensins), which consist of approximately 30 amino acids with a highly conserved backbone of six Cys residues, possess several biological activities, such as antimicrobial, antiviral, cytotoxic, and anti‐adrenocorticotropin (corticostatic) activity in vitro. In the rabbit, six NPs, i.e., NP‐1, ‐2, ‐3A, ‐3B, ‐4, and ‐5, have been isolated, among them, NP‐3A has the most potent corticostatic activity, which involves the inhibition of adrenocorticotropin‐stimulated corticosterone synthesis in adrenal cells. Using a sensitive radioimmunoassay for NP‐3A and reverse‐phase HPLC, we found a novel component in rabbit tissue extracts with NP‐3 A‐like immuno‐reactivity. We further purified and characterized the component and found two novel peptides. One of these peptides, designated NP‐6, has an amino acid sequence identical to that of NP‐3A except for three amino acids, i.e. Leu11, Phe13, and Gln15, in the central portion of the sequence. The other has a sequence corresponding to that of NP‐6 except that the N‐terminal Gly was deleted and is thus named des‐G1‐NP‐6. Using chemically synthesized NP‐6 and des‐G1‐NP‐6, we measured their putative corticostatic activities by a dispersed rat adrenal cell bioassay. NP‐6 showed corticostatic activity comparable to that of NP‐3 A while des‐G1‐NP‐6 showed weak activity. These findings are also compatible with the notion that the N‐terminal Gly, but not the central portion, is important for the anti‐adrenocorticotropin activity of the NPs.