Purification and properties of a peptidase acting on a synthetic collagenase substrate from experimental granuloma tissue in the rat

Abstract

An enzyme which acts on a synthetic collagenase substrate, 4-phenylazobenzyl-oxycarbonyl-l-Pro-l-Leu-Gly-l-Pro-d-Arg (Pz-peptide) has been purified 250-fold from soluble extracts of experimental granuloma tissue of the rat and its properties studied. It is optimally active around pH 7.2. Its apparent K_m value for Pz-peptide is 0.01 mM and V is 100 nmoles/mg protein per min. It is reversibly inhibited by p-hydroxymercuribenzoate (PHMB) and HgCl₂, whereas iodoacetamide does not affect the enzyme activity. Heavy metals like Cu²⁺, Cd²⁺, Ag⁺, Ni²⁺ and Zn²⁺ completely inhibit the enzyme activity while the inhibition by Co²⁺ was only partial. Fe²⁺, Ba²⁺, Mn²⁺, Pb²⁺ and Ca²⁺ did not exert any affect on the activity. Chelating agents like EDTA, sodium diethyl dithiocarbamate and α,α'-dipyridyl do not affect the enzyme activity. Approximate molecular weight of the purified enzyme was estimated to be 56000.