Purification and properties of a peptidase acting on a synthetic substrate for collagenase from monkey kidney

Abstract

A peptidase cleaving a synthetic substrate for collagenase, 4-phenylazobenzyloxycarbonyl-l-Pro-l-Leu-Gly-l-Pro-d-Arg (designated as Pz-peptide) has been purified extensively (about 5200-fold) from a soluble extract of monkey kidney with a view to carrying out studies on its possible physiological role. The purified Pz-peptidase appeared essentially free of collagenase, nonspecific protease and di- and tri-peptidase activities. The properties of the purified Pz-peptidase resemble very much the granuloma enzyme. It is optimally active around pH 7.0. Its apparent K_m value for Pz-peptide is 0.72 mM and V is 10.1 μmol/mg protein/min. It is reversibly inhibited by p-hydroxymercuribenzoate and HgCGl₂, whereas iodoacetamide does not affect the enzyme activity. N-Ethylmaleimide inhibited the enzyme partially (50%). Heavy metals like Cu²⁺, Cd²⁺, Ag⁺, Pb²⁺, Ni²⁺ and Zn²⁺ completely inhibited the enzyme activity, while the inhibition by Co²⁺ was only partial. Fe²⁺ did not exert any effect on the activity. The enzyme activity was completely inhibited by EDTA and was restored almost to the original value by metal ions like Mn²⁺, Mg²⁺, Ca²⁺ and Ba²⁺. The approximate molecular weight of the purified enzyme was estimated to be 56 000.