Cathepsin D of Rat Spleen Affinity Purification and Properties of Two Types of Cathepsin D

Abstract

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...

Involvement of Cathepsin E in Exogenous Antigen Processing in Primary Cultured Murine Microglia

“…In-1 (anti-mouse Ii antibody), anti-mouse Lamp-2 antibody, and anti-mouse transferrin receptor (TfR) antibody were purchased from PharMingen (San Diego, CA). Polyclonal antibodies against purified rat spleen CE and CD were raised in rabbits and purified by affinity chromatography as described previously (32)(33)(34). CE and CD were purified from rat spleen according to the previously described methods (32,33).…”

“…Polyclonal antibodies against purified rat spleen CE and CD were raised in rabbits and purified by affinity chromatography as described previously (32)(33)(34). CE and CD were purified from rat spleen according to the previously described methods (32,33). Cathepsin B (CB) was purified form rat kidney as described (35).…”

Comparison of the Cathepsin D from Mackerel (Scomber australasicus) and Milkfish (Chanos chanos) Muscle

“…Yamamoto et al 36 ) reported that 10mMf3-Me did not activate the cathepsin D of rat spleen, but it did activate 40% activity of that from carp muscle. 29,30) However, 1 mM f3-Me partially inhibited the activity of cathepsin D from banded shrimp i muscle.…”

“…Na +, K +, Ca z +, Zn z +, Ni 2 +, and Cu z + ions activated the cathepsin D from milkfish but did not affect that from mackerel (Table V). According to Takeda et al 45 ) and Yamamoto et al,36,39) cathepsin D from rat spleen and human erythrocyte membrane acid proteinase (EMAP) were inhibited by Hg2 + and Fe 3 +. The Mgz+, Ca 2 +, and Ni 2 + (lOmM) did not affect the activity of cathepsin D from rat spleen, but significantly activated that from tilapia muscle.…”

Role of LAMP-2 in Lysosome Biogenesis and Autophagy

“…The following antibodies were used in this study: Rabbit antiserum against mouse cathepsin D (Pohlmann et al, 1995), rabbit antiserum against rat liver cathepsin D purified from lysosomal contents by affinity chromatography on pepstatin A-sepharose as described (Yamamoto et al, 1979), affinity-purified rabbit antibody against the cytoplasmic tail of MPR46 (MSC1; Klumperman et al, 1993), rabbit antiserum against human MPR46 (II-4; Wenk et al, 1991), rabbit antiserum against rat MPR300 (I-5; Claussen et al, 1995), mouse mAb against ␥-adaptin (Transduction Laboratories, Lexington, KY), and rat monoclonal antibodies against LAMP-2 (ABL93) and LAMP-1 (1D4B; Developmental Studies Hybridoma Bank, Iowa City, Iowa).…”