Cystatins from bovine brain: Purification,some properties,and action on substance P degrading activity

Two cystatins were purified from tissue extract of bovine brain by alkaline treatment, acetone fractionation, gel chromatography on Sephadex G-75, and affinity chromatography on S-carboxymethyl-papain-Sepharose. One of the inhibitors had a relatively high molecular mass, 25 kDa (HMM-cystatin) with pI 4.7, and the other, 11 kDa (LMM-cystatin) with pI 5.23. Both inhibitors showed considerable stability at pH 2 and 80°C. The cystatins inhibited papain, ficin, and cathepsins B and H, but not trypsin, chymotrypsin, thermolysin, nagarse, and cathepsin D. K_i values for the complexes of papain and the inhibitors were estimated to be 2.8×10^–10 M for HMM-cystatin and 1.3×10^–9 M for LMM-cystatin. Both purified cystatins prevented degradation of substance P by soluble fraction and lysosomal extract obtained from synaptosomes, but did not suppress the cleavage of the peptide by synaptosomal plasma membranes.Abbreviations HMM-cystatin high molecular mass inhibitor - LMM-cystatin low molecular mass inhibitor - SP substance P - SPM synaptosomal plasma membranes - p-CMB 4-chloromercuribenzoic acid - BK bradykinin - Bz-Arg-Nap N-benzoyl-dl-arginine--naphthylamide - Arg-Nap dl-arginine--naphthylamide - P-Pxy-Hb hemoglobin initially coupled with pyridoxal-5-phosphate