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 concanava h -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-11, represented about a 900-fold purification and about a 3% recovery. Both enzymes showed four (pl: 4.2, 4.9, 6.1 and 6.5) and three (pl: 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 corrcsponding 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.Cathepsin D is a major proteolytic enzyme of lysosomes [l, 21 and widely distributed in animal tissues. It is presumed that cathepsin D plays an important role in physiological protein degradation and pathological processes [2 -51. However, the precise role of cathepsin D is still unclear, largely because the mechanism of protein degradation is presumably a complex and multistep process. Consequently it is clear that significant progress in understanding cathepsin D function can be made if a reasonably large quantity of pure enzyme can be obtained. For this purpose we used a rapid and convenient method for the purification of cathepsin D, which involved affinity chromatography with pepstatin -Sepharose 4B and concanavalin-A -Sepharose 4B. Although a number of lysosoma1 cathepsin D have been isolated from various tissues, including spleen [6-91, liver [lo-121, uterus [13,14], thyroid [15,16], small intestine [17], lung [18,19] and erythrocytes [20], we have undertaken a study of cathepsin D from rat spleen because cathepsin D from rat spleen has not been extensively studied and a specific activity of the enzyme in this organ is about 5 times higher than that of the liver. Recently several workers have isolated cathepsin D by affinity chromatography using suitable ligands such as substrate [8] and inhibitors [12,21,22]. In the previous work we have shown that rat spleen possesses two types of acid proteinases strongly inhibited by pepstatin [23,24]. One of these was isolated in a pure form by the use of affinity chromatography on pepstatinSepharose 4B and concanavalin-A -Sepharose 4B, and identified as a cathepsin-E-like enzyme that differs from cathepsin D [24]. In the study described here on the isolati...

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.

No abstract

The immunological properties of acid proteinases from rat spleen, two types of cathepsin D and a cathepsin E-like enzyme, were examined. The rabbit antiserum was prepared against the major form of cathepsin D (cathepsin D-I) from rat spleen. The antiserum quantitatively precipitated the enzyme activity from the purified cathepsin D-I preparation. On immunodiffusion analysis, the antiserum showed an identical reaction with the minor form of cathepsin D (cathepsin D-II) from rat spleen. Immunoelectrophoresis showed that the precipitin line with cathepsin D-II ran somewhat faster to the anode than that with cathepsin D-I. The cathepsin E-like acid proteinaspe was neither precipitated nor inhibited by the antiserum to cathepsin D-I, indicating that the cathepsin E-like enzyme is different from cathepsin D. Immunological gel diffusion with the antiserum indicated that rat spleen cathepsin D was immunologically identical with cathepsin D obtained from rat brain, thymus, lungs, heart, liver, kidneys, and adrenals.

No abstract