ISOLATION OF 3 FORMS OF CYSTATIN FROM SUBMANDIBULAR SALIVA OF ISOPROTERENOL-TREATED RATS, ITS PROPERTIES AND KINETIC DATA

Three rat salivary cystatins (designated as RSC-1, RSC-2 and RSC-3) induced by chronic isoproterenol (IPR) treatment, but not detected in normal rats, were purified from submandibular saliva of chronically IPR-treated rats by Mono-Q, hydroxyapatite and TSKgel Phenyl-5PW chromatographies. Their molecular weights (M(r)) and isoelectric points (pI) differed from each other as follows: RSC-1 (M(r) 16500, pI 4.4), RSC-2 (M(r) 15500, pI 4.4) and RSC-3 (M(r) 14500, pI 4.5). The amino acid compositions of these inhibitors were very similar and the three forms showed complete immunological identity in a double immunodiffusion system. The partial amino acid sequence results showed that these inhibitors belonged to family 2 of the cystatin superfamily. These three forms strongly inhibited the enzyme activities of ficin and papain, but not of cathepsin B and trypsin. The inhibition constants (K(i)) of RSC-1, RSC-2 and RSC-3 for ficin were 0.19, 0.50 and 0.012 nM, and for papain were 1.5, 0.93 and 0.03 nM, respectively. RSC-3 inhibited ficin and papain more strongly than did RSC-1 and RSC-2.