Hydrolysis of substance P and bradykinin by black widow spider venom gland extract

Black widow spider venom gland extract was found to contain significant peptidase activity. Aliquots of the venom gland extract incubated at 37 degrees inactivated substance P (SP) and bradykinin but not angiotensin II or the enkephalins. The peptide inactivation was proportional to the duration of the incubation and the amount of extract used. Analysis of the peptides on high pressure liquid chromatography demonstrated that the loss in biological activity of SP and bradykinin in the longitudinal muscle of the guinea pig ileum was correlated with cleavage of the peptides into several fragments. Kinetic studies revealed that SP was initially split into two fragments but that these products underwent further degradation into smaller peptides. The optimal pH for the peptidase activity was 6.5. At 0 degree the enzymatic activity was undetectable, and it was irreversibly destroyed by incubation at 100 degrees for 5 min or by pretreatment of the extract with 100 microM diisopropyl fluorophosphate. In addition, the gland extract preparation hydrolyzed artificial substrates designed to detect trypsin or chymotrypsin-like activity.