Purification of Cathepsin D from Guinea Pig Peritoneal Macrophages

Abstract

Aspartic proteinase was purified from guinea pig peritoneal macrophages (Mφs) by pepstatin-Sepharose, Sephadex G-150 and diethylaminoethyl-cellulose column chromatographies. The purified enzyme was homogeneous on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. In isoelectric focusing, the enzyme was resolved into four peaks which had pIs of 6.04, 6.53, 7.11 and 7.75. The molecular weight of the enzyme was estimated to be 43000 by gel filtration and 45000 by SDS-polyacrylamide gel electrophoresis. The enzyme was adsorbed on a column of concanavalin A-Sepharose and was eluted with α-methyl mannoside, indicating that it is a glycoprotein. The pH optimum of the enzyme for hydrolysis of acid-denatured hemoglobin was around 3.2. The enzyme was completely inhibited by pepstatin, but not by other chemicals tested, including antipain and leupeptin. Since the properties of the enzyme from Mφs resemble those of cathepsins D isolated from the lysosome of other tissues, it can be concluded that this enzyme is cathepsin D (EC 3.4.23.5).