Affinity purification and properties of cathepsin-E-like acid proteinase from rat spleen.

A unique acid proteinase different from cathepsin D was purified from rat spleen by a method involving precipitation at pH 3.5, affinity chromatography on pepstatin-Sepharose 4B and concanavalin A-Sepharose 4B, chromatography on Sephadex G-100 and DEAE-Sephacel, and isoelectric focusing. A purification of 4200-fold over the homogenate was achieved and the yield was 11%. The purified enzyme appeared to be homogeneous on electrophoresis in polyacrylamide gels. The isoelectric point of the enzyme was determined to be 4.1-4.4. The enzyme hydrolyzed hemoglobin with a pH optimum of about 3.1. The molecular weight of the enzyme was estimated to be about 90000 by gel filtration on Sephadex G-100. In sodium dodecylsulfate polyacrylamide gel electrophoresis, the purified enzyme showed a single protein band corresponding to a molecular weight of about 45000. The hydrolysis of bovine hemoglobin by the enzyme was much higher than that of serum albumin. Various synthetic and natural inhibitors of the enzyme were tested. The enzyme was inhbited by Zn2+, Fe3+, Pb2+, cyanide, p-chloromercuribenzoate, iodoacetic acid and pepstatin, whereas 2-mercaptoethanol, phenylmethyl-sulfonyl fluoride and leupeptin showed no effect.