Eur. J . Biochem. 102, 279-289 (1979)
Crystallization and Properties of Cathepsin B from Rat Liver Takae TOWATARI, Yoshimasa KAWABATA, and Nobuhiko KATUNUMA Department of Enzyme Chemistry, Institute for Enzyme Research, School of Medicine, Tokushima University (Received July 9, 1979)
Cathepsin B from rat liver was purified to apparent homogeneity by cell-fractionation, freezing and thawing, acetone treatment, gel filtration, DEAE-Sephadex and CM-Sephadex column chromatography, and was crystallized. The purified enzyme formed spindle-shaped crystals and its homogeneity was proved by disc gel electrophoresis in the presence of sodium dodecyl sulfate and by ultracentrifugical analysis. Its ~ 2 0 value , ~ was 2.8 S and its relative molecular mass was calculated to be 22 500 (k900) by sedimentation equilibrium analysis. Crystalline cathepsin B was shown to consist of four isozymes with isoelectric points between pH 4.9 and 5.3, the main isozyme having an isoelectric point of pH 5.0. The enzyme was irreversibly inactivated by exposure to weak alkali. The pH optimum was 6.0 with a-N-benzoyl-~~-arginine-4-nitroanilide as substrate. Amino acid analysis showed that the enzyme contained hexosamine, glucosamine and galactosamine. Cathepsin B inactivated aldolase, glucokinase, apo-ornithine aminotransferase, and apo-cystathionase, but the rates of inactivation of glucokinase, apo-ornithine aminotransferase, and apocystathionase were lower than that of aldolase. Studies by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate showed that cathepsin B degraded apo-ornithine aminotransferase to two polypeptide chains differing in relative molecular mass and electrophoretic mobility.
Inhibitors of so-called cathepsin B reduce protein degradation in various experimental systems. This has been demonstrated with leupeptin in a system in vitro [l] and in tissue culture [2] and with chloroquine in tissue culture [3]. Therefore, it is thought that cathepsin B is important in intracellular protein degradation. Cathepsin B has been found in various mammalian tissues and its purification has been attempted by many workers. The preparation of cathepsin B of Greenbaum and Fruton [4] was demonstrated by Otto [ 5 ] to consist of two enzymes, cathepsin B (cathepsin B1) and lysosomal carboxypeptidase B (cathepsin B2) [6]. It is now known that cathepsin B of Otto
contains cathepsin B, new cathepsin (cathepsin L) [7-91 and cathepsin H [lo]. We have purified cathepsin B from rat liver and examined its properties in an attempt to understand its role in the liver. We were able to crystallize the enzyme, as described in a preliminary report [ll]. This paper describes details of the method for obtaining crystalline cathepsin B from rat liver and the properties of the enzyme. MATERIALS AND METHODS Muterials Wistar rats fed on standard diet and weighing
200-250 g were used as a source of the enzyme. Abbreviations. PhMeS02F, phenylmethyl sulfonyl fluoride; E-64, N-[N-(~-3-trans-carboxyoxiran-2-carbonyl)-~-leucyl]-agma- Ovalbumin, chymotrypsinogen, and myoglobin, from tin; Bz-Arg-Nan, r-N-benzoyl-~~-arginine-4-nitroanilide. Mann, U.S.A., and bovine serum albumin from Sigma, Enzymes. Cathepsin B (EC 3.4.22.1);carboxypeptidase B (EC U.S.A. were used as markers in the determination of 3.4.17.2);cathepsin L (EC 3.4.22.15);cathepsin H (EC 3.4.22.-); the relative molecular mass. a-N-Benzoyl-DL-argininealdolase (EC 4.1.2.13);glucokinase (EC 2.7.1.2);ornithine aminotransferase (EC 2.6.1.13);cystathionase (EC 4.4.1.1); tyrosine 2-naphthylamide, ~-leucine-2-naphthylamide, a-Naminotransferase (EC 2.6.1.5); glucose-6-phosphate dehydrogenase benzoyl-DL-arginine-4-nitroanihde, N-a-p-tosyl-L-lysine (EC 1.1.1.49);alcohol dehydrogenase (EC 1.1.1.1);malate dechloromethyl ketone . HCl, L-1-tosylamido-2-phenylhydrogenase (EC 1.1.1.37);glyceraldehyde-3-phosphatedehydroethylchloromethyl ketone . HCl, soyben trypsin ingenase (EC 1.2.1.12);glutamate dehydrogenase (EC 1.4.1.2); hibitor, and phenylmethyl sulfonyl fluoride were oblactate dehydrogenase (EC 1.1.1.27).
280
tained from Sigma. a-N-Benzoyl-DL-arginine ethyl ester was from the Peptide Center (Institute for Protein Research, Osaka, Japan). Protease inhibitors of bacterial origin (chymostatin, pepstatin, antipain, and leupeptin) were kindly supplied by Dr Aoyagi and Dr Umezawa (Institute of Microbial Chemistry, Tokyo, Japan). The thiol protease inhibitor, E-64 [12] of Aspergillus japonicus and its synthetic thiol protease inhibitors (monoethylepoxy succinate and monobenzylepoxy succinate) were kindly supplied by Dr Sawada (Taisho Pharmaceutical Co., Japan). Sephadex G-75, DEAE-Sephadex A-50, and CM-Sephadex C-50 were from Pharmacia. All other reagents were standard analytical grade products. Assay of Cathepsin B with Synthetic Substrates Cathepsin B was routinely assayed by the method of Otto and Bhakdi [13]. The cleavages of a-N-benzoyl~~-arginine-2-naphthylamideand ~-1eucine-2-naphthylamide at pH 6.0 were measured by coupling the 2-naphthylamide with fast garnet GBC salts as described by Barrett [14]. The cleavage of a-N-benzoylDL-arginine ethyl ester at pH 6.0 was measured by the procedure of Roberts [15], and the cleavage of a-Nbenzoyl-m-argininamide at pH 6.0 by those-of Seligson and Seligson [16] and Russell [17]. Assay of Cathepsin B with Substrate Enzymes The rates of inactivation of enzymes were measured as follows. The reaction mixture contained 100 pmol potassium phosphate buffer, pH 6.0, 5 pmol 2-mercaptoethanol, 0.5 - 2.0 units of substrate enzyme and a suitable amount of cathepsin B preparation in a final volume of 1.O ml. After incubation for 15- 30 min at 37 "C, samples of 200 pmol were taken for measurement of the remaining enzyme activity. With substrate enzymes one unit of cathepsin B was expressed as the amount giving a first-order rate constant in the inactivating reaction, k = 1 (min-'). Assay of Cuthepsin B with Protein Substrates The proteolytic activities of cathepsin B on casein, acid-denatured hemoglobin [ 181 and bovine serum albumin were assayed by measuring the liberation of free amino acids, recovered in the fraction soluble in trichloroacetic acid, by the ninhydrin method [19]. Preparation and Assay o j Substrate Enzymes Crystalline ornithine aminotransferase was purified from rat liver by the method of Matsuzawa et al. [20], tyrosine aminotransferase by the method of Valeriote et al. [21], cystathionase by the method of Matsuo and Greenberg [22], glucokinase by the method of
Properties of Crystalline Cathepsin B from Rat Liver
Grossman et al. [23], aldolase by that of Rajkumar et al. [24], glucose-6-phosphate dehydrogenase by that of Watanabe and Takeda [25] and lactate dehydrogenase by that of Scopes [26]. Glucose-6-phosphate dehydrogenase and alcohol dehydrogenase from yeast, malate dehydrogenase from pig heart, glyceraldehyde-3-phosphate dehydrogenase and aldolase from rabbit muscle, and glutamate dehydrogenase from beef liver were purchased from Boehringer, Mannheim. Ornithine aminotransferase was assayed by the method of Katunuma et al. [27], tyrosine aminotransferase by that of Rosen et al. [28], cystathionase by that of Matsuo and Greenberg [22], glucokinase by that of Ballard and Oliver [29] and aldolase by that of Blostein and Rutter [30]. Glucose-6-phosphate dehydrogenase was assayed by measuring the rate of increase in absorbance of NADPH at 25 "C [31]. Malate dehydrogenase [32], lactate dehydrogenase [33] and glutamate dehydrogenase [34] were assayed by measuring the rate of decrease in absorbance of NADH at 25 "C. Glyceraldehyde-3-phosphate dehydrogenase was assayed by measuring the coupled reaction with phosphoglycerate kinase 1351. Alcohol dehydrogenase was assayed by measuring the rate of increase in absorbance of NADH at 25 "C [36]. Disc Gel Electrophoresis Polyacrylamide disc gel electrophoresis [37.38] was performed at pH 4.5 and 8.3, using 12.5 "/, separation gel. Samples were applied in 10% sucrose and methyl green and bromophenol blue were used as tracking dyes at pH 4.5 and pH 8.3, respectively. Electrophoresis was carried out at 4 "C at a constant current of 2 mA per tube. The gel was stained with Coomassie blue G-250 and destained with methanol/water/acetic acid (50: 135 : 15, by vol.). Sodium dodecyl sulfate/ polyacrylamide disc gel electrophoresis was performed by the method of Weber and Osborn [39]. Analytical Isoelectric Focusing Isoelectric focusing in polyacrylamide gel was carried out as described by Wrigley [40]. The gel solution contained 8.2 parts of water, 3.0 parts of a solution of 30% acrylamide (w/v), and 0.8% N,N'methylenebisacrylamide (w/v), 0.8 parts of a solution of 1 "/, N,N,N',N'-tetramethylethylenediamine (v/v) and 0.014% riboflavin (w/v) and 0.3 part Ampholine. A mixture of Ampholines of pH 3.5-5, pH 5-7 and pH 3.5 - 10 (40: 40: 20) was used at a final concentration of 1 % (w/v). Tubes of 8-cm length and 0.5-cm diameter were used. Electrophoresis was performed for 4 h at 4 ° C using 0.2% sulfuric acid in the anode vessel and 0.4 % ethanolamine in the cathode vessel and a constant voltage of 300 V. After electrophoresis, protein in the gel was stained with Coomassie blue
28 1
T. Towatari, Y. Kawabata, and N. Katunuma Table 1. Purification of cethepsin B from rat liver One unit of activity is defined as the amount of enzyme liberating 1 pmol p-nitroanilinelmin from Bz-Arg-Nan Purification step
1. Lysosomal extract
2. 3. 4. 5. 6.
Acetone treatment Sephadex (3-75 DEAE-Sephadex A-50 CM-Sephadex C-50 Crystallization
Total volume
Total protein
Total activity
Specific activity
Yield
Purity
ml
mg
units
units/mg
%,
-fold
910 36 18 40 1 1
10700 685 324 24 23 5.6
32.21 24.00 26.43 9.66 9.60 2.65
0.003 0.035 0.085 0.403 0.417 0.473
100 14.5
1 12 27 134 139 158
G-250 (0.4 % in 3 % perchloric acid). For pH determination and measurement of cathepsin B activity, the gels were cut into 40 or more sections and each section was extracted with 1 ml distilled water. Cathepsin B activity was measured as described above.
Determination of Relative Molecular Mass Relative molecular mass was determined by descending gel filtration through a Sephadex G-75 column (2 x 100 cm) equilibrated with 50 mM acetate buffer, pH 5.0, containing 0.1 M sodium chloride and 5 mM 2-mercaptoethanol [41]. The markers used were bovine serum albumin ( M , 67000), ovalbumin ( M , 45 000), chymotrypsinogen ( M , 25 000) and myoglobin ( M , 17800). With sodium dodecyl sulfate gel (run as described above), mobilities with respect to bromophenol blue were plotted against the logarithm of the relative molecular masses using the same markers.
Amino Acid Analysis Amino acid analysis was carried out in a Hitachi amino acid analyzer by the standard procedure [42], except that glucosamine, galactosamine, and tryptophan were measured by the procedure of Liu and Chang [43] using a solution of 3 M p-toluene sulfonic acid containing 0.2 % 3-(2-amino ethyl) indole. Samples in 6 M HCl were hydrolyzed under vacuum at 110 “C for 24 h and 48 h. Half-cystine was determined by performic acid oxidation as described by Moore [44] followed by hydrolysis for 24 h.
Ultracentrifugation Analysis Ultracentrifugation analysis was carried out in a Hitachi 282 analytical ultracentrifuge equipped with a temperature control unit, a monochromator and an absorption scanner, as described by Schachman and Edelstein [45]. Schlieren and absorption optics were used for sedimentation velocity studies and sedimentation equilibrium experiments, The rotor speeds were
82.0 30.0 29.8 8.2
54000 rev./min and 15000 rev./min, respectively, and 20mM acetate buffer, pH5.0, containing 0.1 M sodium chloride was used as solvent. The concentration of protein used was 0.65 mg/ml for sedimentation velocity analysis and 0.33 mg/ml for sedimentation equilibrium analysis. Values were calculated taking the partial specific volume as 0.72 ml/g, based on the amino acid composition.
Determination of Protein Protein concentration was determined by the method of Lowry et al. [46] using bovine serum albumin as a standard. RESULTS
Purification of Cathepsin B Cathepsin B from rat liver was purified to a crystalline form by a modification of the method of Otto [5] and Otto and Riesenkonig [47] with additional procedures. The purification is summarized in Table 1. All purification procedures were performed at 4 “C, using 50 mM acetate buffer, pH 5.0, containing 5 mM 2-mercaptoethanol, referred to as ‘standard buffer’. Extraction. A crude lysosomal fraction was isolated from rat livers as described by Ragab et al. [48], suspended in standard buffer and subjected to sevencycles of freezing and thawing. The mixture was centrifuged at 10000 x g for 30 min and the precipitate was discarded. Acetone Treatment. The supernatant was mixed with cold acetone to a final concentration of 45 % (v/v) with stirring over a period of 5 min and then the mixture was rapidly centrifuged at 8000xg for 5 min. Further cold acetone was added to the supernatant to a final concentration of 75% (v/v). The resulting precipitate was collected by centrifugation and dissolved in a minimum volume of standard buffer. The preparation was centrifuged briefly to remove insoluble material and the supernatant passed through a Sephadex G-25 column (6 x 20 cm) equilibrated with standard buffer and concentrated to a small volume
Properties of Crystalline Cathepsin B from Rat Liver
282
Fig. 1. Pliotomicrogruph of crystuls of cathepsin Bjrom rut liver. Magnification x 400
A
B
C
Fig. 2. Polqacrylamide gel elec!rophorc~sisof crystalline cuthepsin B .from rat liver. Electrophoresis was carried out at pH 4.5 (A), pH 8.3 (B), and with sodium dodecyl sulfate (C) as described under Materials and Methods. Samples of 10 pg protein were used. Migration was from top to bottom of the gel
by ultrafiltration using a PMlO membrane (Amicon, U.S.A.). Gel filtration on a Sephadex G-75 Column. The enzyme solution was divided in half and applied to two identical Sephadex G-75 columns (3.0 x 100 cm) previously equilibrated with standard buffer containing 0.1 M sodium chloride, the columns were eluted with the same buffer. The fraction containing Bz-Arg-Nanhydrolyzing activity with an M , of about 25000 was
eluted from the gel, collected and concentrated to a small volume by ultrafiltration on an Amicon PMlO membrane. Cathepsin B was separated from lysosomal carboxypeptidase and cathepsin D in this step. DEAE-Sephadex A-50 Column Chromatography. The enzyme solution was then applied to a DEAESephadex A-50 column (1.8 x 20 cm) equilibrated with 20 mM acetate buffer, pH 5.0, containing 10 mM sodium chloride and 5 mM 2-mercaptoethanol. The Bz-Arg-Nan-hydrolyzing activity was eluted in two fractions with the same buffer: the fraction eluted first contained cathepsin H activity, which has low sensitivity to leupeptin as described by Davidson andPoole [49]; the fraction eluted second contained cathepsin B, new cathepsin (cathepsin L), and leucine-2-naphthylamide-hydrolyzing enzyme. CM-Sephadex C-50 Column Chromatography. The latter fraction was applied to a CM-Sephadex C-50 column (1.8 x 15 cm) equilibrated with 20 mM acetate buffer, pH 5.0, containing 5 mM 2-mercaptoethanol and 50 mM sodium chloride. The column was washed with the same buffer and then eluted stepwise with 0.1 M, 0.2 M and 0.5 M sodium chloride in 20 mM acetate buffer, pH 5.0, containing 5 mM 2-mercaptoethanol. Cathepsin B was eluted with 0.1 M sodium chloride and concentrated to about 20 mg protein/ml in a collodion bag. The leucine-2-napfithylamidehydrolyzing enzyme was eluted with 50 mM sodium chloride and new cathepsin (cathepsin L) with 0.5 M sodium chloride. Crystallization. Solid ammonium sulfate was added gradually to the concentrated enzyme solution until just before a slight turbidity appeared in the cold and then the solution was stored for several days in the cold. The spindle-shaped crystals formed are shown in Fig. 1.
T. Towatari, Y. Kawabata, and N. Katunuma
283
Crystals, Homogeneity and Relative Molecular Mass Crystalline cathepsin B was subjected to polyacrylamide gel electrophoresis. As shown in Fig. 2, the crystalline enzyme migrated as a single protein at pH 4.5, but a minor component was separated at pH 8.3. Similar results were obtained with the mother liquor. It gave one major protein band when subjected to gel electrophoresis in the presence of sodium dodecyl sulfate and had an M , of 23000. It gave a single symmetrical boundary on ultracentrifugation (Fig. 3), and its sedimentation coefficient was determined to be 2.8 S. Its relative molecular mass was calculated to be 22 500 ( * 900) by sedimentation equilibrium centrifugation, and 26000 by gel filtration on a Sephadex G-75 column (Fig. 4). Fig. 3 . Ultracentrijiugul patterns of crystalline cathepsin B from rat livrr. Sedimentation velocities were measured at 54000 rev./min at 21 C. The enzyme (11.4 mg/ml) was dissolved in 20 mM acetate buffer, pH 5.0, containing 0.1 M sodium chloride. The picture was taken 54 min after attaining the maximal speed
Analytical Isoelectric Focusing On isoelectric focusing in polyacrylamide gel with Ampholine pH 3.5-8.0, the cathepsin B gave four protein bands with isoelectric points between pH 4.9 and 5.3 (Fig. 5), the main protein band having an isoelectric point of pH 5.0. All the protein bands corresponded with bands of activity and stained with periodic acid/Schiff reagent, indicating that cathepsin B from rat liver consists of four isozymes and is a glycoprotein. Amino Acid Composition
0
50
60
70
80
90
Fraction number
Fig. 4. Determination o/ relative moleculur muss. A column ( 2 x 1OOcm) of Sephadex (3-75 was equilibrated with 50 mM acetate buffer, p H 5.0, containing 0.1 M sodium chloride and 5 rnM 2-rnercaptoethanol. Standard proteins (5 mg) were applied in a volume of 1.5 ml: (1) bovine serum albumin; (2) ovalbumin; ( 3 ) chymotrypsinogen; (4) myoglobin. The amount of cathepsin B applied formed 3.5 Fmol p-nitronilinelmin from Bz-Arg-Nan. Fractions of 2.5 ml were collected at a flow rate of 25 mlih
Cathepsin B has been defined as a protease with an M , of about 25000 and an optimum pH of about 6.0 that inactivates various high-M, proteins, such as aldolase, hydrolyzes synthetic substrates, such as BzArg-Nan, and does not hydrolyze L-leucine-2-naphthylamide [50 - 531. These findings were confirmed in the following experiments.
The amino acid composition of crystalline cathepsin B from rat liver is shown in comparison with that of human cathepsin B [54] in Table 2. The most characteristic features are that it contains considerable amounts of glucosamine and galactosamine. These findings also show that cathepsin B is a glycoprotein, like the other lysosomal enzymes that have been characterized. The contents of glycine, aspartic acid, and glutamic acid are high and the content of methionine is low, as in human cathepsin B, although the values for individual amino acid residues/molecule ( M , 25000) differ from those of human cathepsin B. Optimum p H As shown in Fig. 6, the activity of the cathepsin B with Bz-Arg-Nan as substrate was maximal at pH 6.0 and decreased markedly above pH 7.0 and below pH 4.0.
Stability Fig.7 shows the affects of preincubation at pH 4.0 - 7.5 at 37 "C for 30 min on cathepsin B. The
Properties of Crystalline Cathepsin B from Rat Liver
284
A
B
+
?
I
8.0 100 %O
.->
A c
.-" c
61)
m
I,
a .-*m 1
50
50
d
4.0
3.0 0
0
10
20 Gel slice number
30
40
Fig. 5. Analytical isoelectric focusing of crystalline ccitkrpsin B ji-om rat liver. pH (0-0); enzyme activity ( 0 2 ) .Analytical conditions were as described under Materials and Methods. 54 Fg enzyme protein were used in gel stained for protein and 108 Kg in gel stained for carbohydrate. Gels were stained for Drotein with Coomassie blue G-250 and for carbohydrate with periodate/Schiff procedure. (A) Carbohydrate; (B) protein
cathepsin B was stable between pH 4.0 and pH 6.5, but it was very unstable at alkaline pH values. It was stable on heat treatment at 35 "C, 42 "C, and 47 "C for 15 min at pH 6.0.
tease inhibitor from Aspergillus japonicus, and by its synthetic analogues, monoethylepoxy succinate and monobenzylepoxy succinate. Substrate Specificity
Effects of Activators and Inhibitors As shown in Table 3, the cathepsin B required the presence of a sulfhydryl compound plus EDTA for maximal activity. In the presence of EDTA, 1 mM cysteine, 0.25 mM dithiothreitol or 8 mM 2-mercaptoethanol was required for maximal activity. EDTA alone was ineffective, but a sulfhydryl compound alone caused some activation. Cathepsin B was inhibited by monoiodoacetate at low concentration, showing that it possesses an essential thiol group [51]. It was also strongly inhibited by the microbial inhibitors, leupeptin, chymostatin, and antipain [53] and N-cc-ptosyl-L-lysinechloromethyl ketone and L-l-tosylamido2-phenylethylchloromethyl ketone [55]. Cathepsin B was also inhibited by E-64 [12], which is a thiol pro-
As shown in Table 4, cathepsin B from rat liver hydrolyzed Bz-Arg-Nan, a-N-benzoyl-~~-arginine-2naphthylamide, a-N-benzoyl-DL-argininamide,and a-N-benzoyl-DL-arginine ethyl ester, but not L-leucine2-naphthylamide. Results of the inactivations of various enzymes by cathepsin B are shown in Table 5. Cathepsin B inactivated aldolases (muscle and liver) and glucokinase (liver), which are reported to be attacked by cathepsin B [50]. It inactivated aldolase significantly more rapidly than glucokinase, although it did not cause more than 50 inactivation of aldolase. It slightly inactivated apo-cystathionase and apoornithine aminotransferase. The mode of its inactivation of apo-ornithine aminotransferase was studied in detail by measuring the decrease in enzyme activity,
T . Towatari, Y. Kawabata, and N. Katunuma
285
Table 2. Amino m i d composition of cathepsin B,from rut liver Values are residues/molecule (Mr 25000) Amino acid
Residues after 24 h
Tryptophanb Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cystine (halod Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Glucosamine Galactosamine
Calculated number of residues
Human cathepsin B"
6.5 8.1 4.6 6.2 19.6 9.9 16.2 19.9 12.0 28.6 11.7 9.4 12.0 3.0' 11.8 8.2 9.0 6.9 8.1 9.0
7 10 8 8 23 10 17 23 17 29 13 12 15 4 13 10 13 7 3
220.7
242
48 h
5.4 f 0.9 8.3 f 1.0 4.0 f 0.1 6.0 (2) 20.2 f 1.3 9.2 f 1.2 16.5 k 0.2 19.6 f 0.7 12.6 & 1.9 28.9 & 1.7 11.7 f 0.2 9.4 (2) 11.9 k 1.0 2.6 0.4 8.9 f 0.8 6.3 f 0.5 8.7 (2) 7.0 (2) 7.8 f 0.1 8.9 f 0.8
(3) (3) (3)
4.2 f 0.1 (3) 7.9 (2) 5.2 (2) 6.4 (2) 19.0 & 0.4 (3) 10.5 k 0.2 (3) 15.8 i 0.3 (3) 20.1 k 0.3 (3) 11.3 f 0.4 (3) 28.3 f 0.1 (3) 11.6 f 0 (3)
(4) (4) (4) (4) (4) (4) (4) (4) (4) (4) (4)
12.0 2.4 11.8 8.2 9.2 6.7 8.3 9.1
(3) (3)
*f 0.20.1 (3)(3) f 0.1 (3) f 0 (3) f 0.1 (3) f 0.2 (3) f 0 (3) k 0.1 (3)
Total * Taken from Barrett's human cathepsin B [54].
Hydrolyzed in 3 M p-tuluene sulfonic acid containing 0.2 3-(2-aminoethyl)indole at 110 "C Values extrapolated to zero-hour hydrolysis. Oxidized with performic acid before hydrolysis in 6 M HCI at 110°C for 24 h.
4.0
4.5
5.0
5.5
60
6.5
70
PH
4.0
5.0
60
TO
8.0
PH
Fig. 6. pHIActivity curve of cathepsin B from rat liver. The buffers used at final concentrations of 0.1 M were acetate buffer for p H 4.6- 5.6 (+O) and potassium phosphate buffer for p H 5.7- 7.2 (-0). Bz-Arg-Nan was used as substrate
Fig. 7. pH-dependent stabilities of cathepsin B f r o m rat liver. The enzyme was preincubated for 30 min at 3 7 ' C in 0.1 M buffers of various p H values and then remaining activities were assayed using Bz-Arg-Nan. Acetate buffer p H 4.0-5.6 (&-0); potassium phosphate buffer p H 5.7-7.5 (-0)
changes in relative molecular mass of the products separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and changes in mobility of the native subunit and products by polyactylamide gel electrophoresis. Fig. 8 shows that the loss of activity
corresponded quantitatively to the loss of material migrating in the position of the native subunit and that two products appeared which migrated in different position on polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate.
Properties of Crystalline Cathepsin B from Rat Liver
286 Table 3. Effects of’uctivators and inhihitors on cathepsin B,from rat liver The enzyme was incubated with various effectors for 5 min and then assayed as described under Materials and Methods using Bz-ArgNan as substrate. Experiment 2 was performed in the presence of 2 m M EDTA. Experiment 3 was performed in the presence of 2 mM EDTA and 4 mM cysteine
Table 4. Activities of cathepsin B on synthetic substrates Reaction mixtures contained 5 pmol synthetic substrate (except a-N-benzoyl-DL-argininamide, which was added at 50 pmol), 100 pmol potassium phosphate buffer, pH 6.0, 2 pmol EDTA, 4 pmol cysteine, and a suitable amount of enzyme in a final volume of 1.0 ml. Assays were performed at 37 ’C for 15 and 30 min as described under Materials and Methods
Expt Effectors
Substrate
Final concn
Relative activity
PM
7i
4000 4000 2000 4000 2000
100 210 100
Cathepsin B activity pmol x min-’ x mg-’
1.
Control Cysteine EDTA Cysteine
+ EDTA
__ Control Dithiothreitol Cyyterne ~
2.
~~
-
-
2-Mercaptoethanol
250 500 1000 8000 500 8000
100 329.4 376.4 323.5 323.5 132.1 342.9
-~
3
Control Monoiodoacetate Leupeptin Chymostatin L-I-Tosylamide-2-phenylethylchloro-methyl ketone N-a-p-Tosyl-L-lysine chloromethyl ketone Antipain Pepstatin PhMeSOzF Trypsin inhibitor (soybean) E-64 Monoethylepoxy succinate Monobenzylepoxy succinate
a-N-Benzoyl-~~-arginine-4-nitroanilide0.40 a-N-Benzoyl-~~-arginine-2-naphthylamide 2.75 a-N-Benzoyl-~~-arginine ethyl ester 1.04 a-N-Benzo yl-uL-argininamide 2.12 ~-Leucine-2-naphthylamide 0
1.0 0.1 6.2
100 62 100 87.7
3.0
53.3
1.o 1.o 10 100 14 0.28 50.0 3.8
71.4 60 100 100 100 63.8 19.6 28.3
The relative molecular mass of the products were estimated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis using markers. That of the native subunit, referred to as the A form, was 45000 and those of the B and C forms were 27000 and 18000, respectively. These results suggest that cathepsin B inactivates the apo-form of ornithine aminotransferase by limited proteolysis and degrades it to the B and C forms. Glucose-6-phosphate dehydrogenases (yeast and rat liver), apo-tyrosine aminotransferase (rat liver), glyceraldehyde-3-phosphate dehydrogenase (rabbit muscle), malate dehydrogenase (pig heart), lactate dehydrogenase (rat liver), glutamate dehydrogenase (beef liver), and alcohol dehydrogenase (yeast) were not inactivated at all by cathepsin B. Of the protein substrates tested, acid-denatured hemoglobin, casein, and bovine serum albumin were hydrolyzed, as reported earlier [50,51]. Therefore, the inactivation of
Table 5. Activities of cathepsin B on enzyme substrates The reaction conditions were as described under Materials and Methods. Values show inactivations of substrate enzymes as percentages of that of aldolase from rabbit muscle. Cathepsin B activity on aldolase (muscle) was 11.6 units/mg Substrate
Source
Cathepsin B activity 0, :r,
Aldolase Aldolase Glucokinase Cystathionase (apo-) Ornithine aminotransferase (apo-1
rabbit muscle rat liver rat liver rat liver rat liver
100 182 8 2 1
various enzymes by cathepsin B seems to result from their proteolysis.
DISCUSSION Cathepsin B has been identified as a lysosomal thiol enzyme present in numerous animal tissues including liver. Previously we isolated a new lysosomal protease, named new cathepsin [7], from the cathepsin B fraction of rat liver and reported that it may be identical with cathepsin L of Kirschke et al. [9]. Recently Singh and Kalnitsky [59] separated a new x-N-benzoylarginine-2-naphthylamidehydrolase from rabbit lung, and clearly differentiated it from new cathepsin (cathepsin L), cathepsin B, and a-N-benzoylarginine-2-naphthylamide aminohydrolase [60] from rat liver on thebasis ofits substrate specificity, pH optimum, relative molecular mass and sensitivity to leu-
1
2
1
2
3
4
5
6
7
8
C)
A-
B-
C-
3
4
5
6
7
8
Fig. 8. Mode ofproreolysis of’apo-ornithineurninorrunsferase by cuthcpsin Bfrorn rat liver. (a) Time course of proteolysis of apo-ornithine aminotransferase by cathepsin B. Apo-ornithine aminotransferase (0.46 mg) was incubated at 37 ‘C with cathepsin B (0.22 mg) in 1 ml of 0.1 M sodium phosphate buffer (pH 6.0) containing 10 mM 2-mercaptoethanol and 1 mM EDTA. 20O-pl samples were removed at the times indicated for assay of ornithine aminotransferase activity and for analysis by polyacrylamide gel electrophoresis. Reactions were stopped by adding 10nmol leupeptin. Complete system (0-0); control system in which apo-ornithine aminotransferase was incubated with watef instead of cathepsin B (0-0). (b) Polyacrylamide gel electrophoregrams of the products of apo-ornithine aminotransferase formed with cathepsin B. Gels 1-4 show results with the complete system after 0, 1, 3 and 6 h ; gels 5 and 6 show results in the apo-ornithine aminotransferase control system after 0 and 6 h ; gels 7 and 8, show results in the cathepsin B control system after 0 and 6 h. (c) Sodium dodecyl sulfateipolyacrylamide gel electrophoregrams of the products of apo-ornithine aminotransferase formed with cathepsin B. Conditions were the same as for (b)
288
peptin. Rabbit lung a-N-benzoylarginine-2-naphthylamide hydrolase is, in some respects, similar to cathepsin H recently isolated from rat liver [lo]. Recently we isolated cathepsin B in crystalline form from rat liver and determined its properties. The relative molecular mass of crystalline cathepsin B was calculated to be 26000 by Sephadex G-75 column chromatography, 22 500 by sedimentation equilibrium analysis, and 23 000 by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. These values are similar to that of 24000 reported for the enzyme from human liver [52] and that of 25000 reported for the enzymes of bovine spleen [5] and liver [56], and rat liver, kidney, spleen, and lung [51]. Moreover it has been reported that ovine [57], bovine [58], and human cathepsin B have multiple forms that can be separated by ion-exchange chromatography. The forms differ in isoelectric points, and plvalues ranging from pH 4.5 to 5.5 were reported for the human enzyme, the major form having a p l value of pH 5.0 to 5.2. Recently cathepsin B from rabbit lung was shown to consist of four isozymes with isoelectric points ranging from pH 5.0 to 5.5 [59]. Warwas and Dobryszycka demonstrated by isoelectric focusing in polyacrylamide gel that there are three isozymes of cathepsin B in the human fetal placenta membrane, chorion, differing in pl, whereas there is only one isoenzyme activity in the amnion [61]. As shown in Fig. 5, crystalline cathepsin B from rat liver also consisted of four isozymes with isoelectric points ranging from pH 4.9 to 5.3, the major form having an isoelectric point of pH 5.0. All the forms stained with periodic acid/Schiff reagent. The crystalline form and the enzyme in the mother liquor both migrated as a single protein on polyacrylamide disc gel electrophoresis in the presence of sodium dodecyl sulfate. Amino acid analysis showed that crystalline cathepsin B contained hexosamine, and that the contents of glycine, aspartic acid, and glutamic acid are high and the content of methionine is low, as in cathepsin B from human liver [54]. Since the charge heterogeneity of cathepsin B was not changed by crystallization of the enzyme, and since N-terminal leucine has been found in several forms of bovine enzyme by Franklin and Metrione [58] and Keilova and Tomasek [62], the multiple forms may result from changes in carbohydrate content during the solubilization step, as is the case with some other lysosomal enzymes. This possibility needs further investigation. Crystalline cathepsin B was inhibited by E-64 and its synthetic analogues. The enzyme inactivated apoornithine aminotransferase and apo-cystathionase to some extent, Recently Aronson and Barret showed that cathepsin B from rat and human liver and bovine spleen degraded glucagon by sequential cleavage of dipeptides from the C-terminal end of the molecule [63]. Nakai et al. reported that inactivation of aldolase
Properties of Crystalline Cathepsin B from Rat Liver
(rabbit muscle) by cathepsin B in vitro was due to release of one mole of the dipeptide alanyl-tyrosine from the C-terminal end of the molecule [64]. We found that cathepsin B split apo-ornithine aminotransferase into two polypeptide chains. This difference in the modes of proteolysis by cathepsin B seems to be due to differences in the conformation of the protein molecules. Glucose-6-phosphate dehydrogenase, apotyrosine aminotransferase and glyceraldehyde-3-phosphate dehydrogenase, which are good substrates for the new cathepsin [S], were not inactivated by cathepsin B from rat liver. It has been reported that thiol protease in lysosomes can inactivate native enzymes, such as aldolase [50], glucokinase [50], pyruvate kinase [51], tyrosine aminotransferase [65], arginase [66], alanine aminotransferase [66], and glyceraldehyde3-phosphate dehydrogenase [67]. These findings suggest that various lysosomal proteases have specific actions on substrate enzymes and play important roles in intracellular protein degradation. We would like to thank Mr R. Kunai for help with Ultracentrifugal analysis and M r H. Miyai for assistance in amino acid analysis. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan.
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20. Matsuzawa, T., Katsunuma, T. Sr Katunuma, N. (1968) Biochem. Biophys. Res. Commun. 32, 161 -166. 21. Valeriote, F. A,, Auricchio, F., Tomlins, G. M. & Reley, D. (1969) J . Biol. Chem. 244, 361 8 - 3624. 22. Matsuo, Y. & Greenberg, D. M . (1958) J . Biol. Chem. 230, 560- 571, 23. Grossman, S. W.,Dorn, C. C . Sr Potter, V. R. (1974) J . Biol. Chem. 249, 3055 - 3060. 24. Rajkumar, T. V., Woodfin, B. M. Sr Rutter, W. J. (1966) Methods Enzymol. 9,491 -498. 25. Watanabe, A. Sr Takeda, K. (1972) J . Biochem. (Tokyo) 72, 1277-1280. 26. Scopes, R. K. (1977) Biochem. J . 161, 253-263. 27. Katunuma, N., Okada, M., Matsuzawa, T. Sr Otuka, Y. (1965) J . Biochem. (Tokyo) 57, 446-449. 28. Rosen, F., Harding, H. R., Milholland, R. J. Sr Nichol, C. A. (1963) J . Biol. Chem. 238, 3725 - 3729. 29. Ballard, F. J. Sr Oliver, I. T. (1964) Biochem. J . YO, 261 -268. 30. Blostein, R . Sr Rutter, W. T. (1963) J . B i d . Chem. 238, 32803285. 31. Kornberg, A. Sr Horecker, B. L. (1955) Methods Enzymol. I , 323- 327. 32. Ochoa, S. (1955) Methods Enzymol. I , 735-739. 33. Kornberg, A. (1955) Methods Enzymol. I , 441 -443. 34. Fahien, L. A. & Cohen, P. P. (1970) Methods Enzymol. 17A, 839 - 844. 35. Kuehl, L. Sr Sumsion, E. N. (1970) J . Biol. Chem. 245, 66166623. 36. Bonnichsen, R. K . Sr Brink, N. G. (1955) h4ethod.s Enzymol. I, 495 - 500. 37. Davis, B. J. (1964) Ann. N.Y. Acad. Sci. 121, 404-427. 38. Reisfeld, R. A., Lewis, U. T. Sr Williams, D. E. (1962) Nature (Lond.) 195,281 -283. 39. Weber, K . Sr Osborn, M. (1969) J . Biol. Chem. 244, 44064412. 40. Wrigley, C . W . (1969) Sci. Tools, 15, 17-23. 41. Andrews, P. (1964) Biochem. J . 91, 222-233. 42. Moore, S. & Stein, W. H . (1967) Methods Enzymol. 19, 213283. 43. Liu, T. Y . s( Chang, Y . H. (1971) J . Biol. Chem. 246, 28422848. 44. Moore, S. (1963) J . Biol. Chem. 238, 235-237. 45. Schachrnan, H . K. Sr Edelstein, S. J. (1973) Methods Enzymol. 27, 3-59.
46. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. Sr Randall, R. J. (1951) J . Biol. Chem. 193, 265-275. 47. Otto, K. Sr Riesenkonig, H. (1975) Biochim. Biophys. Acra, 379,462 - 475. 48. Ragab, H., Beck, S. Dillard, C. & Tapple, A. L. (1967) Biochim. Biophys. Acta, 148, 501 - 505. 49. Davidson, E. Sr Poole, B. (1975) Biochim. Biophys. Acfa, 3Y7, 437 - 442. 50. Otto, K . & Schepers, P. (1967) Hoppe-Seyler’s Z. Physiol. Chem. 348,482-490. 51. Otto, K. (1971) in Tissue Proteinuses (Barrett, A. J. & Dingle, J. T., eds) pp. 1-28, North-Holland, Amsterdam. 52. Barrett, A. J. (1973) Biochem. J . 131, 809-822. 53. Aoyagi, T., Takeuchi, T., Matsuzaki, A,, Kawamura, K., Kondo, S., Hamada. M., Maeda, K . Sr Umezawa, H. (1969) J . Antibiot. ( T o k y o ) 22, 283-286. 54. Barrett, A. J. (1977) in Proteinase in Mammaliun Cells and Tissues (Barrett, A. J., ed.) pp. 181 -208, North-Holland, Amsterdam. 55. Swanson, A. A,, Martin, B. J. Sr Spicer, S. S. (1974) Biochem. J . 137,223 - 228. 56. Snellman, 0. (1969) Biochrm. J . 114, 673-678. 57. Suominen, J. Sr Hopsu-Havu, U. K. (1971) Aria Chem. Scand. 25,2531 - 2540. 58. Franklin, S. G. Sr Metrione, R. M . (1972) Biochem. J . 127, 207-213. 59. Singh, H. Sr Kalnitsky, G. (1978) J . Biol. Chem. 253, 43194326. 60. de Lumen, B. 0. & Tappel, A. L. (1972) J . Biol. Chem. 247, 3552- 3557. 61. Warwas, M . Sr Dobryszycka, W . (1976) Biochim. Biophys. Acta, 429, 573 -580. 62. Keilova, H. Sr Tomasek, V. (1973) FEBS Lett. 29, 335-338. 63. Aronson, N. N., J r Sr Barrett, A. J. (1978) Biochem. J . 171, 759 - 765. 64. Nakai, N., Wada, K., Kobashi, K. Sr Hase, J. (1978) Biochim. Biophys. Acta, 83, 881 -885. 65. Auricchio, F., Mollica, L. s( Liguori, A. (1972) Biochem. J . 129, 1131-1138. 66. Haider, M. Sr Segal, H. L. (1972) Arch. Biochem. Biophys. 148, 228 - 237. 67. Johnson, L. W. & Velick, S. F. (1972) J . Biol. Chem. 247. 41 38 - 4143.
T. Towatari, Y . Kawabata, and N . Katunuma, Department of Enzyme Chemistry, Institute for Enzyme Research, Tokushima University School of Medicine, 3 Kuramoto-cho, Tokushima-shi, Tokushima-ken, Japan 770
Crystallization and Properties of Cathepsin B from Rat Liver Takae TOWATARI, Yoshimasa KAWABATA, and Nobuhiko KATUNUMA Department of Enzyme Chemistry, Institute for Enzyme Research, School of Medicine, Tokushima University (Received July 9, 1979)
Cathepsin B from rat liver was purified to apparent homogeneity by cell-fractionation, freezing and thawing, acetone treatment, gel filtration, DEAE-Sephadex and CM-Sephadex column chromatography, and was crystallized. The purified enzyme formed spindle-shaped crystals and its homogeneity was proved by disc gel electrophoresis in the presence of sodium dodecyl sulfate and by ultracentrifugical analysis. Its ~ 2 0 value , ~ was 2.8 S and its relative molecular mass was calculated to be 22 500 (k900) by sedimentation equilibrium analysis. Crystalline cathepsin B was shown to consist of four isozymes with isoelectric points between pH 4.9 and 5.3, the main isozyme having an isoelectric point of pH 5.0. The enzyme was irreversibly inactivated by exposure to weak alkali. The pH optimum was 6.0 with a-N-benzoyl-~~-arginine-4-nitroanilide as substrate. Amino acid analysis showed that the enzyme contained hexosamine, glucosamine and galactosamine. Cathepsin B inactivated aldolase, glucokinase, apo-ornithine aminotransferase, and apo-cystathionase, but the rates of inactivation of glucokinase, apo-ornithine aminotransferase, and apocystathionase were lower than that of aldolase. Studies by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate showed that cathepsin B degraded apo-ornithine aminotransferase to two polypeptide chains differing in relative molecular mass and electrophoretic mobility.
Inhibitors of so-called cathepsin B reduce protein degradation in various experimental systems. This has been demonstrated with leupeptin in a system in vitro [l] and in tissue culture [2] and with chloroquine in tissue culture [3]. Therefore, it is thought that cathepsin B is important in intracellular protein degradation. Cathepsin B has been found in various mammalian tissues and its purification has been attempted by many workers. The preparation of cathepsin B of Greenbaum and Fruton [4] was demonstrated by Otto [ 5 ] to consist of two enzymes, cathepsin B (cathepsin B1) and lysosomal carboxypeptidase B (cathepsin B2) [6]. It is now known that cathepsin B of Otto
contains cathepsin B, new cathepsin (cathepsin L) [7-91 and cathepsin H [lo]. We have purified cathepsin B from rat liver and examined its properties in an attempt to understand its role in the liver. We were able to crystallize the enzyme, as described in a preliminary report [ll]. This paper describes details of the method for obtaining crystalline cathepsin B from rat liver and the properties of the enzyme. MATERIALS AND METHODS Muterials Wistar rats fed on standard diet and weighing
200-250 g were used as a source of the enzyme. Abbreviations. PhMeS02F, phenylmethyl sulfonyl fluoride; E-64, N-[N-(~-3-trans-carboxyoxiran-2-carbonyl)-~-leucyl]-agma- Ovalbumin, chymotrypsinogen, and myoglobin, from tin; Bz-Arg-Nan, r-N-benzoyl-~~-arginine-4-nitroanilide. Mann, U.S.A., and bovine serum albumin from Sigma, Enzymes. Cathepsin B (EC 3.4.22.1);carboxypeptidase B (EC U.S.A. were used as markers in the determination of 3.4.17.2);cathepsin L (EC 3.4.22.15);cathepsin H (EC 3.4.22.-); the relative molecular mass. a-N-Benzoyl-DL-argininealdolase (EC 4.1.2.13);glucokinase (EC 2.7.1.2);ornithine aminotransferase (EC 2.6.1.13);cystathionase (EC 4.4.1.1); tyrosine 2-naphthylamide, ~-leucine-2-naphthylamide, a-Naminotransferase (EC 2.6.1.5); glucose-6-phosphate dehydrogenase benzoyl-DL-arginine-4-nitroanihde, N-a-p-tosyl-L-lysine (EC 1.1.1.49);alcohol dehydrogenase (EC 1.1.1.1);malate dechloromethyl ketone . HCl, L-1-tosylamido-2-phenylhydrogenase (EC 1.1.1.37);glyceraldehyde-3-phosphatedehydroethylchloromethyl ketone . HCl, soyben trypsin ingenase (EC 1.2.1.12);glutamate dehydrogenase (EC 1.4.1.2); hibitor, and phenylmethyl sulfonyl fluoride were oblactate dehydrogenase (EC 1.1.1.27).
280
tained from Sigma. a-N-Benzoyl-DL-arginine ethyl ester was from the Peptide Center (Institute for Protein Research, Osaka, Japan). Protease inhibitors of bacterial origin (chymostatin, pepstatin, antipain, and leupeptin) were kindly supplied by Dr Aoyagi and Dr Umezawa (Institute of Microbial Chemistry, Tokyo, Japan). The thiol protease inhibitor, E-64 [12] of Aspergillus japonicus and its synthetic thiol protease inhibitors (monoethylepoxy succinate and monobenzylepoxy succinate) were kindly supplied by Dr Sawada (Taisho Pharmaceutical Co., Japan). Sephadex G-75, DEAE-Sephadex A-50, and CM-Sephadex C-50 were from Pharmacia. All other reagents were standard analytical grade products. Assay of Cathepsin B with Synthetic Substrates Cathepsin B was routinely assayed by the method of Otto and Bhakdi [13]. The cleavages of a-N-benzoyl~~-arginine-2-naphthylamideand ~-1eucine-2-naphthylamide at pH 6.0 were measured by coupling the 2-naphthylamide with fast garnet GBC salts as described by Barrett [14]. The cleavage of a-N-benzoylDL-arginine ethyl ester at pH 6.0 was measured by the procedure of Roberts [15], and the cleavage of a-Nbenzoyl-m-argininamide at pH 6.0 by those-of Seligson and Seligson [16] and Russell [17]. Assay of Cathepsin B with Substrate Enzymes The rates of inactivation of enzymes were measured as follows. The reaction mixture contained 100 pmol potassium phosphate buffer, pH 6.0, 5 pmol 2-mercaptoethanol, 0.5 - 2.0 units of substrate enzyme and a suitable amount of cathepsin B preparation in a final volume of 1.O ml. After incubation for 15- 30 min at 37 "C, samples of 200 pmol were taken for measurement of the remaining enzyme activity. With substrate enzymes one unit of cathepsin B was expressed as the amount giving a first-order rate constant in the inactivating reaction, k = 1 (min-'). Assay of Cuthepsin B with Protein Substrates The proteolytic activities of cathepsin B on casein, acid-denatured hemoglobin [ 181 and bovine serum albumin were assayed by measuring the liberation of free amino acids, recovered in the fraction soluble in trichloroacetic acid, by the ninhydrin method [19]. Preparation and Assay o j Substrate Enzymes Crystalline ornithine aminotransferase was purified from rat liver by the method of Matsuzawa et al. [20], tyrosine aminotransferase by the method of Valeriote et al. [21], cystathionase by the method of Matsuo and Greenberg [22], glucokinase by the method of
Properties of Crystalline Cathepsin B from Rat Liver
Grossman et al. [23], aldolase by that of Rajkumar et al. [24], glucose-6-phosphate dehydrogenase by that of Watanabe and Takeda [25] and lactate dehydrogenase by that of Scopes [26]. Glucose-6-phosphate dehydrogenase and alcohol dehydrogenase from yeast, malate dehydrogenase from pig heart, glyceraldehyde-3-phosphate dehydrogenase and aldolase from rabbit muscle, and glutamate dehydrogenase from beef liver were purchased from Boehringer, Mannheim. Ornithine aminotransferase was assayed by the method of Katunuma et al. [27], tyrosine aminotransferase by that of Rosen et al. [28], cystathionase by that of Matsuo and Greenberg [22], glucokinase by that of Ballard and Oliver [29] and aldolase by that of Blostein and Rutter [30]. Glucose-6-phosphate dehydrogenase was assayed by measuring the rate of increase in absorbance of NADPH at 25 "C [31]. Malate dehydrogenase [32], lactate dehydrogenase [33] and glutamate dehydrogenase [34] were assayed by measuring the rate of decrease in absorbance of NADH at 25 "C. Glyceraldehyde-3-phosphate dehydrogenase was assayed by measuring the coupled reaction with phosphoglycerate kinase 1351. Alcohol dehydrogenase was assayed by measuring the rate of increase in absorbance of NADH at 25 "C [36]. Disc Gel Electrophoresis Polyacrylamide disc gel electrophoresis [37.38] was performed at pH 4.5 and 8.3, using 12.5 "/, separation gel. Samples were applied in 10% sucrose and methyl green and bromophenol blue were used as tracking dyes at pH 4.5 and pH 8.3, respectively. Electrophoresis was carried out at 4 "C at a constant current of 2 mA per tube. The gel was stained with Coomassie blue G-250 and destained with methanol/water/acetic acid (50: 135 : 15, by vol.). Sodium dodecyl sulfate/ polyacrylamide disc gel electrophoresis was performed by the method of Weber and Osborn [39]. Analytical Isoelectric Focusing Isoelectric focusing in polyacrylamide gel was carried out as described by Wrigley [40]. The gel solution contained 8.2 parts of water, 3.0 parts of a solution of 30% acrylamide (w/v), and 0.8% N,N'methylenebisacrylamide (w/v), 0.8 parts of a solution of 1 "/, N,N,N',N'-tetramethylethylenediamine (v/v) and 0.014% riboflavin (w/v) and 0.3 part Ampholine. A mixture of Ampholines of pH 3.5-5, pH 5-7 and pH 3.5 - 10 (40: 40: 20) was used at a final concentration of 1 % (w/v). Tubes of 8-cm length and 0.5-cm diameter were used. Electrophoresis was performed for 4 h at 4 ° C using 0.2% sulfuric acid in the anode vessel and 0.4 % ethanolamine in the cathode vessel and a constant voltage of 300 V. After electrophoresis, protein in the gel was stained with Coomassie blue
28 1
T. Towatari, Y. Kawabata, and N. Katunuma Table 1. Purification of cethepsin B from rat liver One unit of activity is defined as the amount of enzyme liberating 1 pmol p-nitroanilinelmin from Bz-Arg-Nan Purification step
1. Lysosomal extract
2. 3. 4. 5. 6.
Acetone treatment Sephadex (3-75 DEAE-Sephadex A-50 CM-Sephadex C-50 Crystallization
Total volume
Total protein
Total activity
Specific activity
Yield
Purity
ml
mg
units
units/mg
%,
-fold
910 36 18 40 1 1
10700 685 324 24 23 5.6
32.21 24.00 26.43 9.66 9.60 2.65
0.003 0.035 0.085 0.403 0.417 0.473
100 14.5
1 12 27 134 139 158
G-250 (0.4 % in 3 % perchloric acid). For pH determination and measurement of cathepsin B activity, the gels were cut into 40 or more sections and each section was extracted with 1 ml distilled water. Cathepsin B activity was measured as described above.
Determination of Relative Molecular Mass Relative molecular mass was determined by descending gel filtration through a Sephadex G-75 column (2 x 100 cm) equilibrated with 50 mM acetate buffer, pH 5.0, containing 0.1 M sodium chloride and 5 mM 2-mercaptoethanol [41]. The markers used were bovine serum albumin ( M , 67000), ovalbumin ( M , 45 000), chymotrypsinogen ( M , 25 000) and myoglobin ( M , 17800). With sodium dodecyl sulfate gel (run as described above), mobilities with respect to bromophenol blue were plotted against the logarithm of the relative molecular masses using the same markers.
Amino Acid Analysis Amino acid analysis was carried out in a Hitachi amino acid analyzer by the standard procedure [42], except that glucosamine, galactosamine, and tryptophan were measured by the procedure of Liu and Chang [43] using a solution of 3 M p-toluene sulfonic acid containing 0.2 % 3-(2-amino ethyl) indole. Samples in 6 M HCl were hydrolyzed under vacuum at 110 “C for 24 h and 48 h. Half-cystine was determined by performic acid oxidation as described by Moore [44] followed by hydrolysis for 24 h.
Ultracentrifugation Analysis Ultracentrifugation analysis was carried out in a Hitachi 282 analytical ultracentrifuge equipped with a temperature control unit, a monochromator and an absorption scanner, as described by Schachman and Edelstein [45]. Schlieren and absorption optics were used for sedimentation velocity studies and sedimentation equilibrium experiments, The rotor speeds were
82.0 30.0 29.8 8.2
54000 rev./min and 15000 rev./min, respectively, and 20mM acetate buffer, pH5.0, containing 0.1 M sodium chloride was used as solvent. The concentration of protein used was 0.65 mg/ml for sedimentation velocity analysis and 0.33 mg/ml for sedimentation equilibrium analysis. Values were calculated taking the partial specific volume as 0.72 ml/g, based on the amino acid composition.
Determination of Protein Protein concentration was determined by the method of Lowry et al. [46] using bovine serum albumin as a standard. RESULTS
Purification of Cathepsin B Cathepsin B from rat liver was purified to a crystalline form by a modification of the method of Otto [5] and Otto and Riesenkonig [47] with additional procedures. The purification is summarized in Table 1. All purification procedures were performed at 4 “C, using 50 mM acetate buffer, pH 5.0, containing 5 mM 2-mercaptoethanol, referred to as ‘standard buffer’. Extraction. A crude lysosomal fraction was isolated from rat livers as described by Ragab et al. [48], suspended in standard buffer and subjected to sevencycles of freezing and thawing. The mixture was centrifuged at 10000 x g for 30 min and the precipitate was discarded. Acetone Treatment. The supernatant was mixed with cold acetone to a final concentration of 45 % (v/v) with stirring over a period of 5 min and then the mixture was rapidly centrifuged at 8000xg for 5 min. Further cold acetone was added to the supernatant to a final concentration of 75% (v/v). The resulting precipitate was collected by centrifugation and dissolved in a minimum volume of standard buffer. The preparation was centrifuged briefly to remove insoluble material and the supernatant passed through a Sephadex G-25 column (6 x 20 cm) equilibrated with standard buffer and concentrated to a small volume
Properties of Crystalline Cathepsin B from Rat Liver
282
Fig. 1. Pliotomicrogruph of crystuls of cathepsin Bjrom rut liver. Magnification x 400
A
B
C
Fig. 2. Polqacrylamide gel elec!rophorc~sisof crystalline cuthepsin B .from rat liver. Electrophoresis was carried out at pH 4.5 (A), pH 8.3 (B), and with sodium dodecyl sulfate (C) as described under Materials and Methods. Samples of 10 pg protein were used. Migration was from top to bottom of the gel
by ultrafiltration using a PMlO membrane (Amicon, U.S.A.). Gel filtration on a Sephadex G-75 Column. The enzyme solution was divided in half and applied to two identical Sephadex G-75 columns (3.0 x 100 cm) previously equilibrated with standard buffer containing 0.1 M sodium chloride, the columns were eluted with the same buffer. The fraction containing Bz-Arg-Nanhydrolyzing activity with an M , of about 25000 was
eluted from the gel, collected and concentrated to a small volume by ultrafiltration on an Amicon PMlO membrane. Cathepsin B was separated from lysosomal carboxypeptidase and cathepsin D in this step. DEAE-Sephadex A-50 Column Chromatography. The enzyme solution was then applied to a DEAESephadex A-50 column (1.8 x 20 cm) equilibrated with 20 mM acetate buffer, pH 5.0, containing 10 mM sodium chloride and 5 mM 2-mercaptoethanol. The Bz-Arg-Nan-hydrolyzing activity was eluted in two fractions with the same buffer: the fraction eluted first contained cathepsin H activity, which has low sensitivity to leupeptin as described by Davidson andPoole [49]; the fraction eluted second contained cathepsin B, new cathepsin (cathepsin L), and leucine-2-naphthylamide-hydrolyzing enzyme. CM-Sephadex C-50 Column Chromatography. The latter fraction was applied to a CM-Sephadex C-50 column (1.8 x 15 cm) equilibrated with 20 mM acetate buffer, pH 5.0, containing 5 mM 2-mercaptoethanol and 50 mM sodium chloride. The column was washed with the same buffer and then eluted stepwise with 0.1 M, 0.2 M and 0.5 M sodium chloride in 20 mM acetate buffer, pH 5.0, containing 5 mM 2-mercaptoethanol. Cathepsin B was eluted with 0.1 M sodium chloride and concentrated to about 20 mg protein/ml in a collodion bag. The leucine-2-napfithylamidehydrolyzing enzyme was eluted with 50 mM sodium chloride and new cathepsin (cathepsin L) with 0.5 M sodium chloride. Crystallization. Solid ammonium sulfate was added gradually to the concentrated enzyme solution until just before a slight turbidity appeared in the cold and then the solution was stored for several days in the cold. The spindle-shaped crystals formed are shown in Fig. 1.
T. Towatari, Y. Kawabata, and N. Katunuma
283
Crystals, Homogeneity and Relative Molecular Mass Crystalline cathepsin B was subjected to polyacrylamide gel electrophoresis. As shown in Fig. 2, the crystalline enzyme migrated as a single protein at pH 4.5, but a minor component was separated at pH 8.3. Similar results were obtained with the mother liquor. It gave one major protein band when subjected to gel electrophoresis in the presence of sodium dodecyl sulfate and had an M , of 23000. It gave a single symmetrical boundary on ultracentrifugation (Fig. 3), and its sedimentation coefficient was determined to be 2.8 S. Its relative molecular mass was calculated to be 22 500 ( * 900) by sedimentation equilibrium centrifugation, and 26000 by gel filtration on a Sephadex G-75 column (Fig. 4). Fig. 3 . Ultracentrijiugul patterns of crystalline cathepsin B from rat livrr. Sedimentation velocities were measured at 54000 rev./min at 21 C. The enzyme (11.4 mg/ml) was dissolved in 20 mM acetate buffer, pH 5.0, containing 0.1 M sodium chloride. The picture was taken 54 min after attaining the maximal speed
Analytical Isoelectric Focusing On isoelectric focusing in polyacrylamide gel with Ampholine pH 3.5-8.0, the cathepsin B gave four protein bands with isoelectric points between pH 4.9 and 5.3 (Fig. 5), the main protein band having an isoelectric point of pH 5.0. All the protein bands corresponded with bands of activity and stained with periodic acid/Schiff reagent, indicating that cathepsin B from rat liver consists of four isozymes and is a glycoprotein. Amino Acid Composition
0
50
60
70
80
90
Fraction number
Fig. 4. Determination o/ relative moleculur muss. A column ( 2 x 1OOcm) of Sephadex (3-75 was equilibrated with 50 mM acetate buffer, p H 5.0, containing 0.1 M sodium chloride and 5 rnM 2-rnercaptoethanol. Standard proteins (5 mg) were applied in a volume of 1.5 ml: (1) bovine serum albumin; (2) ovalbumin; ( 3 ) chymotrypsinogen; (4) myoglobin. The amount of cathepsin B applied formed 3.5 Fmol p-nitronilinelmin from Bz-Arg-Nan. Fractions of 2.5 ml were collected at a flow rate of 25 mlih
Cathepsin B has been defined as a protease with an M , of about 25000 and an optimum pH of about 6.0 that inactivates various high-M, proteins, such as aldolase, hydrolyzes synthetic substrates, such as BzArg-Nan, and does not hydrolyze L-leucine-2-naphthylamide [50 - 531. These findings were confirmed in the following experiments.
The amino acid composition of crystalline cathepsin B from rat liver is shown in comparison with that of human cathepsin B [54] in Table 2. The most characteristic features are that it contains considerable amounts of glucosamine and galactosamine. These findings also show that cathepsin B is a glycoprotein, like the other lysosomal enzymes that have been characterized. The contents of glycine, aspartic acid, and glutamic acid are high and the content of methionine is low, as in human cathepsin B, although the values for individual amino acid residues/molecule ( M , 25000) differ from those of human cathepsin B. Optimum p H As shown in Fig. 6, the activity of the cathepsin B with Bz-Arg-Nan as substrate was maximal at pH 6.0 and decreased markedly above pH 7.0 and below pH 4.0.
Stability Fig.7 shows the affects of preincubation at pH 4.0 - 7.5 at 37 "C for 30 min on cathepsin B. The
Properties of Crystalline Cathepsin B from Rat Liver
284
A
B
+
?
I
8.0 100 %O
.->
A c
.-" c
61)
m
I,
a .-*m 1
50
50
d
4.0
3.0 0
0
10
20 Gel slice number
30
40
Fig. 5. Analytical isoelectric focusing of crystalline ccitkrpsin B ji-om rat liver. pH (0-0); enzyme activity ( 0 2 ) .Analytical conditions were as described under Materials and Methods. 54 Fg enzyme protein were used in gel stained for protein and 108 Kg in gel stained for carbohydrate. Gels were stained for Drotein with Coomassie blue G-250 and for carbohydrate with periodate/Schiff procedure. (A) Carbohydrate; (B) protein
cathepsin B was stable between pH 4.0 and pH 6.5, but it was very unstable at alkaline pH values. It was stable on heat treatment at 35 "C, 42 "C, and 47 "C for 15 min at pH 6.0.
tease inhibitor from Aspergillus japonicus, and by its synthetic analogues, monoethylepoxy succinate and monobenzylepoxy succinate. Substrate Specificity
Effects of Activators and Inhibitors As shown in Table 3, the cathepsin B required the presence of a sulfhydryl compound plus EDTA for maximal activity. In the presence of EDTA, 1 mM cysteine, 0.25 mM dithiothreitol or 8 mM 2-mercaptoethanol was required for maximal activity. EDTA alone was ineffective, but a sulfhydryl compound alone caused some activation. Cathepsin B was inhibited by monoiodoacetate at low concentration, showing that it possesses an essential thiol group [51]. It was also strongly inhibited by the microbial inhibitors, leupeptin, chymostatin, and antipain [53] and N-cc-ptosyl-L-lysinechloromethyl ketone and L-l-tosylamido2-phenylethylchloromethyl ketone [55]. Cathepsin B was also inhibited by E-64 [12], which is a thiol pro-
As shown in Table 4, cathepsin B from rat liver hydrolyzed Bz-Arg-Nan, a-N-benzoyl-~~-arginine-2naphthylamide, a-N-benzoyl-DL-argininamide,and a-N-benzoyl-DL-arginine ethyl ester, but not L-leucine2-naphthylamide. Results of the inactivations of various enzymes by cathepsin B are shown in Table 5. Cathepsin B inactivated aldolases (muscle and liver) and glucokinase (liver), which are reported to be attacked by cathepsin B [50]. It inactivated aldolase significantly more rapidly than glucokinase, although it did not cause more than 50 inactivation of aldolase. It slightly inactivated apo-cystathionase and apoornithine aminotransferase. The mode of its inactivation of apo-ornithine aminotransferase was studied in detail by measuring the decrease in enzyme activity,
T . Towatari, Y. Kawabata, and N. Katunuma
285
Table 2. Amino m i d composition of cathepsin B,from rut liver Values are residues/molecule (Mr 25000) Amino acid
Residues after 24 h
Tryptophanb Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cystine (halod Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Glucosamine Galactosamine
Calculated number of residues
Human cathepsin B"
6.5 8.1 4.6 6.2 19.6 9.9 16.2 19.9 12.0 28.6 11.7 9.4 12.0 3.0' 11.8 8.2 9.0 6.9 8.1 9.0
7 10 8 8 23 10 17 23 17 29 13 12 15 4 13 10 13 7 3
220.7
242
48 h
5.4 f 0.9 8.3 f 1.0 4.0 f 0.1 6.0 (2) 20.2 f 1.3 9.2 f 1.2 16.5 k 0.2 19.6 f 0.7 12.6 & 1.9 28.9 & 1.7 11.7 f 0.2 9.4 (2) 11.9 k 1.0 2.6 0.4 8.9 f 0.8 6.3 f 0.5 8.7 (2) 7.0 (2) 7.8 f 0.1 8.9 f 0.8
(3) (3) (3)
4.2 f 0.1 (3) 7.9 (2) 5.2 (2) 6.4 (2) 19.0 & 0.4 (3) 10.5 k 0.2 (3) 15.8 i 0.3 (3) 20.1 k 0.3 (3) 11.3 f 0.4 (3) 28.3 f 0.1 (3) 11.6 f 0 (3)
(4) (4) (4) (4) (4) (4) (4) (4) (4) (4) (4)
12.0 2.4 11.8 8.2 9.2 6.7 8.3 9.1
(3) (3)
*f 0.20.1 (3)(3) f 0.1 (3) f 0 (3) f 0.1 (3) f 0.2 (3) f 0 (3) k 0.1 (3)
Total * Taken from Barrett's human cathepsin B [54].
Hydrolyzed in 3 M p-tuluene sulfonic acid containing 0.2 3-(2-aminoethyl)indole at 110 "C Values extrapolated to zero-hour hydrolysis. Oxidized with performic acid before hydrolysis in 6 M HCI at 110°C for 24 h.
4.0
4.5
5.0
5.5
60
6.5
70
PH
4.0
5.0
60
TO
8.0
PH
Fig. 6. pHIActivity curve of cathepsin B from rat liver. The buffers used at final concentrations of 0.1 M were acetate buffer for p H 4.6- 5.6 (+O) and potassium phosphate buffer for p H 5.7- 7.2 (-0). Bz-Arg-Nan was used as substrate
Fig. 7. pH-dependent stabilities of cathepsin B f r o m rat liver. The enzyme was preincubated for 30 min at 3 7 ' C in 0.1 M buffers of various p H values and then remaining activities were assayed using Bz-Arg-Nan. Acetate buffer p H 4.0-5.6 (&-0); potassium phosphate buffer p H 5.7-7.5 (-0)
changes in relative molecular mass of the products separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and changes in mobility of the native subunit and products by polyactylamide gel electrophoresis. Fig. 8 shows that the loss of activity
corresponded quantitatively to the loss of material migrating in the position of the native subunit and that two products appeared which migrated in different position on polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate.
Properties of Crystalline Cathepsin B from Rat Liver
286 Table 3. Effects of’uctivators and inhihitors on cathepsin B,from rat liver The enzyme was incubated with various effectors for 5 min and then assayed as described under Materials and Methods using Bz-ArgNan as substrate. Experiment 2 was performed in the presence of 2 m M EDTA. Experiment 3 was performed in the presence of 2 mM EDTA and 4 mM cysteine
Table 4. Activities of cathepsin B on synthetic substrates Reaction mixtures contained 5 pmol synthetic substrate (except a-N-benzoyl-DL-argininamide, which was added at 50 pmol), 100 pmol potassium phosphate buffer, pH 6.0, 2 pmol EDTA, 4 pmol cysteine, and a suitable amount of enzyme in a final volume of 1.0 ml. Assays were performed at 37 ’C for 15 and 30 min as described under Materials and Methods
Expt Effectors
Substrate
Final concn
Relative activity
PM
7i
4000 4000 2000 4000 2000
100 210 100
Cathepsin B activity pmol x min-’ x mg-’
1.
Control Cysteine EDTA Cysteine
+ EDTA
__ Control Dithiothreitol Cyyterne ~
2.
~~
-
-
2-Mercaptoethanol
250 500 1000 8000 500 8000
100 329.4 376.4 323.5 323.5 132.1 342.9
-~
3
Control Monoiodoacetate Leupeptin Chymostatin L-I-Tosylamide-2-phenylethylchloro-methyl ketone N-a-p-Tosyl-L-lysine chloromethyl ketone Antipain Pepstatin PhMeSOzF Trypsin inhibitor (soybean) E-64 Monoethylepoxy succinate Monobenzylepoxy succinate
a-N-Benzoyl-~~-arginine-4-nitroanilide0.40 a-N-Benzoyl-~~-arginine-2-naphthylamide 2.75 a-N-Benzoyl-~~-arginine ethyl ester 1.04 a-N-Benzo yl-uL-argininamide 2.12 ~-Leucine-2-naphthylamide 0
1.0 0.1 6.2
100 62 100 87.7
3.0
53.3
1.o 1.o 10 100 14 0.28 50.0 3.8
71.4 60 100 100 100 63.8 19.6 28.3
The relative molecular mass of the products were estimated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis using markers. That of the native subunit, referred to as the A form, was 45000 and those of the B and C forms were 27000 and 18000, respectively. These results suggest that cathepsin B inactivates the apo-form of ornithine aminotransferase by limited proteolysis and degrades it to the B and C forms. Glucose-6-phosphate dehydrogenases (yeast and rat liver), apo-tyrosine aminotransferase (rat liver), glyceraldehyde-3-phosphate dehydrogenase (rabbit muscle), malate dehydrogenase (pig heart), lactate dehydrogenase (rat liver), glutamate dehydrogenase (beef liver), and alcohol dehydrogenase (yeast) were not inactivated at all by cathepsin B. Of the protein substrates tested, acid-denatured hemoglobin, casein, and bovine serum albumin were hydrolyzed, as reported earlier [50,51]. Therefore, the inactivation of
Table 5. Activities of cathepsin B on enzyme substrates The reaction conditions were as described under Materials and Methods. Values show inactivations of substrate enzymes as percentages of that of aldolase from rabbit muscle. Cathepsin B activity on aldolase (muscle) was 11.6 units/mg Substrate
Source
Cathepsin B activity 0, :r,
Aldolase Aldolase Glucokinase Cystathionase (apo-) Ornithine aminotransferase (apo-1
rabbit muscle rat liver rat liver rat liver rat liver
100 182 8 2 1
various enzymes by cathepsin B seems to result from their proteolysis.
DISCUSSION Cathepsin B has been identified as a lysosomal thiol enzyme present in numerous animal tissues including liver. Previously we isolated a new lysosomal protease, named new cathepsin [7], from the cathepsin B fraction of rat liver and reported that it may be identical with cathepsin L of Kirschke et al. [9]. Recently Singh and Kalnitsky [59] separated a new x-N-benzoylarginine-2-naphthylamidehydrolase from rabbit lung, and clearly differentiated it from new cathepsin (cathepsin L), cathepsin B, and a-N-benzoylarginine-2-naphthylamide aminohydrolase [60] from rat liver on thebasis ofits substrate specificity, pH optimum, relative molecular mass and sensitivity to leu-
1
2
1
2
3
4
5
6
7
8
C)
A-
B-
C-
3
4
5
6
7
8
Fig. 8. Mode ofproreolysis of’apo-ornithineurninorrunsferase by cuthcpsin Bfrorn rat liver. (a) Time course of proteolysis of apo-ornithine aminotransferase by cathepsin B. Apo-ornithine aminotransferase (0.46 mg) was incubated at 37 ‘C with cathepsin B (0.22 mg) in 1 ml of 0.1 M sodium phosphate buffer (pH 6.0) containing 10 mM 2-mercaptoethanol and 1 mM EDTA. 20O-pl samples were removed at the times indicated for assay of ornithine aminotransferase activity and for analysis by polyacrylamide gel electrophoresis. Reactions were stopped by adding 10nmol leupeptin. Complete system (0-0); control system in which apo-ornithine aminotransferase was incubated with watef instead of cathepsin B (0-0). (b) Polyacrylamide gel electrophoregrams of the products of apo-ornithine aminotransferase formed with cathepsin B. Gels 1-4 show results with the complete system after 0, 1, 3 and 6 h ; gels 5 and 6 show results in the apo-ornithine aminotransferase control system after 0 and 6 h ; gels 7 and 8, show results in the cathepsin B control system after 0 and 6 h. (c) Sodium dodecyl sulfateipolyacrylamide gel electrophoregrams of the products of apo-ornithine aminotransferase formed with cathepsin B. Conditions were the same as for (b)
288
peptin. Rabbit lung a-N-benzoylarginine-2-naphthylamide hydrolase is, in some respects, similar to cathepsin H recently isolated from rat liver [lo]. Recently we isolated cathepsin B in crystalline form from rat liver and determined its properties. The relative molecular mass of crystalline cathepsin B was calculated to be 26000 by Sephadex G-75 column chromatography, 22 500 by sedimentation equilibrium analysis, and 23 000 by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. These values are similar to that of 24000 reported for the enzyme from human liver [52] and that of 25000 reported for the enzymes of bovine spleen [5] and liver [56], and rat liver, kidney, spleen, and lung [51]. Moreover it has been reported that ovine [57], bovine [58], and human cathepsin B have multiple forms that can be separated by ion-exchange chromatography. The forms differ in isoelectric points, and plvalues ranging from pH 4.5 to 5.5 were reported for the human enzyme, the major form having a p l value of pH 5.0 to 5.2. Recently cathepsin B from rabbit lung was shown to consist of four isozymes with isoelectric points ranging from pH 5.0 to 5.5 [59]. Warwas and Dobryszycka demonstrated by isoelectric focusing in polyacrylamide gel that there are three isozymes of cathepsin B in the human fetal placenta membrane, chorion, differing in pl, whereas there is only one isoenzyme activity in the amnion [61]. As shown in Fig. 5, crystalline cathepsin B from rat liver also consisted of four isozymes with isoelectric points ranging from pH 4.9 to 5.3, the major form having an isoelectric point of pH 5.0. All the forms stained with periodic acid/Schiff reagent. The crystalline form and the enzyme in the mother liquor both migrated as a single protein on polyacrylamide disc gel electrophoresis in the presence of sodium dodecyl sulfate. Amino acid analysis showed that crystalline cathepsin B contained hexosamine, and that the contents of glycine, aspartic acid, and glutamic acid are high and the content of methionine is low, as in cathepsin B from human liver [54]. Since the charge heterogeneity of cathepsin B was not changed by crystallization of the enzyme, and since N-terminal leucine has been found in several forms of bovine enzyme by Franklin and Metrione [58] and Keilova and Tomasek [62], the multiple forms may result from changes in carbohydrate content during the solubilization step, as is the case with some other lysosomal enzymes. This possibility needs further investigation. Crystalline cathepsin B was inhibited by E-64 and its synthetic analogues. The enzyme inactivated apoornithine aminotransferase and apo-cystathionase to some extent, Recently Aronson and Barret showed that cathepsin B from rat and human liver and bovine spleen degraded glucagon by sequential cleavage of dipeptides from the C-terminal end of the molecule [63]. Nakai et al. reported that inactivation of aldolase
Properties of Crystalline Cathepsin B from Rat Liver
(rabbit muscle) by cathepsin B in vitro was due to release of one mole of the dipeptide alanyl-tyrosine from the C-terminal end of the molecule [64]. We found that cathepsin B split apo-ornithine aminotransferase into two polypeptide chains. This difference in the modes of proteolysis by cathepsin B seems to be due to differences in the conformation of the protein molecules. Glucose-6-phosphate dehydrogenase, apotyrosine aminotransferase and glyceraldehyde-3-phosphate dehydrogenase, which are good substrates for the new cathepsin [S], were not inactivated by cathepsin B from rat liver. It has been reported that thiol protease in lysosomes can inactivate native enzymes, such as aldolase [50], glucokinase [50], pyruvate kinase [51], tyrosine aminotransferase [65], arginase [66], alanine aminotransferase [66], and glyceraldehyde3-phosphate dehydrogenase [67]. These findings suggest that various lysosomal proteases have specific actions on substrate enzymes and play important roles in intracellular protein degradation. We would like to thank Mr R. Kunai for help with Ultracentrifugal analysis and M r H. Miyai for assistance in amino acid analysis. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan.
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T. Towatari, Y . Kawabata, and N . Katunuma, Department of Enzyme Chemistry, Institute for Enzyme Research, Tokushima University School of Medicine, 3 Kuramoto-cho, Tokushima-shi, Tokushima-ken, Japan 770
