BOVINE SPLEEN CATHEPSIN A - CHARACTERIZATION AND COMPARISON WITH THE PROTECTIVE PROTEIN

Cathepsin A was purified approximately 550-fold with an overall yield of 4% from bovine spleen crude extracts by successive chromatographies on DEAE-Sephadex A-50, phenyl-Toyopearl 650C, and Con A-agarose, PAGE of the purified enzyme without 8-mercaptoethanol revealed an apparent molecular size of 110 kDa, and SDS-PAGE with 2-mercaptoethanol gave t wo polypeptide bands corresponding to 32 and 25 kDa and without 2-merc aptoethanol a single polypeptide 52 kDa band, These results indicate t hat the enzyme has an (alpha beta)(2) tetrameric structure in which th e alpha (32 kDa) and beta (25 kDa) subunits are linked by disulfide bo nd(s), The enzyme exhibited peptidase activities, hydrolyzing various Z-dipeptides with optimum pHs between 5.0 and 5.8, The hydrolytic rate for Z-Phe-Ala was 15 times higher than that for Z-Glu-Tyr, the tradit ional cathepsin A substrate, The enzyme also catalyzed the hydrolysis of the C-terminal amino acids of RCM-RNase A and showed esterase activ ity toward BTEE at pH around 7.5. DFP and TPCK completely inhibited bo th peptidase and esterase activities, and [1,3-H-3]DFP was bound to th e alpha subunit, All these results support the fact that the enzyme is a serine carboxypeptidase. The N-terminal amino acid sequences of the alpha and beta subunits are highly homologous to those of the human p rotective protein in galactosialidosis, strongly supporting the identi ty between cathepsin A and the protective protein.