Purification of the main somatostatin‐degrading proteases from rat and pig brains, their action on other neuropeptides, and their identification as endopeptidases 24.15 and 24.16

The main somatostatin‐degrading proteases were purified from rat and pig brain homogenates and characterized as thiol‐and metal‐dependent endoproteases. Two types of proteases with apparent native and subunit molecular masses of 70 kDa and 68 kDa could be differentiated in both species. Beside somatostatin, both hydrolyzed several other neuropeptides with chain lengths between 8 and 30 amino acid residues. Cleavage sites were generally similar or identical, but some clear exceptions were observed for enzymes from both species which could be used to differentiate between the two proteases. The 68‐kDa protease cleaved somatostatin at three bonds (Asn5‐Phe6, Phe6‐Phe7 and Thr10‐Phe11) and neurotensin only at the Arg8‐Arg9 bond, whereas the 70‐kDa protease digested somatostatin at only two bonds (Phe6‐Phe7 and Thr10‐Phe11) and neurotensin as well as acetylneurotensin‐(8–13) additionally (pig protease) or almost exclusively (rat protease) at the Pro10‐Tyr11 bond. Relative rates for the digestions of various peptides were, however, more dependent on the species than on the type of protease. Cleavage sites for angiotensin II, bradykinin, dynorphin, gonadoliberin and substance P were, apart from different rates, identical for both proteases. In both species the 68‐kDa protease was found to be mainly, but not exclusively, soluble and not membrane‐associated, whereas the inverse was detected for the 70‐kDa protease. Based on distinct molecular and catalytic properties, the 68‐kDa protease is supposed to be congruent with the endopeptidase 24.15 (EC 3.4.24.15), the 70‐kDa protease with endopeptidase 24.16 (EC 3.4.24.16, neurotensindegrading endopeptidase). This investigation demonstrates that both proteases hydrolyze various neuropeptides with similar cleavage sites, but with species‐dependent activity. Species‐independent distinctions are the exclusive action of endopeptidase 24.16 on acetylneurotensin‐(8–13) and liberation of free Phe from somatostatin only by endopeptidase 24.15.