CATHEPSIN-D OF RAT SPLEEN - AFFINITY PURIFICATION AND PROPERTIES OF 2 TYPES OF CATHEPSIN-D

Two types of cathepsin D were purified from rat spleen by a rapid procedure involving an acid precipitation of tissue extract, affinity chromatography with pepstatin–Sepharose 4B and concanavalin‐A–Sepharose 4B, and chromatography on Sephadex G‐100 and DEAE‐Sephacel. The purified major enzyme (85% of the cathepsin D activity after DEAE‐Sephacel chromatography), termed cathepsin D‐I, represented about a 1000‐fold purification over the homogenate and about a 20% recovery. The purified minor enzyme (15%), termed cathepsin D‐II, represented about a 900‐fold purification and about a 3% recovery. Both enzymes showed four (PI: 4.2, 4.9, 6.1 and 6.5) and three (pI: 4.6, 5.6 and 5.8) multiple forms after isoelectric focusing, respectively. The purified enzymes appeared homogeneous on electrophoresis in polyacrylamide gel and had a molecular weight of about 44000. In sodium dodecylsulfate/polyacrylamide gel electrophoresis both enzymes showed a single protein band corresponding to a molecular weight of 44000. The enzymes had similar amino acid compositions except for serine, proline and methionine. Cathepsin D‐I contained 6.6% carbohydrate, consisting of mannose, glucose, galactose, fucose and glucosamine in a ratio of 8:2:1:1:5 with a trace of sialic acid. The properties of purified enzymes were also compared. Copyright © 1979, Wiley Blackwell. All rights reserved