Eur. J. Biochem. 95,459-467 (1979)
Cathepsin D of Rat Spleen Affinity Purification and Properties of Two Types of Cathepsin D Kenji YAMAMOTO, Nobuo KATSUDA, Masaru HIMENO, and Keitaro KATO Department of Pharmacology, Faculty of Dentistry and Department of Physiological Chemistry, F x u l t y of Pharmaceutical Sciences, Kyushu bnivehiLy (Received October 21, 1978/January 4, 1979)
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 Enzyyme. Cathepsin D (EC 3.4.23.5).
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 isolation of another acid proteinase, cathepsin D, we succeeded in separating two types of cathepsin D, cathepsin D-I and D-11, by DEAE-Sephacel chromatography. Both enzymes revealed 4 and 3 multiple forms respectively after isoelectric focusing, as reported previously by several workers [7,8,11]. Cethepsin D is also an interesting enzyme because of its value in an investigation of the biosynthetic route and mechanism of the packaging of lysosomal enzymes. It is now evident that almost all lysosomal enzymes are glycoproteins. Rat liver lysosomal 8-glucuronidase [25] and cc-L-fucosidase [26] were shown to contain five sugars; mannose, glucosamine, glucose, galactose and fucose. Therefore, the carbohydrate composition of cathepsin D is of special interest, and
Cathepsin D of Rat Spleen
460
in order to clarify the role of carbohydrate moieties in the translocation of lysosomal enzymes, cathepsin D purified from rat spleen was also subjected to chemical analysis. In the present paper we describe the purification and properties of the two enzymes, cathepsin D-I and D-11. We also determined the carbohydrate composition of cathepsin D-I and compared the carbohydrate moiety with those of other lysosomal enzymes [25,26].
MATERIALS AND METHODS Materials Chemicals were obtained from the following sources : bovine hemoglobin (Typ II), bovine serum albumin (fraction V) and phenylmethylsulfonyl fluoride from Sigma Chemical Co. Pepstatin and leupeptin were from The Protein Research Foundation (Osaka, Japan). Sephadex G-100, DEAE-Sephacel, AH-Sepharose 4B and concanavalin-A - Sepharose 4B were from Pharmacia. Ampholytes were from LKB Instruments Inc. All other chemicals were of reagent grade, purchased from various commercial sources.
of Davis [29]. Gels were stained for protein with Coomassie brilliant blue and destained in 7 % acetic acid and for carbohydrate by the periodate/Schiff procedure described by Zacharius et al. [30]. Sodium dodecylsulfate/polyacrylamide gels were prepared by a slight modification of the method of Fairbanks et al. [31] as described previously [24]. Enzyme samples were solubilized by incubation at 100 "C for 3 min after addition of the following compounds (to the indicated final concentration); 1 % sodium dodecylsulfate, 1 % 2-mercaptoethanol, 10 % sucrose, 0.01 M Tris-HC1, pH 8.0, and 0.001 M EDTA. Gels were stained with Coomassie brilliant blue followed by complete destaining. Staining for carbohydrate was done by the method of Zacharius et al.
POI. Molecular Weight Determination A Sephadex G-100 column (1.6 x 90 cm) for molecular weight determinations was used as described by Andrews [32], except that 0.005 M sodium phosphate buffer, pH 7.0, containing 0.2 M NaCl was used. Subunit molecular weight was determined by sodium dodecylsulfate/polyacrylamide gel electrophoresis as mentioned above.
Enzyme Assay Cathepsin D activity was determined by a modification of the method of Anson [27]. The reaction mixture contained 0.5 mlO.1 M sodium acetate buffer, pH 3.8, 1.O ml buffered hemoglobin solution (2.5 %, w/v, in 0.1 M sodium acetate buffer, pH 3.8). To this was added 5- 100 pl of enzyme solution and the final volume was adjusted to 2.0 ml with distilled water. After a 40-min incubation at 37 "C, the reaction was stopped by plunging the tubes into ice and adding 2.0 ml of 5 % ice-cold trichloroacetic acid solution. The precipitated mixture was allowed to stand for 10 min in ice and then centrifuged. The liberated peptides in the supernatant (1.0 ml) were measured by the FolinLowry reaction [28]. The enzyme activity was determined with reference to a tyrosine standard curve. The enzyme unit, U, was defined as pg tyrosine solubilized/ min. The absorbance at 660nm in the assay was 0.08-0.40 in order to retain linearity with respect to enzyme concentration. Protein Determination Protein was determined by the method of Lowry et al. [28] with bovine serum albumin as standard.
Other Column Chromatography Concanavalin-A - Sepharose 4B, Sephadex G-100, and DEAE-Sephacel were used as recommended by the manufacturer's instructions. Preparation of Affinity Column oj' Pepstatin-Sepharose 4B Pepstatin was coupled to AH-Sepharose 4B by the method of Murakami and Inagami [33]. Isoelectric Focusing Sucrose gradient isoelectric focusing was carried out in a LKB 8101 column (1 10 ml capacity) according to the manufacturer's instructions. The pH gradient was stabilized with a stepwise sucrose gradient of 0-56% (w/v). The samples of cathepsin D were layered in the middle of the gradient and electrofocused at 600 V for 60 h. After isoelectric focusing fractions of 2 ml were collected, and the cathepsin D activity and pH of each fraction were measured. Heat Inactivation
Polyacrylamide Gel Electrophoresis Polyacrylamide gel electrophoresis was performed in 12 % gels in Tris-HC1 buffer, pH 8.9, by the method
Enzyme samples were adjusted to pH 7.0 (0.1 M sodium phosphate buffer) and pH 3.8 (0.1 M sodium acetate buffer) respectively. Samples (0.1 ml, 100 pg protein/ml) of each buffered enzyme solution were
46 1
K. Yamamoto, N. Katsuda, M. Himeno, and K. Kato
Table 1. Purification of cathepsin D from rat spleen Starting material was 370 g (wet weight) spleen. The proteolytic activity was determined under the conditions described in the text. The first six rows contain the combined activities of cathepsin-D and E-like enzymes, whereas the last three rows refer to cathepsin D only. Values in parentheses are those corrected by calculating the quantity of cathepsin D in the homogenate to be 60 % Step
Homogenate Spleen extract Ammonium sulfate Acid treatment Pepstatin - Sepharose 4B Concanavalin-A - Sepharose 4B Sephadex G-I00 DEAE-Sephacel : peak-I (cathepsin D-I) peak-I1 (cathepsin D-11)
Protein
Total activity
Specific activity
Yield
Purification factor
mg
mU
U/mg protein
%
-fold
88995 56 794 18808 5 836 115 60 28
801.9 152.9 685.9 486.5 428.2 350.3 186.7
9 13 36 83 3732 5838 6668
100 94 86 61 53 44 23 (38)
1 1.4 4.0 9.2 414.6 648.7 740.9
10 2
93.4 18.7
9526 8118
12 (20) 2 (3)
1058.4 902.0
incubated at 37 "C or 60 "C for periods of up to 90 min. Then they were chilled and remaining enzyme activity was measured. Determination of Sialic Acid Sialic acid was determined by the method of Warren [34] with N-acetylneuraminic acid as a standard. Determination of Hexose and Hexosamine Neutral sugar was determined by the phenol/sulfuric acid method of Dubois et al. [35]. The purified cathepsin D-I was hydrolyzed in 2.5 M trifluoroacetic acid for 8 h at 100°C. The hydrolysate was passed through an Amberlite C G 4 B (CH&OO-) column (0.8 x 12 cm) and an Amberlite CG-120 (H') column (0.8 x 12 cm) in order to separate neutral and amino sugars. For identification and quantitative analysis of each monosaccharide in the carbohydrate moiety, gas-liquid chromatography was carried out after reduction of monosaccharides to the corresponding alditols and hexosaminols, followed by trifluoroacetylation, according to the methods of Matsui et al. [36] and Tamura et al. [37]. Gas chromatography was performed on a Schimazu GC-3BF unit. Amino Acid Analysis The enzyme samples were dialyzed against distilled water and hydrolyzed under a vacuum in 6 M HCI for 24, 48 and 72 h at 110°C. Amino acids were analyzed on a Hitachi model 835 zutomatic aminoacid analyzer by the method of Spackman et al. [38]. Tryptophan content was determined spectrophotometrically [39].
RESULTS PURIFICATION OF CATHEPSIN D
Enzyme Extraction and Partial Purification by Ammonium Sulfate Fractionation, Precipitation at p H 3 3 , and Affinity Chromatography with Pepstatin - Sepharose 4B and Concanavalin-A - Sepharose 4 B In the present work the purification procedures of cathepsin D up to the step of concanavalin-A - Sepharose 4B affinity chromatography were carried out under the same conditions as previously employed for the purification of the cathepsin-E-like enzyme from rat spleen [24].
Gel Filtration The enzyme fractions obtained by concanavalinA- Sepharose 4B affinity chromatography (Fig. 1) were concentrated and dialyzed against 0.02 M sodium phosphate buffer, pH 7.0, and then applied to a column, 2.5 x 90 cm, of Sephadex G-100, which had been equilibrated with 0.02 M sodium phosphate buffer, pH 7.0. The column was eluted with the same buffer at a rate of 6 ml/h and fractions of 3 ml were collected. The elution pattern is presented in Fig.2. Two peaks of the proteolytic activity that hydrolyzed bovine hemoglobin at pH 3.8 were found. The first peak, having a molecular weight of about 90000, contained about 40 % of the total activity and was identified as a cathepsin-E-like acid proteinase as described previously [24]. Cathepsin D activity was recovered in the second peak. The fractions between 65 and 75 were collected, concentrated and then dialyzed against 0.005 M sodium phosphate buffer, pH 7.0. At this step the specific activity was about 6700 units, indi-
Cathepsin D of Rat Spleen
462 OiI
0.3
d 0.2
p"
0.1
0
o
2
o
m
~
~
o
~
r
n
m
w
m
0
Fraction number Fraction number
Fig. 1. Concanavalin-A - Sepharose 4 B affinity chromatographv of the enzyme fraction obtained by pepstatin-Sepharose 4 B affinty chroIriutography. The enzyme solution after pepstatin-Sepharose 4B ;il.linity chromatography was applied to a column, 2.5 x 5.0 cm, of concanavalin-A - Sepharose 4B equilibrated with 0.02 M sodium phosphate buffer, pH 7.0, containing 1 M NaCI. After exhaustive washing, 0.2 M methyl r-D-glucoside was added to the buffer at the point indicated by the arrow. The flow rate was 9 mlih and fractions of 5 ml were collected. ( 0 )Cathepsin activity; (----) protein
I4 1500
-E
I
. .-
33
u)
I
- K)oO c 3
ZI
c ._ 2. .c
0.2
m
9
D
.-c
B m
0 1
r I
3 0
0
5 3 6 0 7 0 8 0 9 0 Fraction number
Fig. 2. Gel filtration on Scphudex GI00 of' the enzyme fraction obtained by concanavalin-A - Sepharose 4 B affinity chromatography. The enzyme solution after concanavalin-A -Sepharose 4B affinity chromatography was applied to a column, 2.5 x 90 cm, equilibrated with 0.02 M sodium phosphate buffer, pH 7.0. The flow rate was 6 ml/h and fractions of 3 ml were collected. (0)Cathepsin activity; (----) protein
cating about 740-fold purification over the homogenate. Since about 40 % of the enzyme activity present in rat spleen extract is due to cathepsin-E-like enzyme, the yield of cathepsin D is practically about 38%. Therefore, the yields in all subsequent steps were corrected by calculating a quantity of cathepsin D in the homogenate to be 60 %.
D EA E-Sephacel Chromatography The enzyme preparation from the previous step was applied to a column, 1.6 x 12 cm, of DEAE-Seph-
Fig. 3. DEAE-Sephacel column cliromutography of' the enzyme fraction obtained by Sephadex G-100 chromatography. The enzyme fraction after gel filtration on Sephadex G-100 was applied to a column, 1.6 x 12 cm, of DEAE-Sephacel equilibrated with 0.005 M sodium phosphate buffer, pH 7.0. Elution was carried out with the same buffer. The arrows indicate the change of sodium chloride concentration: (1); 0.02 M, (2); 0.05 M. The flow rate was 6 ml/h and fractions of 3 ml were collected. ( 0 ) Cathepsin activity; (----) protein
ace1 equilibrated with 0.005 M sodium phosphate buffer, pH 7.0. The column was washed with the same buffer and the enzyme was then eluted stepwise with NaCl in the buffer. Two peaks of enzyme activity were obtained by elution at 0.05 M NaCl. A typical elution profile is shown in Fig. 3 . The major peak that represented 70 - 80 % yield of the applied activity was designated as 'cathepsin D-1'. The minor peak that represented 10- 15 % yield was designated as 'cathepsin D-II'. These two peaks of enzyme activity marked with the bar were pooled separately and concentrated with a collodion bag. The purification ratios of cathepsin D-I and D-I1 were about 1000-fold and 900-fold respectively over the homogenate. Further purification procedure using isoelectric focusing neither increased the specific activity nor changed the polyacrylamide gel electrophoretic pattern of each enzyme. DEAESephacel chromatography also achieved the complete separation of cathepsin D from the cathepsin-E-like enzyme that was eluted at 0.3 M NaCl under the same condition. A summary of a representative purification is shown in Table 1.
PROPERTIES OF THE PURIFIED ENZYMES
Polyucrylumide Gel Electrophoresis On polyacrylamide gel electrophoresis of purified cathepsin D-I and D-11, each revealed a single protein band when stained for protein with Coomassie brilliant blue and for carbohydrate by the periodate/Schiff procedure (Fig. 4). Cathepsin D-I1 moved somewhat faster than cathepsin D-I.
463
K. Yamamoto, N. Katsuda, M. Himeno, and K. Kato 10 *
-In -x L
Cathepin D - I and D - I I 5.
3
p
4. 3.
U
Q 2 1 0.1
0.2
0.3
0.4 Relative
Q5 Q6 mobility
07
00
Fig. 6 . Detrwninu/ion uf thc .\iihiiiii/ n ~ i ~ l keeights ~ ~ ~ of u cathepsin l ~ ~ ~ ~ D-I and D-II by dectrophoresis on sodium dodecylsulfate/polyacr~lamidegels. The purified cathepsin D-I and D-I1 and marker proteins were subjected to electrophoresis in polyacrylamide gels (7.5 %) containing 0.1 sodium dodecylsulfate. Other details are the same as described in Fig.5. The molecular weights of proteins were plotted on a semilogarithmic scale against the distance of migration from the top of the gel, relative to that of a tracking dye
Fig. 4. Polyacrylamide gel electrophoresis of cathepsin D-Iand D-II. Polyacrylamide gel electrophoresis was performed in 12 % polyacrylamide gel in Tris-HCl buffer, pH 8.9. Gels were stained for protein with Coomassie brilliant blue (A and B) and for carbohydrate with periodate-Schiff procedure (C and D). (A and C) Cathepsin D-I; (B and D) cathepsin D-I1
Polyacrylamide gel electrophoresis of the purified enzymes in the presence of sodium dodecylsulfate revealed a single protein band in each case (Fig.5A and B). These bands also gave positive periodate/ Schiff reaction (Fig.5C and D). Occasionally a very faint additional protein band was seen around a position of molecular weight of 25 000 on sodium dodecylsulfate/polyacrylamide gel electrophoresis of both enzymes. Molecular Weight
The estimation of the molecular weight of the purified enzymes was made with the calibrated column of Sephadex G-100. The activity peak of cathepsin D-I coincided with that of cathepsin D-11, corresponding to a molecular weight of about 44000. Molecular Weight of Subunits
The purified enzymes were subjected to electrophoresis in polyacrylamide gels containing 0.1 % sodium dodecylsulfate. Both enzymes migrated as a single protein band with a molecular weight of about 44000 (Fig. 6). Isoelectric Focusing
Fig. 5. Sodium d o d e c ~ l s u l f h t e ~ ~ o l y a c ~ ~ gel vlam electrophoresis i~~~~ of cathepsin D-I and D-II. The enzyme protein (30 pg protein each) was subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate after denaturation at 100°C after 3 min in 1 ‘%;sodium dodecylsulfate/l % mercaptoethanol. Electrophoresis was carried out in 7.5 % acrylamide gel containing 0.1 I,:,, sodium dodecylsulfate. Gels were stained for protein with Coomussic brilliant blue (A and B) and for carbohydrate with the pcrtodaic Schiff procedure (C and D). (A and C) cathepsin D-I; ( B and D) cathepsin D-I1
Cathepsin D-I and D-I1 were subjected to isoelectric focusing on the LKB 8101 column with a pH gradient of 4- 8 across the column. Fig. 7 shows the isoelectric focusing pattern of cathepsin D-I. Four distinct peaks of enzyme activity were resolved, which were designated as forms a,p, y and 6, having isoelectric points (pl) of about 4.2, 4.9, 6.1 and 6.5 respectively. The distribution of enzyme activity in the a, B, ’J and 6 forms accounted for about 12 %, 22 %, 35 ”/, and 10% of the total activity, respectively. The results indicated heterogeneity in cathepsin D-I. The resolu-
Cathepsin D of Rat Spleen
464
'I
Table 2. Enzyme action of cathepsin D-I and D-11 towards various protein substrates Enzyme activity towards bovine hemoglobin as substrate is 100%
0
1
0
2
0
3
0
4
0
5
Substrate in 0.1 M acetate buffer, pH 3.5
Concn
Bovine hemoglobin Bovine serum albumin Bovine serum albumin Human serum albumin Human serum albumin
1.25 2.5 5.0 2.5 5.0
Enzyme activity
D-I
D-I1
100 5 17 21 25
100 7 18 6 9
Substrate Specificity
0
Fraction number
Fig. 7. Isoelectr-ic, focir.sirig O/ (crthc,p.\in D-I. The enzyme solution was subjected 10 the column electrophoresis at 2 -C with a sucrose density gradient containing 1.75% pH3.5-10 and 0.75% p H 4 - 6 ampholytes. Fractions of 2 ml were collected after isoelectric focusing for 60 h at a constant voltage (600 V). The enzyme activity ( 0 )and pH (0)of each fraction were determined
I
9
Effect of Substrate Concentration
8
The effect of substrate concentration on the enzymes was determined at pH 3.8. Both enzyme activities were essentially maximal at 1.25 % substrate concentration. With increase in concentration above 2.5 %, appreciable inhibition was observed for both enzymes. This type of inhibition with the same substrate has been also shown in the cathepsin-E-like enzyme [24]. At present we can not find out a reason for the inhibition. Consequently the K, values for both enzymes were determined by employing substrate concentrations up to 2.5%. The K , values with bovine hemoglobin as substrate were 0.34 % for cathepsin D-I and 0.28 % for cathepsin D-11.
-00
7
6
5% 4 3 2 1
O
D
2
0
3
0 4 Fraction number
0
5
Table 2 gave the results of the proteolytic action on various proteins. The preferential substrate for both enzymes was bovine hemoglobin. These enzymes were also capable of splitting human and bovine serum albumin, although far less efficiently than bovine hemoglobin.
0
Fig. 8. lsoelectricfi,cusirig O/ c~rfhep.sinD-Il. Fractions of 2 ml were collected after isoelectric focusing for 60 h at a constant voltage (600 V). The enzyme activity ( 0 )and pH (0)of each fraction were determined. Other details are the same as described in Fig. 7
tion of these forms was always good and their relative positions were reproducible. Fig. 8 shows the isoelectric focusing pattern of cathepsin D-11. This enzyme also exhibited heterogeneity with 3 peaks whose p l values were about 4.6, 5.6 and 5.8. Ejject of p H The pH optimum for digestion of bovine hemoglobin was tested in both purified cathepsin D-I and D-11. The pH optima were around 3.8 for cathepsin D-I and around 3.5 for cathepsin D-11.
Temperature Optimum The temperature for optimum hydrolysis of bovine hemoglobin was close to 55 C for cathepsin D-I and 45 "C for cathepsin D-11. Heat Inactivation Fig.9 shows the heat inactivation of the purified enLqmes at 37 C and 60 C at different pH values (3.8 and 7.0). In the presence of sodium phosphate buffer, pH 7.0, 60% of the initial enzyme activities of both enzymes remained after 90 min at 37 "C, although in earlier incubation periods cathepsin D-I showed somewhat greater loss of activity than cathcpain 0-11. Replacement of sodium phosphate buffer, pH 7.0, by sodium acetate buffer, pH 3.8, caused faster loss
465
K. Yamamoto, N. Katsuda, M . Himeno, and K. Kato
B
A
__t
"
20 40 Incubatlon time
0
60
80
ao
100
a t 3 7 O C (min)
100
I n c u b a t i o n time at W"' (min)
Fig.9. Hrrrt inactivation of cathepsin D-I and D - f / . The enzyme activities of cathepsin D-I ( 0 )and D-I1 (0)are plotted as a function of the Sodium phosphate duraii(3n of heat treatment at 37°C (A) or 60°C (B). Activities are expressed as percentage of the initial activity. (-) bullci-. pll 7.0; (----) sodium acetate buffer, pH 3.8
Table 3. The effects of various compounds on the proteolytic activity of cathepsin D-I and D-II Eeach purified enzyme (12 pg) was preincubated with each compound at the respective concentrations for 5 min at 37°C. before the 40-min incubation with bovine hemoglobin. The values are means of at least four determinations. n.d., not determined
Concn
Compoiind
Enzyme activity D-I
D-I1
~~
%
Control NaCl MgClz ZnC12 CaC12 FeC13 Pb(N03)z KCN p-Chloromercuribenzoate Iodoacetic acid 2-Mercaptoethanol Phenylmethylsulfonyl fluoride Concanavalin A 7-Amino-I-chloro-3-tosylamido-~-heptan-2-one Na2EDTA Leupeptin Pepstatin Urea
-
10 m M 10 m M 10 m M 10 m M 10 m M 10 m M 10 mM 1 mM 10 mM 10 m M 10 mM 5 mM 1.0 mg 1 mM 10 m M 10 Pg 40 nM 6M
__ 100 100 91 100 97 13 92 100 92 41 86 100 t 00
110
100 100
92 0 3
100 100 100 137 90 25 41 97 95 65 89 92 n.d. 105 100 100 100
0 11
of the activities of both enzymes than at pH 7.0. At 60°C a large portion of both enzyme activities was lost within 10 min in the presence of sodium phosphate buffer, pH 7.0, or sodium acetate buffer, pH 3.8. Regardless of pH or temperature, the rate of inactiva-
tion of cathepsin D-I was faster than that of cathepsin D-11.
Effects of Activators and Inhibitors The effects of various activators and inhibitors on the activities of the purified enzymes are shown in Table 3. Fe3+ was a potent inhibitor of both enzymes. Cathepsin D-11 was inhibited by Pb2+ at a given concentration, whereas cathepsin D-I was hardly affected by it. Although Zn2+ had no effect on cathepsin D-I, cathepsin D-I1 was somewhat activated by it. The effect of preincubation with compounds which predominatly affect the thiol groups of proteins was investigated. The effects of cyanide, p-chloromercuribenzoate at a concentration of 1 mM, iodoacetic acid, and 2-mercaptoethanol were not observed in both enzymes. However, the appreciable inhibition by p-chloromercuribenzoate of both enzymes was found by the increasing the concentration up to 10 mM. Pepstatin and urea were powerful inhibitors of both enzymes. Amino Acid Composition The amino acid compositions of the purified cathepsin D-I and D-I1 calculated assuming the molecular weights to be 44000 are shown in Table 4. These enzymes had similar amino acid compositions, except for serine, proline, and methionine. Carbohydrate Composition qf Cathepsin D-I The carbohydrate composition of the major enzyme, cathepsin D-I, is shown in Table 5. The phenol/ sulfuric acid method indicated a neutral sugar content
Cathepsin D of Rat Spleen
466 Table 4. Amino acid compositions of cathepsin D-I and D-11 from rat spleen Results are based on a molecular weight of 44000 for each enzyme. n.d., not determined Amino acid
Cathepsin D-I
Cathepsin D-I1
pmol/mg protein
0.73 0.48 0.80 0.73 0.50 0.89 0.48 0.23 0.48 0.07 0.34 0.57 0.36 0.34 0.39 0.1 1 0.11 0.23
Aspartic acid Threonine" Serine" Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine" Phenylalanine Lysine Histidine Arginine Tryptophan
0.77 0.54 0.61 0.79 0.64 0.93 0.52 0.25 0.55 0.18 0.38 0.63 0.30 0.39 0.43 0.11 0.14 n.d.
a The values were obtained from extrapolation to zero time of hydrolysis. Determined spectrophotometrically 1391.
Table 5. Carbohydrate composition of rai spleen cathepsin D-I ______~___
Carbohydrate moiety
Mannose Galactose Glucose Fucose Glucosamine Sialic acid
~
~~
Proportion of cathepsin D-I by weight
molar ratio
9/100 g
mol/mol
3.36 0.30 0.64 0.34 1.93 0.05
8 1 2 1 5 -
of about 6 "/,. Qualitative and quantitative analyses of the carbohydrate moiety were performed by gas chromatography. Cathepsin D-I contained mannose, glucose, galactose, fucose and glucosamine in a ratio of 8 :2: 1 : 1 :5. A trace amount of sialic acid was detected in this enzyme by the method of Warren [34].About 6.6 "/,carbohydrate was found in the purified cathepsin D-I.
DISCUSSION In the present study the purification method using affinity chromatography with pepstatin - Sepharose 4B and concanavalin-A - Sepharose 4B makes the iso-
lation of cathepsin D from rat spleen much faster and larger in scale. Special attention should be given to the isolation of cathepsin D in order to minimize the proteolytic destruction. In view of partial degradation of cathepsin D during isolation, several other workers have reported that the isolated enzyme preparations sometimes contain a variety of polypeptide chains [8,14]. We !lave bcen able to isolate a pure and stable form of cathcp4n D without substantial proteolytic degradation. Itc affinity chromatography on pepstatin -- Sepharose 4B was the most important stepand facilitated the subsequent purification procedures. We also succeeded in separating two types of the enzyme, termed cathepsin D-I and D-I1 after DEAE-Sephacel chromatography. Overall yields of cathepsin D-I and D-I1 were about 20 % and 3 "/, with about 1000 and 900-fold increases in the specific activity respectively, (Table 1). The homogeneity of both enzymes was ascertained by the single protein band observed in polyacrylamide gel electrophoresis with (Fig. 5) and without (Fig. 4) sodium dodecylsulfate. Only on sodium dodecylsulfate/polyacrylamide gel electrophoresis, occasionally, was a very faint additional band seen around a position of molecular weight of 25000. This is presumably due to a partial degradation of the enzyme in vitro or in vivo. The molecular weights of both enzymes were estimated to be about 44000 by Sephadex G-100 chromatography. Analyses of both enzymes by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate showed that subunit molecular weights of the enzymes were about 44000, indicating that the enzymes may consist of a single polypeptide chain (Fig. 6). Both purified enzymes showed the multiple forms after isoelectric focusing. The isoelectric points of cathepsin D-I were about 4.2,4.9, 6.1 and 6.5 (Fig. 7), whereas those of cathepsin D-I1 were about 4.6, 5.6 and 5.8 (Fig. 8). Barrett [lI ] has described the presence of multiple forms of cathepsin D in chicken and human liver by isoelectric focusing. The isoelectric points of these enzymes were 5.1, 5.4, 5.7 and 5.7, 6.0, 6.5, respectively. Ferguson et al. [7] have reported that bovine spleen cathepsin D contained three multiple forms with p l values 6.1, 6.3 and 6.7. Furthermore, Smith and Turk [8] have also shown three multiple forms of cathepsin D with p l values 5.6, 5.9 and 6.4 after isolation of the enzyme from bovine spleen and thymus by the use of affinity chromatography with hemoglobin-Agarose resin. The amino acid composition of cathepsin D-I was similar to that of cathepsin D-I1 except for differences in serine, proline and methionine contents (Table 4). However, the amino acid composition of both enzymes were fairly different from that of the cathepsinE-like enzyme previously obtained from the same sources [24].
467
K. Yamamoto, N. Katsuda, M. Himeno, and K. Kato
The data obtained with various effectors, except for Zn2+ and Pb2+, indicated the resemblance of both enzymes (Table 3). Cathepsin D-I1 was somewhat activated by Zn2+ and inhibited by Pb 2 +, whereas cathepsin D-I was not affected by them. The effects of sulfhydryl reagents and urea on both enzymes were fairly different from those on the cathepsin-E-like enzyme [24]. Fe3+, pepstatin and urea were very powerful inhibitors of both cathepsin D-I and D-11. Both purified enzymes were apparently glycoproteins because of their binding to concanavalin-A Sepharose 4B and their positive staining with periodate/Schiff procedure on polyacrylamide gel electrophoresis in the absence and presence of sodium dodecylsulfate (Fig. 4 and 5). Accordingly we have analyzed the carbohydrate composition of cathepsin D-I, which comprises the major portion of cathepsin D activity in rat spleen. 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. Although a number of cathepsin D preparations have been isolated from various tissues, the carbohydrate composition of the enzyme has not yet been elucidated. Some investigators showed the presence of glucosamine in cathepsin D by the technique of amino acid analysis [2,16]. It is noteworthy thai rat liver lysosomal ,&glucuronidase [25] and WLfucosidase [26] contain the same five sugars. The content of sialic acid in cathepsin D-I was only 0.05 %. However, this value may be increased by determination of sialic acid in the more acidic forms of the enzyme resolved by isoelectric focusing, as reported by Himeno et al. [25]. They showed the presence of sialic acid (0.11 %) in the more acidic forms of lysosoma1 8-glucuronidase purified from rat liver. Probably, a small part of cathepsin D-I may be a sialoglycoprotein.
REFERENCES 1. Barrett, A. J. (1969) in Lysosomes in Biology and Pathology (Dingle, J . T. & Fell, H. B., eds) 2nd edn, pp. 245-312, North Holland Publishing Co., Amsterdam 2. Barrett, A. J. (1977) in Proteinases in Mammalian Cells and Tissues (Barrett, A. J., ed.) pp. 209-248, North Holland Publishing Co., Amsterdam 3 . SchiiiiLc. R . T. ( 1975) in Iitrracellular Prorein Tirriiover (Schimke, R. T. h K:it,uXiuiiia. N., eds) pp. 173-183, Academic Press. Nc\\ j ' o c h . 4. Gordon, A. H . (1973) i n L.,iwsomes in Biology and Pathology (Dingle, J . T., ed.) 3rd edn, pp. 89- 137, North Holland Publishing Co., Amsterdam 5. Woessner, J. F., Jr (1971) in Tissue Proteinases (Barrett, A. J. & Dingle. J. T., eds) pp. 291 -308, North Holland Publishing Co., Amsterdam.
6. Press, E. M., Porter, R. R. & Cebra, J. (1960) Biochem. J . 74, 501-514. 7. Ferguson, J. B., Andrews, J. R., Voynick, I. M. & Fruton, J. S. (1973) J . Biol. Chem. 248. 6701 -6708. 8. Smith, R. & Turk, V. (1974) E w . J . Biocheni. 4s'. 245--254. 9. Cunningham, M. & Tans. J . ( 1 976) .I. Riol. Chcru. 2 5 / . 3528 4536. 10. Barrett, A. J. (1967) Biochem. J . 104, 601 -608. 11. Barrett, A. J. (1970) Biochem. J . 117, 601 -607. 12. Kazakova, 0. V. & Orekhovich, V. N. (1976) Biochem. Biophys. Res. Commun. 72,747-752. 13. Woessner, J. F., Jr & Shamberger, R. J., Jr (1971) J . Biol. Chem. 246,1951 - 1960. 14. Sapolsky, A. I. & Woessner, J. F., Jr (1972) J . Biol. Chem. 247, 2069 - 2076. 15. Kress, L. F., Peanasky, R. J . & Klitgaard, H. M. (1966) Biochim. Biophys. Acta, 113, 375 - 389. 16. Smith, G. D., Murray, L. W., Nichol, L. W. & Trikojus, V. M. (1969) Biochim. Biophys. Acta, 171, 288-298. 17. Kregar, I., Turk, V. & Lebez, D. (1967) Enzymologia, 33, 80-88. 18. Rojas-Espinosa, O., Dannenberg, A. M., Jr, Murphy, P. A,, Straat, P. A,, Huang, P. C. & James, S. P. (1973) Infect. Immun. 8, 1000- 1008. 19. Moriyama, A. & Takahashi, K. (1976) J . Biochem. (Tokyo) 83, 441-451. 20. Reichelt, D., Jacobsohn, E. & Haschen, R. J. (1974) Biochim. Biophys. Acta, 341, 15- 26. 21. Gubenstk, F., Barstow, L., Kregar, I. & Turk, V. (1976) FEBS Lett. 71,42-44. 22. Kregar, I., Urh. I . _ Smith, R., Umezawa. H. & Turk, V. (1977) in Intracellului. t'ivtein Catabolism (Turk, V. & Marks, N., eds) 2nd edn. pp. 250-254, Plenum Press, New York. 23. Yamamoto, K., Katsuda, N. & Kato, K. (1978) Seikagaku, 50, 889- 890. 24. Yamamoto, K., Katsuda, N. & Kato, K. (1978) Eur. J . Biochem. 92, 499-508. 25. Himeno, M., Nishimura,Y., Takahashi,K. & Kato, K. (1978) J . Biochem. (Tokyo) 83, 511-518. 26. Opheim, D. J. & Touster, 0. (1977) J . Biol. Chem. 252, 739743. 27. Anson, M. L. (1939) J . Gen. Physiol. 22, 79-89. 28. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J . Biol. Chem. 193, 265-275 29. Davis, B. J. (1964) Ann. N . Y. Acad. Sci. 121, 404-427. 30. Zacharius, R. M., Zell, T. E., Morrison, J. H. & Woodlock, J. J. (1969) Anal. Biochem. 30, 148- 152. 31. Fairbanks, G., Steck, T. L. & Wallach, D. F. H. (1971) Biochemistry, 10, 2606 - 2617. 32. Andrews, P. (1964) Biochem. J . 91, 222-233. 33. Murakami, K. & Inagami, T. (1975) Biochem. Biophys. Res. Commun. 62,757 - 763. 34. Warren, L. (1959) J . Biol. Chem. 234, 1971-1975. 35. Dubois, M., Gilles, K. A,, Hamilton, J. K., Rebers, P. A. & Smith. F. (1956) Anal. Chem. 28, 350-356. 36. Matsui, M., Okada, M., Imanari, T. & Tamura, Z. (1968) Chem. Pharm. Bull. 16, 1383- 1387. 37. Tamura, Z., Imanari, T. & Akazawa, Y. (1968) Chem. Pharm. Bull. 16, 1864-1865. 38. Spackman, D. H., Stein, W. H. & Moore, S. (1958) Anal. Chem. 30, 1190-1206. 39. Goodwin, T. W. & Morton, R. A. (1946) Biochem. J . 40, 628 - 632.
K. Yamamoto and N. Katsuda, Department of Pharmacology, Faculty of Dentistry, Kyushu University, Higashi-ku, Fukuoka-shi, Fukuoka-ken, Japan 812 M. Himeno and K . Kato, Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyushu University, Higashi-ku, Fukuoka-shi, Fukuoka-ken, Japan 812
Cathepsin D of Rat Spleen Affinity Purification and Properties of Two Types of Cathepsin D Kenji YAMAMOTO, Nobuo KATSUDA, Masaru HIMENO, and Keitaro KATO Department of Pharmacology, Faculty of Dentistry and Department of Physiological Chemistry, F x u l t y of Pharmaceutical Sciences, Kyushu bnivehiLy (Received October 21, 1978/January 4, 1979)
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 Enzyyme. Cathepsin D (EC 3.4.23.5).
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 isolation of another acid proteinase, cathepsin D, we succeeded in separating two types of cathepsin D, cathepsin D-I and D-11, by DEAE-Sephacel chromatography. Both enzymes revealed 4 and 3 multiple forms respectively after isoelectric focusing, as reported previously by several workers [7,8,11]. Cethepsin D is also an interesting enzyme because of its value in an investigation of the biosynthetic route and mechanism of the packaging of lysosomal enzymes. It is now evident that almost all lysosomal enzymes are glycoproteins. Rat liver lysosomal 8-glucuronidase [25] and cc-L-fucosidase [26] were shown to contain five sugars; mannose, glucosamine, glucose, galactose and fucose. Therefore, the carbohydrate composition of cathepsin D is of special interest, and
Cathepsin D of Rat Spleen
460
in order to clarify the role of carbohydrate moieties in the translocation of lysosomal enzymes, cathepsin D purified from rat spleen was also subjected to chemical analysis. In the present paper we describe the purification and properties of the two enzymes, cathepsin D-I and D-11. We also determined the carbohydrate composition of cathepsin D-I and compared the carbohydrate moiety with those of other lysosomal enzymes [25,26].
MATERIALS AND METHODS Materials Chemicals were obtained from the following sources : bovine hemoglobin (Typ II), bovine serum albumin (fraction V) and phenylmethylsulfonyl fluoride from Sigma Chemical Co. Pepstatin and leupeptin were from The Protein Research Foundation (Osaka, Japan). Sephadex G-100, DEAE-Sephacel, AH-Sepharose 4B and concanavalin-A - Sepharose 4B were from Pharmacia. Ampholytes were from LKB Instruments Inc. All other chemicals were of reagent grade, purchased from various commercial sources.
of Davis [29]. Gels were stained for protein with Coomassie brilliant blue and destained in 7 % acetic acid and for carbohydrate by the periodate/Schiff procedure described by Zacharius et al. [30]. Sodium dodecylsulfate/polyacrylamide gels were prepared by a slight modification of the method of Fairbanks et al. [31] as described previously [24]. Enzyme samples were solubilized by incubation at 100 "C for 3 min after addition of the following compounds (to the indicated final concentration); 1 % sodium dodecylsulfate, 1 % 2-mercaptoethanol, 10 % sucrose, 0.01 M Tris-HC1, pH 8.0, and 0.001 M EDTA. Gels were stained with Coomassie brilliant blue followed by complete destaining. Staining for carbohydrate was done by the method of Zacharius et al.
POI. Molecular Weight Determination A Sephadex G-100 column (1.6 x 90 cm) for molecular weight determinations was used as described by Andrews [32], except that 0.005 M sodium phosphate buffer, pH 7.0, containing 0.2 M NaCl was used. Subunit molecular weight was determined by sodium dodecylsulfate/polyacrylamide gel electrophoresis as mentioned above.
Enzyme Assay Cathepsin D activity was determined by a modification of the method of Anson [27]. The reaction mixture contained 0.5 mlO.1 M sodium acetate buffer, pH 3.8, 1.O ml buffered hemoglobin solution (2.5 %, w/v, in 0.1 M sodium acetate buffer, pH 3.8). To this was added 5- 100 pl of enzyme solution and the final volume was adjusted to 2.0 ml with distilled water. After a 40-min incubation at 37 "C, the reaction was stopped by plunging the tubes into ice and adding 2.0 ml of 5 % ice-cold trichloroacetic acid solution. The precipitated mixture was allowed to stand for 10 min in ice and then centrifuged. The liberated peptides in the supernatant (1.0 ml) were measured by the FolinLowry reaction [28]. The enzyme activity was determined with reference to a tyrosine standard curve. The enzyme unit, U, was defined as pg tyrosine solubilized/ min. The absorbance at 660nm in the assay was 0.08-0.40 in order to retain linearity with respect to enzyme concentration. Protein Determination Protein was determined by the method of Lowry et al. [28] with bovine serum albumin as standard.
Other Column Chromatography Concanavalin-A - Sepharose 4B, Sephadex G-100, and DEAE-Sephacel were used as recommended by the manufacturer's instructions. Preparation of Affinity Column oj' Pepstatin-Sepharose 4B Pepstatin was coupled to AH-Sepharose 4B by the method of Murakami and Inagami [33]. Isoelectric Focusing Sucrose gradient isoelectric focusing was carried out in a LKB 8101 column (1 10 ml capacity) according to the manufacturer's instructions. The pH gradient was stabilized with a stepwise sucrose gradient of 0-56% (w/v). The samples of cathepsin D were layered in the middle of the gradient and electrofocused at 600 V for 60 h. After isoelectric focusing fractions of 2 ml were collected, and the cathepsin D activity and pH of each fraction were measured. Heat Inactivation
Polyacrylamide Gel Electrophoresis Polyacrylamide gel electrophoresis was performed in 12 % gels in Tris-HC1 buffer, pH 8.9, by the method
Enzyme samples were adjusted to pH 7.0 (0.1 M sodium phosphate buffer) and pH 3.8 (0.1 M sodium acetate buffer) respectively. Samples (0.1 ml, 100 pg protein/ml) of each buffered enzyme solution were
46 1
K. Yamamoto, N. Katsuda, M. Himeno, and K. Kato
Table 1. Purification of cathepsin D from rat spleen Starting material was 370 g (wet weight) spleen. The proteolytic activity was determined under the conditions described in the text. The first six rows contain the combined activities of cathepsin-D and E-like enzymes, whereas the last three rows refer to cathepsin D only. Values in parentheses are those corrected by calculating the quantity of cathepsin D in the homogenate to be 60 % Step
Homogenate Spleen extract Ammonium sulfate Acid treatment Pepstatin - Sepharose 4B Concanavalin-A - Sepharose 4B Sephadex G-I00 DEAE-Sephacel : peak-I (cathepsin D-I) peak-I1 (cathepsin D-11)
Protein
Total activity
Specific activity
Yield
Purification factor
mg
mU
U/mg protein
%
-fold
88995 56 794 18808 5 836 115 60 28
801.9 152.9 685.9 486.5 428.2 350.3 186.7
9 13 36 83 3732 5838 6668
100 94 86 61 53 44 23 (38)
1 1.4 4.0 9.2 414.6 648.7 740.9
10 2
93.4 18.7
9526 8118
12 (20) 2 (3)
1058.4 902.0
incubated at 37 "C or 60 "C for periods of up to 90 min. Then they were chilled and remaining enzyme activity was measured. Determination of Sialic Acid Sialic acid was determined by the method of Warren [34] with N-acetylneuraminic acid as a standard. Determination of Hexose and Hexosamine Neutral sugar was determined by the phenol/sulfuric acid method of Dubois et al. [35]. The purified cathepsin D-I was hydrolyzed in 2.5 M trifluoroacetic acid for 8 h at 100°C. The hydrolysate was passed through an Amberlite C G 4 B (CH&OO-) column (0.8 x 12 cm) and an Amberlite CG-120 (H') column (0.8 x 12 cm) in order to separate neutral and amino sugars. For identification and quantitative analysis of each monosaccharide in the carbohydrate moiety, gas-liquid chromatography was carried out after reduction of monosaccharides to the corresponding alditols and hexosaminols, followed by trifluoroacetylation, according to the methods of Matsui et al. [36] and Tamura et al. [37]. Gas chromatography was performed on a Schimazu GC-3BF unit. Amino Acid Analysis The enzyme samples were dialyzed against distilled water and hydrolyzed under a vacuum in 6 M HCI for 24, 48 and 72 h at 110°C. Amino acids were analyzed on a Hitachi model 835 zutomatic aminoacid analyzer by the method of Spackman et al. [38]. Tryptophan content was determined spectrophotometrically [39].
RESULTS PURIFICATION OF CATHEPSIN D
Enzyme Extraction and Partial Purification by Ammonium Sulfate Fractionation, Precipitation at p H 3 3 , and Affinity Chromatography with Pepstatin - Sepharose 4B and Concanavalin-A - Sepharose 4 B In the present work the purification procedures of cathepsin D up to the step of concanavalin-A - Sepharose 4B affinity chromatography were carried out under the same conditions as previously employed for the purification of the cathepsin-E-like enzyme from rat spleen [24].
Gel Filtration The enzyme fractions obtained by concanavalinA- Sepharose 4B affinity chromatography (Fig. 1) were concentrated and dialyzed against 0.02 M sodium phosphate buffer, pH 7.0, and then applied to a column, 2.5 x 90 cm, of Sephadex G-100, which had been equilibrated with 0.02 M sodium phosphate buffer, pH 7.0. The column was eluted with the same buffer at a rate of 6 ml/h and fractions of 3 ml were collected. The elution pattern is presented in Fig.2. Two peaks of the proteolytic activity that hydrolyzed bovine hemoglobin at pH 3.8 were found. The first peak, having a molecular weight of about 90000, contained about 40 % of the total activity and was identified as a cathepsin-E-like acid proteinase as described previously [24]. Cathepsin D activity was recovered in the second peak. The fractions between 65 and 75 were collected, concentrated and then dialyzed against 0.005 M sodium phosphate buffer, pH 7.0. At this step the specific activity was about 6700 units, indi-
Cathepsin D of Rat Spleen
462 OiI
0.3
d 0.2
p"
0.1
0
o
2
o
m
~
~
o
~
r
n
m
w
m
0
Fraction number Fraction number
Fig. 1. Concanavalin-A - Sepharose 4 B affinity chromatographv of the enzyme fraction obtained by pepstatin-Sepharose 4 B affinty chroIriutography. The enzyme solution after pepstatin-Sepharose 4B ;il.linity chromatography was applied to a column, 2.5 x 5.0 cm, of concanavalin-A - Sepharose 4B equilibrated with 0.02 M sodium phosphate buffer, pH 7.0, containing 1 M NaCI. After exhaustive washing, 0.2 M methyl r-D-glucoside was added to the buffer at the point indicated by the arrow. The flow rate was 9 mlih and fractions of 5 ml were collected. ( 0 )Cathepsin activity; (----) protein
I4 1500
-E
I
. .-
33
u)
I
- K)oO c 3
ZI
c ._ 2. .c
0.2
m
9
D
.-c
B m
0 1
r I
3 0
0
5 3 6 0 7 0 8 0 9 0 Fraction number
Fig. 2. Gel filtration on Scphudex GI00 of' the enzyme fraction obtained by concanavalin-A - Sepharose 4 B affinity chromatography. The enzyme solution after concanavalin-A -Sepharose 4B affinity chromatography was applied to a column, 2.5 x 90 cm, equilibrated with 0.02 M sodium phosphate buffer, pH 7.0. The flow rate was 6 ml/h and fractions of 3 ml were collected. (0)Cathepsin activity; (----) protein
cating about 740-fold purification over the homogenate. Since about 40 % of the enzyme activity present in rat spleen extract is due to cathepsin-E-like enzyme, the yield of cathepsin D is practically about 38%. Therefore, the yields in all subsequent steps were corrected by calculating a quantity of cathepsin D in the homogenate to be 60 %.
D EA E-Sephacel Chromatography The enzyme preparation from the previous step was applied to a column, 1.6 x 12 cm, of DEAE-Seph-
Fig. 3. DEAE-Sephacel column cliromutography of' the enzyme fraction obtained by Sephadex G-100 chromatography. The enzyme fraction after gel filtration on Sephadex G-100 was applied to a column, 1.6 x 12 cm, of DEAE-Sephacel equilibrated with 0.005 M sodium phosphate buffer, pH 7.0. Elution was carried out with the same buffer. The arrows indicate the change of sodium chloride concentration: (1); 0.02 M, (2); 0.05 M. The flow rate was 6 ml/h and fractions of 3 ml were collected. ( 0 ) Cathepsin activity; (----) protein
ace1 equilibrated with 0.005 M sodium phosphate buffer, pH 7.0. The column was washed with the same buffer and the enzyme was then eluted stepwise with NaCl in the buffer. Two peaks of enzyme activity were obtained by elution at 0.05 M NaCl. A typical elution profile is shown in Fig. 3 . The major peak that represented 70 - 80 % yield of the applied activity was designated as 'cathepsin D-1'. The minor peak that represented 10- 15 % yield was designated as 'cathepsin D-II'. These two peaks of enzyme activity marked with the bar were pooled separately and concentrated with a collodion bag. The purification ratios of cathepsin D-I and D-I1 were about 1000-fold and 900-fold respectively over the homogenate. Further purification procedure using isoelectric focusing neither increased the specific activity nor changed the polyacrylamide gel electrophoretic pattern of each enzyme. DEAESephacel chromatography also achieved the complete separation of cathepsin D from the cathepsin-E-like enzyme that was eluted at 0.3 M NaCl under the same condition. A summary of a representative purification is shown in Table 1.
PROPERTIES OF THE PURIFIED ENZYMES
Polyucrylumide Gel Electrophoresis On polyacrylamide gel electrophoresis of purified cathepsin D-I and D-11, each revealed a single protein band when stained for protein with Coomassie brilliant blue and for carbohydrate by the periodate/Schiff procedure (Fig. 4). Cathepsin D-I1 moved somewhat faster than cathepsin D-I.
463
K. Yamamoto, N. Katsuda, M. Himeno, and K. Kato 10 *
-In -x L
Cathepin D - I and D - I I 5.
3
p
4. 3.
U
Q 2 1 0.1
0.2
0.3
0.4 Relative
Q5 Q6 mobility
07
00
Fig. 6 . Detrwninu/ion uf thc .\iihiiiii/ n ~ i ~ l keeights ~ ~ ~ of u cathepsin l ~ ~ ~ ~ D-I and D-II by dectrophoresis on sodium dodecylsulfate/polyacr~lamidegels. The purified cathepsin D-I and D-I1 and marker proteins were subjected to electrophoresis in polyacrylamide gels (7.5 %) containing 0.1 sodium dodecylsulfate. Other details are the same as described in Fig.5. The molecular weights of proteins were plotted on a semilogarithmic scale against the distance of migration from the top of the gel, relative to that of a tracking dye
Fig. 4. Polyacrylamide gel electrophoresis of cathepsin D-Iand D-II. Polyacrylamide gel electrophoresis was performed in 12 % polyacrylamide gel in Tris-HCl buffer, pH 8.9. Gels were stained for protein with Coomassie brilliant blue (A and B) and for carbohydrate with periodate-Schiff procedure (C and D). (A and C) Cathepsin D-I; (B and D) cathepsin D-I1
Polyacrylamide gel electrophoresis of the purified enzymes in the presence of sodium dodecylsulfate revealed a single protein band in each case (Fig.5A and B). These bands also gave positive periodate/ Schiff reaction (Fig.5C and D). Occasionally a very faint additional protein band was seen around a position of molecular weight of 25 000 on sodium dodecylsulfate/polyacrylamide gel electrophoresis of both enzymes. Molecular Weight
The estimation of the molecular weight of the purified enzymes was made with the calibrated column of Sephadex G-100. The activity peak of cathepsin D-I coincided with that of cathepsin D-11, corresponding to a molecular weight of about 44000. Molecular Weight of Subunits
The purified enzymes were subjected to electrophoresis in polyacrylamide gels containing 0.1 % sodium dodecylsulfate. Both enzymes migrated as a single protein band with a molecular weight of about 44000 (Fig. 6). Isoelectric Focusing
Fig. 5. Sodium d o d e c ~ l s u l f h t e ~ ~ o l y a c ~ ~ gel vlam electrophoresis i~~~~ of cathepsin D-I and D-II. The enzyme protein (30 pg protein each) was subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate after denaturation at 100°C after 3 min in 1 ‘%;sodium dodecylsulfate/l % mercaptoethanol. Electrophoresis was carried out in 7.5 % acrylamide gel containing 0.1 I,:,, sodium dodecylsulfate. Gels were stained for protein with Coomussic brilliant blue (A and B) and for carbohydrate with the pcrtodaic Schiff procedure (C and D). (A and C) cathepsin D-I; ( B and D) cathepsin D-I1
Cathepsin D-I and D-I1 were subjected to isoelectric focusing on the LKB 8101 column with a pH gradient of 4- 8 across the column. Fig. 7 shows the isoelectric focusing pattern of cathepsin D-I. Four distinct peaks of enzyme activity were resolved, which were designated as forms a,p, y and 6, having isoelectric points (pl) of about 4.2, 4.9, 6.1 and 6.5 respectively. The distribution of enzyme activity in the a, B, ’J and 6 forms accounted for about 12 %, 22 %, 35 ”/, and 10% of the total activity, respectively. The results indicated heterogeneity in cathepsin D-I. The resolu-
Cathepsin D of Rat Spleen
464
'I
Table 2. Enzyme action of cathepsin D-I and D-11 towards various protein substrates Enzyme activity towards bovine hemoglobin as substrate is 100%
0
1
0
2
0
3
0
4
0
5
Substrate in 0.1 M acetate buffer, pH 3.5
Concn
Bovine hemoglobin Bovine serum albumin Bovine serum albumin Human serum albumin Human serum albumin
1.25 2.5 5.0 2.5 5.0
Enzyme activity
D-I
D-I1
100 5 17 21 25
100 7 18 6 9
Substrate Specificity
0
Fraction number
Fig. 7. Isoelectr-ic, focir.sirig O/ (crthc,p.\in D-I. The enzyme solution was subjected 10 the column electrophoresis at 2 -C with a sucrose density gradient containing 1.75% pH3.5-10 and 0.75% p H 4 - 6 ampholytes. Fractions of 2 ml were collected after isoelectric focusing for 60 h at a constant voltage (600 V). The enzyme activity ( 0 )and pH (0)of each fraction were determined
I
9
Effect of Substrate Concentration
8
The effect of substrate concentration on the enzymes was determined at pH 3.8. Both enzyme activities were essentially maximal at 1.25 % substrate concentration. With increase in concentration above 2.5 %, appreciable inhibition was observed for both enzymes. This type of inhibition with the same substrate has been also shown in the cathepsin-E-like enzyme [24]. At present we can not find out a reason for the inhibition. Consequently the K, values for both enzymes were determined by employing substrate concentrations up to 2.5%. The K , values with bovine hemoglobin as substrate were 0.34 % for cathepsin D-I and 0.28 % for cathepsin D-11.
-00
7
6
5% 4 3 2 1
O
D
2
0
3
0 4 Fraction number
0
5
Table 2 gave the results of the proteolytic action on various proteins. The preferential substrate for both enzymes was bovine hemoglobin. These enzymes were also capable of splitting human and bovine serum albumin, although far less efficiently than bovine hemoglobin.
0
Fig. 8. lsoelectricfi,cusirig O/ c~rfhep.sinD-Il. Fractions of 2 ml were collected after isoelectric focusing for 60 h at a constant voltage (600 V). The enzyme activity ( 0 )and pH (0)of each fraction were determined. Other details are the same as described in Fig. 7
tion of these forms was always good and their relative positions were reproducible. Fig. 8 shows the isoelectric focusing pattern of cathepsin D-11. This enzyme also exhibited heterogeneity with 3 peaks whose p l values were about 4.6, 5.6 and 5.8. Ejject of p H The pH optimum for digestion of bovine hemoglobin was tested in both purified cathepsin D-I and D-11. The pH optima were around 3.8 for cathepsin D-I and around 3.5 for cathepsin D-11.
Temperature Optimum The temperature for optimum hydrolysis of bovine hemoglobin was close to 55 C for cathepsin D-I and 45 "C for cathepsin D-11. Heat Inactivation Fig.9 shows the heat inactivation of the purified enLqmes at 37 C and 60 C at different pH values (3.8 and 7.0). In the presence of sodium phosphate buffer, pH 7.0, 60% of the initial enzyme activities of both enzymes remained after 90 min at 37 "C, although in earlier incubation periods cathepsin D-I showed somewhat greater loss of activity than cathcpain 0-11. Replacement of sodium phosphate buffer, pH 7.0, by sodium acetate buffer, pH 3.8, caused faster loss
465
K. Yamamoto, N. Katsuda, M . Himeno, and K. Kato
B
A
__t
"
20 40 Incubatlon time
0
60
80
ao
100
a t 3 7 O C (min)
100
I n c u b a t i o n time at W"' (min)
Fig.9. Hrrrt inactivation of cathepsin D-I and D - f / . The enzyme activities of cathepsin D-I ( 0 )and D-I1 (0)are plotted as a function of the Sodium phosphate duraii(3n of heat treatment at 37°C (A) or 60°C (B). Activities are expressed as percentage of the initial activity. (-) bullci-. pll 7.0; (----) sodium acetate buffer, pH 3.8
Table 3. The effects of various compounds on the proteolytic activity of cathepsin D-I and D-II Eeach purified enzyme (12 pg) was preincubated with each compound at the respective concentrations for 5 min at 37°C. before the 40-min incubation with bovine hemoglobin. The values are means of at least four determinations. n.d., not determined
Concn
Compoiind
Enzyme activity D-I
D-I1
~~
%
Control NaCl MgClz ZnC12 CaC12 FeC13 Pb(N03)z KCN p-Chloromercuribenzoate Iodoacetic acid 2-Mercaptoethanol Phenylmethylsulfonyl fluoride Concanavalin A 7-Amino-I-chloro-3-tosylamido-~-heptan-2-one Na2EDTA Leupeptin Pepstatin Urea
-
10 m M 10 m M 10 m M 10 m M 10 m M 10 m M 10 mM 1 mM 10 mM 10 m M 10 mM 5 mM 1.0 mg 1 mM 10 m M 10 Pg 40 nM 6M
__ 100 100 91 100 97 13 92 100 92 41 86 100 t 00
110
100 100
92 0 3
100 100 100 137 90 25 41 97 95 65 89 92 n.d. 105 100 100 100
0 11
of the activities of both enzymes than at pH 7.0. At 60°C a large portion of both enzyme activities was lost within 10 min in the presence of sodium phosphate buffer, pH 7.0, or sodium acetate buffer, pH 3.8. Regardless of pH or temperature, the rate of inactiva-
tion of cathepsin D-I was faster than that of cathepsin D-11.
Effects of Activators and Inhibitors The effects of various activators and inhibitors on the activities of the purified enzymes are shown in Table 3. Fe3+ was a potent inhibitor of both enzymes. Cathepsin D-11 was inhibited by Pb2+ at a given concentration, whereas cathepsin D-I was hardly affected by it. Although Zn2+ had no effect on cathepsin D-I, cathepsin D-I1 was somewhat activated by it. The effect of preincubation with compounds which predominatly affect the thiol groups of proteins was investigated. The effects of cyanide, p-chloromercuribenzoate at a concentration of 1 mM, iodoacetic acid, and 2-mercaptoethanol were not observed in both enzymes. However, the appreciable inhibition by p-chloromercuribenzoate of both enzymes was found by the increasing the concentration up to 10 mM. Pepstatin and urea were powerful inhibitors of both enzymes. Amino Acid Composition The amino acid compositions of the purified cathepsin D-I and D-I1 calculated assuming the molecular weights to be 44000 are shown in Table 4. These enzymes had similar amino acid compositions, except for serine, proline, and methionine. Carbohydrate Composition qf Cathepsin D-I The carbohydrate composition of the major enzyme, cathepsin D-I, is shown in Table 5. The phenol/ sulfuric acid method indicated a neutral sugar content
Cathepsin D of Rat Spleen
466 Table 4. Amino acid compositions of cathepsin D-I and D-11 from rat spleen Results are based on a molecular weight of 44000 for each enzyme. n.d., not determined Amino acid
Cathepsin D-I
Cathepsin D-I1
pmol/mg protein
0.73 0.48 0.80 0.73 0.50 0.89 0.48 0.23 0.48 0.07 0.34 0.57 0.36 0.34 0.39 0.1 1 0.11 0.23
Aspartic acid Threonine" Serine" Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine" Phenylalanine Lysine Histidine Arginine Tryptophan
0.77 0.54 0.61 0.79 0.64 0.93 0.52 0.25 0.55 0.18 0.38 0.63 0.30 0.39 0.43 0.11 0.14 n.d.
a The values were obtained from extrapolation to zero time of hydrolysis. Determined spectrophotometrically 1391.
Table 5. Carbohydrate composition of rai spleen cathepsin D-I ______~___
Carbohydrate moiety
Mannose Galactose Glucose Fucose Glucosamine Sialic acid
~
~~
Proportion of cathepsin D-I by weight
molar ratio
9/100 g
mol/mol
3.36 0.30 0.64 0.34 1.93 0.05
8 1 2 1 5 -
of about 6 "/,. Qualitative and quantitative analyses of the carbohydrate moiety were performed by gas chromatography. Cathepsin D-I contained mannose, glucose, galactose, fucose and glucosamine in a ratio of 8 :2: 1 : 1 :5. A trace amount of sialic acid was detected in this enzyme by the method of Warren [34].About 6.6 "/,carbohydrate was found in the purified cathepsin D-I.
DISCUSSION In the present study the purification method using affinity chromatography with pepstatin - Sepharose 4B and concanavalin-A - Sepharose 4B makes the iso-
lation of cathepsin D from rat spleen much faster and larger in scale. Special attention should be given to the isolation of cathepsin D in order to minimize the proteolytic destruction. In view of partial degradation of cathepsin D during isolation, several other workers have reported that the isolated enzyme preparations sometimes contain a variety of polypeptide chains [8,14]. We !lave bcen able to isolate a pure and stable form of cathcp4n D without substantial proteolytic degradation. Itc affinity chromatography on pepstatin -- Sepharose 4B was the most important stepand facilitated the subsequent purification procedures. We also succeeded in separating two types of the enzyme, termed cathepsin D-I and D-I1 after DEAE-Sephacel chromatography. Overall yields of cathepsin D-I and D-I1 were about 20 % and 3 "/, with about 1000 and 900-fold increases in the specific activity respectively, (Table 1). The homogeneity of both enzymes was ascertained by the single protein band observed in polyacrylamide gel electrophoresis with (Fig. 5) and without (Fig. 4) sodium dodecylsulfate. Only on sodium dodecylsulfate/polyacrylamide gel electrophoresis, occasionally, was a very faint additional band seen around a position of molecular weight of 25000. This is presumably due to a partial degradation of the enzyme in vitro or in vivo. The molecular weights of both enzymes were estimated to be about 44000 by Sephadex G-100 chromatography. Analyses of both enzymes by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate showed that subunit molecular weights of the enzymes were about 44000, indicating that the enzymes may consist of a single polypeptide chain (Fig. 6). Both purified enzymes showed the multiple forms after isoelectric focusing. The isoelectric points of cathepsin D-I were about 4.2,4.9, 6.1 and 6.5 (Fig. 7), whereas those of cathepsin D-I1 were about 4.6, 5.6 and 5.8 (Fig. 8). Barrett [lI ] has described the presence of multiple forms of cathepsin D in chicken and human liver by isoelectric focusing. The isoelectric points of these enzymes were 5.1, 5.4, 5.7 and 5.7, 6.0, 6.5, respectively. Ferguson et al. [7] have reported that bovine spleen cathepsin D contained three multiple forms with p l values 6.1, 6.3 and 6.7. Furthermore, Smith and Turk [8] have also shown three multiple forms of cathepsin D with p l values 5.6, 5.9 and 6.4 after isolation of the enzyme from bovine spleen and thymus by the use of affinity chromatography with hemoglobin-Agarose resin. The amino acid composition of cathepsin D-I was similar to that of cathepsin D-I1 except for differences in serine, proline and methionine contents (Table 4). However, the amino acid composition of both enzymes were fairly different from that of the cathepsinE-like enzyme previously obtained from the same sources [24].
467
K. Yamamoto, N. Katsuda, M. Himeno, and K. Kato
The data obtained with various effectors, except for Zn2+ and Pb2+, indicated the resemblance of both enzymes (Table 3). Cathepsin D-I1 was somewhat activated by Zn2+ and inhibited by Pb 2 +, whereas cathepsin D-I was not affected by them. The effects of sulfhydryl reagents and urea on both enzymes were fairly different from those on the cathepsin-E-like enzyme [24]. Fe3+, pepstatin and urea were very powerful inhibitors of both cathepsin D-I and D-11. Both purified enzymes were apparently glycoproteins because of their binding to concanavalin-A Sepharose 4B and their positive staining with periodate/Schiff procedure on polyacrylamide gel electrophoresis in the absence and presence of sodium dodecylsulfate (Fig. 4 and 5). Accordingly we have analyzed the carbohydrate composition of cathepsin D-I, which comprises the major portion of cathepsin D activity in rat spleen. 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. Although a number of cathepsin D preparations have been isolated from various tissues, the carbohydrate composition of the enzyme has not yet been elucidated. Some investigators showed the presence of glucosamine in cathepsin D by the technique of amino acid analysis [2,16]. It is noteworthy thai rat liver lysosomal ,&glucuronidase [25] and WLfucosidase [26] contain the same five sugars. The content of sialic acid in cathepsin D-I was only 0.05 %. However, this value may be increased by determination of sialic acid in the more acidic forms of the enzyme resolved by isoelectric focusing, as reported by Himeno et al. [25]. They showed the presence of sialic acid (0.11 %) in the more acidic forms of lysosoma1 8-glucuronidase purified from rat liver. Probably, a small part of cathepsin D-I may be a sialoglycoprotein.
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K. Yamamoto and N. Katsuda, Department of Pharmacology, Faculty of Dentistry, Kyushu University, Higashi-ku, Fukuoka-shi, Fukuoka-ken, Japan 812 M. Himeno and K . Kato, Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyushu University, Higashi-ku, Fukuoka-shi, Fukuoka-ken, Japan 812
