Eur. J . Biochem. 55,211-219 (1975)
Broad-Specificity Proteinase Inhibitors in Scopolia japonica (Solanaceae) Cultured Cells Isolation, Physicochemical Properties, and Inhibition Kinetics Kuniaki SAKATO, Hozumi TANAKA, and Masanaru MISAWA Tokyo Research Laboratory, Kyowa Hakko Kogyo Co., Ltd., Tokyo (Received October 11, 1974 / March 10, 1975)
Proteinase inhibitors were isolated from Scopoliu juponicu cultured cells. Isolation procedures involve concentration by a hydrophobic resin of Diaion HP-20, decolorization by Duolite A-7, affinity chromatography on trypsin-Sepharose, and Bio-Gel P-4 chromatography. It was found that the proteinase inhibitors from S.juponicu cells are a mixture of at least five components. For the inhibitory components except one, amino acid analyses, measurements of sedimentation equilibrium and optical rotatory dispersion (ORD) were carried out. The inhibitors were shown to be the polypeptides with molecular weights in the range of approximately 4000 to 6000. In addition, one of them was found to have approximately 15% a-helical conformation by the MoffittYang analysis of ORD data. The inhibitors were found to have potent inhibitory activity for trypsin, chymotrypsin, plasmin, kallikrein and pepsin but not for papain with synthetic and natural substrates. These inhibitors formed stable complexes with trypsin and chymotrypsin in an equimolar ratio, and their inhibitory mechanisms for both enzymes were of non-competitive type.
sin-kallikrein inhibitor of Kunitz [3] and 4-aminomethylcyclohexane carboxylic acid [4] have reached to the stage of clinical uses. An assay method for inhibition of plasmin was designed in our laboratory for screening a useful plasmin inhibitor from natural sources [ 5 ] . This method has the following advantages : (a) inhibitory activities for plasmin and thrombin can be estimated simultaneously; (b) the crude material can be assayed in small quantities. By this method, the screening of new inhibitors for plasmin were extensively performed with plant calli, microbial cultures and synthetic compounds. In the screening, aqueous extracts from several calli were found to inhibit plasmin activity [5,6]. An extract from Scopolia japonicu (family Solanaceae) showed the strongest inhibition among Abbreviations. Bz-nL-Arg-ONan, a-N-benzoyl-DL-arginine-p- them. The cultural conditions for cell growth and nitroanilide; Bz-L-Arg-ONan, a-N-benzoyl-L-arginine-p-nitroanilide; Glt-Phe-ONan, a-N-glutaryl-L-phenylalanyl-p-nitroanilide; accumulation of the inhibitor were examined in detail with S.juponicu cell suspensions [ 5 ] .As a result, Bz-Arg-OEt, u-N-benzoyl-L-arginine ethyl ester; Ac-Phe-Tyr(I,), a-N-acetyl-~-phenylalanyl-~-3,5-diiodotyrosine ; ORD, optical rotaS.juponicu cultured cells were found to accumulate tory dispersion. higher amounts of the inhibitor than the intact plant. Enzyrnes(CBN Recommendations 1972).Trypsin (EC 3.4.21.4); Cell cultures of higher plants have been of much chymotrypsin (EC 3.4.21.1): plasmin (EC 3.4.21.7): kallikrein interest for the biosynthetic potential of various (EC 3.4.ii.8); pepsin (EC 3.4.23.1); papain (EC 3.4.22.2); thrombin metabolites because of the possibility for the produc(EC 3.4.21.5).
In living organisms, the action of proteolytic enzymes is controlled by many ingenius ways. One of control mechanisms is to use the proteinase inhibitor, although very little has been known about its physiological functions. Proteinase inhibitors occur naturally as polypeptides, proteins, glycoproteins or polysaccharides [l]. More recently they have been isolated from not only plants and animals but also microorganisms [2]. This type of control has made possible the therapeutic application of the pathophysiological processes due to the blood coagulation, the fibrinolysis and the liberation of kinins [l]. Many proteinase inhibitors have been found in natural sources and also have been synthesized for clinical uses [ 2 ] .As the result, a basic bovine pancreatic tryp-
Eur. J. Biochem. 55 (1975)
212
tion of useful constituents. However, very few has been reported on the production of important metabolites by plant cell cultures. This may be ascribed mainly to the fact that the accumulation of metabolites, especially secondary metabolites such as alkaloids, decreases remarkably in dedifferentiated plants. We have induced a variety of plant calli and have examined the production of important metabolites by their cells in cultures. These results will be reviewed elsewhere [6]. The present paper deals with the isolation procedures and physicochemical properties of the proteinase inhibitor from S. japonica cultured cells together with their inhibitory activities for several proteolytic enzymes.
Proteinase Inhibitors from S . .japonir-a Cultured Cells
[8] containing 1.O mg/l of 2,4-dichlorophenoxyacetic acid, 0.02 mg/l of kinetin and 3 % sucrose, using a gas bubble fermentor (20 1) with an aeration of 10 l/min at 28 “C [ 5 ] . After 14 days of culture, 5.8 kg of fresh cells was harvested. Extraction Fresh cells (5.8 kg) were homogenized with 11.6 1 of deionized water in a homogenizer for approximately 10 min. The suspension was heated in boiling water for approximately 10 min. This treatment enhanced remarkably the inhibitory activity for plasmin. Then, the suspension was filtered through a Miracloth (Chicopee Manufacturing Co., U.S.A.) to remove insoluble materials.
EXPERIMENTAL PROCEDURE Muteriuh Porcine trypsin (grade V) was obtained from MilesSeravac Co., {J.S.A. As trypsin substrates, a-N-benzoyl-DL-arginine-p-nitroanilide (Bz-DL-Arg-ONan) (Bz-L-Argand a-N-benzoyl-L-arginine-p-nitroanilide ONan) were obtained from Merck AG, Germany and the Protein Research Foundation, Japan, respectively. Bovine x-chymotrypsin was obtained from Worthington Biochemicals, Corporation, U.S.A. As an a-chymotrypsin substrate, a-N-glutaryl-L-phenylalanine-p-nitroanilide (Glt-Phe-ONan) was obtained from Merck AG, Germany. Plasminogen was prepared from human plasma fraction I11 according to the method of Kline [7]. Streptokinase, obtained from Lederle Laboratories, U.S.A., was used for activation of plasminogen. As plasmin substrates, Bz-DL-ArgONan and casein “Hammersten” (Merck AG) were used. Porcine pancreas kallikrein for clinical use is the product of Bayer Co., Germany, and a-N-benzoylL-arginine ethyl ester (Bz-Arg-OEt) from Tokyo Kasei Kogyo Co., Japan was used as its substrate. Porcine pepsin was obtained from Pentex Biochemicals, U.S.A., and its activity was determined with a-Nacetyl-~-phenylalanyl-~-3,5-diiodotyrosine [Ac-PheTyr(I,)] from Merck AG. Papain was purchased from Worthington Biochemicals Corporation, U.S.A., and was employed as a substrate. A basic bovine pancreatic trypsin-kallikrein inhibitor, which is available commercially as Trasylol, was obtained from Bayer Co., Germany. All other chemicals used were of reagent grade. Suspension Cultures A cell line derived from the stem of S . japonica was incubated in 17 1 of the Murashige and Skoog’ medium
Hydrophobic Resin Chromatography Inhibitory substances in the filtrate were concentrated with the hydrophobic resin, Diaion HP-20 (Mitsubishi Chemical Industries Ltd., Japan). Inhibitor solutions were passed through a column (2 1) of Diaion HP-20 which was washed with deionized water. After washing the column with deionized water, the inhibitor was eluted with 80% methanol. The eluate was evaporated to dryness at 40 “C and was dissolved in a small volume of water. Duolite A-7 Chromatography Inhibitor solution was discharged by passing through a column of Duolite A-7 C1- (Chemical Process Co., U.S.A.). The effluent was neutralized immediately with 0.1 N NaOH for prevention against inactivation of the inhibitor by acid. Affinity Chromatography on Trypsin-Sepharose Inhibitors were separated completely from impurities with an affinity chromatography on trypsinSepharose which was prepared by the method of Axen and Ernback [9]. Sepharose 4B activated by cyanogen bromide was obtained from Pharmacia, Sweden. Trypsin-Sepharose was packed into a column (2.0 x 20.0 cm) in 0.05 M Tris-HC1 buffer (pH 8.6) containing 0.02 M CaC1,. Approximately 2 g of crude inhibitor was dissolved in 100 ml of the same buffer and insoluble materials were removed by centrifugation. The supernatant was applied to the column and washed thoroughly with 0.05 M Tris-HC1 buffer (pH 8.6) containing 1 M NaCl and then with deionized water. The inhibitor was eluted rapidly with 0.01 N HC1 and the eluate was neutralized immediately. Eur. J. Biochem. 55 (1975)
213
K. Sakato, H. Tanaka, and M. Misawa
Bio-Gel P-4 Chromatography The inhibitor separated by affinity chromatography was fractionated into five components by the chromatography on Bio-Gel P-4 (Bio-Rad Laboratories, U.S.A.) equilibrated with deionized water. Each fraction was further purified by rechromatography on the same column (2.5 x 100 cm) and lyophilized. Amino Acid Analysis Approximately 2 m g of each component was hydrolyzed with 6 N HC1 for 24 h at 110 "C in an evacuated, sealed tube. Analyses were performed in a JEOL model JLC-5AH amino acid analyzer (Japan Electron Optics Laboratories Co., Japan) with a JEOL model DK digital integrator. Tryptophan was determined spectrophotometrically by the method of Beaven and Holiday [lo]. Sedimentation Equilibrium Molecular weight of purified inhibitor was determined by sedimentation equilibrium measurements in a Hitachi model UCA-1 ultracentrifuge (Hitachi Ltd, Japan) at a rotor speed of 19780rev./min and 20 "C according to the short-column method of van Holde and Baldwin [11,12]. Increase of the protein concentrations was determined with the Rayleigh interference optical system. The inhibitor solutions with three different concentrations were centrifuged simultaneously with a six-channel centerpiece. Inhibitor concentration prior to centrifugation was determined with a double-sector synthetic boundary cell. Measurements were made in 0.05 M Tris-HC1 buffer (pH 8.6) containing 0.5 M NaCl. Inhibitor solutions could not be equilibrated against the same buffer by dialysis because of their low molecular weights. Accordingly, inhibitor solutions were almost equilibrated by passing through a column of Sephadex G-25 pre-equilibrated with the buffer used. Density measurements could not be made due to shortage of samples. We used therefore the partial specific volume V calculated from amino acid composition (Table 3). The V values for components 1.1,1.3,1.4 and 1.5 were 0.699, 0.703, 0.695 and 0.712 ml/g, respectively [13]. Optical Rotatory Dispersion Optical rotatory dispersion (ORD) was measured for each component in the wavelength range from 240 to 500 nm with a Jasco model ORD/UV-5 spectropolarimeter (Japan Spectroscopic Co., Japan). The ORD data were expressed in terms of [m'],the reduced Eur. J. Biochem. 55 (1975)
mean residue rotation. The ORD data were analyzed in terms of the two-term equation proposed by Moffitt and Yang [14]. Determinat ion of Enzyme Activity and Inhibitory Activity Hydrolytic activity of trypsin was determined with Bz-Arg-ONan as a substrate at 25 "C in 0.2 M triethanolamine buffer (pH 7.8) containing 0.01 M CaC1, [15,16]. One inhibitor milliunit (1 mIU) corresponds to a reduction of 1 pmol of hydrolysis of Bz-Arg-ONan per min and also is equivalent to a decrease of absorbance at 405nm, 0.00332 per min. For the assay of inhibition, trypsin was pre-incubated with the inhibitor in the period from 1 to 30 min. The level of inhibition was independent of the pre-incubation time. Hydrolytic activity of chymotrypsin was determined with Glt-Phe-ONan as a substrate at 25 "C in 0.2 M triethanolamine buffer (pH 7.8) containing 0.02 M CaC12 [17]. One inhibitor milliunit (1 mIU) corresponds to a reduction of 1 pmol of hydrolysis Glt-Phe-ONan per min and also to a decrease of absorbance at 405 nm, 0.00332 per min. The level of inhibition was independent of the pre-incubation time of chymotrypsin and inhibitor in the period from 1 to 30 min. Plasmin activity was measured spectrophotometrically by a similar procedure to the measurement of trypsin activity using Bz-Arg-ONan as a substrate in 0.02 M triethanolamine buffer (pH 8.2) at 25 "C [17]. Plasminogen was activated by incubating with streptokinase (5000 units/ml) at 25 "C for 5 min. Hydrolytic activity of plasmin was also determined with casein as a substrate in 0.02 M phosphate buffer (pH 7.4) at 25 "C [18]. For the assay of inhibition, plasmin was pre-incubated with the inhibitor for 5 min at 25 "C before addition of the substrate. Esterolytic activity of kallikrein was determined colorimetrically with Bz-Arg-OEt as a substrate by using Roberts' modification of the Hestrin method in 0.05 M triethanolamine buffer (pH 8.2) at 25 "C [18]. The inhibitor was pre-incubated with kallikrein ( 5 KU) for 5 min at 25 "C before addition of Bz-Arg-OEt. Hydrolytic activity of pepsin was determined colorimetrically with the ninhydrin method for diiodotyrosine formed in the hydrolysis of Ac-Phe-Tyr(1,) at 25 "C [20]. Inhibitory activity was estimated after pepsin and inhibitor were incubated for 5 min. Hydrolytic activity of papain was determined spectrometrically by hydrolysis of Bz-Arg-ONan in 0.05 M Tris-HC1 buffer (pH 7.5) containing 0.05 M cysteine and 0.002 M EDTA at 25 "C [21]. The procedure used is similar to that for measurement of trypsin activity.
214
In the inhibition spectrum for various proteinases, inhibitory activity of each inhibitory component was estimated as the inhibitor amount (pg/ml) required for 50 inhibition of each proteinase activity. In the kinetic experiments for inhibitions of trypsin and chymotrypsin, the initial rates of hydrolyses of Bz-L-Arg-ONan and Glt-Phe-ONan were assessed by increases in absorbance at 405 nm with a Hitachi model 139 spectrophotometer (Hitachi Ltd, Japan), in which the temperature of the cell could be kept constant to 95 & 0.1 "C with the use of the thermomodule. The inhibition constant Ki was determined by the Lineweaver-Burk method.
RESULTS Isolation und Purification of Inhibitors
Inhibitors in the extract of S. japonica cultured cells were adsorbed on a column of Diaion HP-20 and eluted with 80:, methanol. The active eluate was evaporated to dryness, dissolved in small amount of deionized water and passed through a column of Duolite A-7 C1- . The effluent was lyophilized to a yellow powder. It was further purified with the affinity chromatography on trypsin-Sepharose followed by elution with 0.01 N HCl (Fig. 1). The eluate was lyophilized and a white powder with higher inhibitory activity was obtained. The inhibitor was separated into five components, designated as 1.1 to 1.5, by chromatography on Bio-Gel P-4 equilibrated with deionized water (Fig. 2). In the gel filtration with 0.05 M ammonium formate as eluant, this separation into each component did not occur on the same column. Individual components obtained were further purified by rechromatography on the same column. Separation into five components was shown also by ion-exchange chromatography on SP-Sephadex C-25 (Pharmacia, Sweden), in which the inhibitor components were eluted with both linear gradients of pH and ionic strength using two buffers, 0.01 M NaH,PO, and 0.01 M Na,HPO, containing 0.1 M NaCl. A schematic diagram of the whole procedure of isolation and purification is described in detail in Table 1. The yield and inhibitory activity for trypsin at each step of isolation are shown typically in Table 2. In the table, it must be noted that the total activity increases in the course of purification but only in the step of Duolite A-7 the total and specific activity decreases to a large extent. The former is considered due to the fact that the colored materials exist more or less in active fractions in the steps prior to Bio-Gel P-4 chromatography and therefore reduce the inhibitory potency for trypsin. The latter can be ascribed clearly to the pH stability of the inhibitor. The inhibitory
Proteinase Inhibitors from S . japonica Cultured Cells
I
i -I I 1M NaCl
Sample
water
0.01 N HCI
Elution
Fig. 1. Purification of the proteinase inhibitor .from S . japonica cultured cells by affinity chromatography oti a tryysin-Sepharose column. The column was equilibrated with 0.05 M Tris-HCI buffer (pH 8.6) containing 0.02 M CaCI,. Approximately 2 g of the crude inhibitor was dissolved in the same buffer and the solution was centrifuged at 31 000 x g for 20 min. The supernatant was applied to the column. After washing the column with 0.05 M Tris-HCI buffer (pH 8.6) containing 1 M NaCI. the inhibitor (horizontal bar) was eluted with 0.01 N HCI. The solid line represents the absorbancy at 280 nm
g o.lol N
I
1.1
I .3
1.2
I .4
1.5
Fraction number
Fig. 2. Separation oftheproteinuse inhibitorfroin S. japonica cultured cells into its components by Bio-Gel P-4 chromatography. The column was equilibrated with deionized water adjusted to pH 7.0 and the elution was run at a flow rate of 50 mljh. Components separated are shown by horizontal bars and by absorbances at 780 nm
activity for plasmin was remarkably stable in the range of pH 4 to 9.6. The pH value of the effluent from Duolite A-7 fell to approximately 2, and the inhibitor was considered to be inactivated by acid. Amino Acid Composition
Four inhibitor components except 1.2 were determined their amino acid composition (Table 3). The absence of tryptophan was confirmed by the spectrophotometric procedure of Beaven and Holiday [lo]. As seen in Table 3, the contents of aspartic acid (or asparagine), serine, glutamic acid and glycine exist at relatively higher level but those of aromatic amino acids are lower as a whole. In addition, there are some similarities in amino acid composition among three components except 1.1; each component contains one Eur. J . Biochem. 55 (1975)
K. Sakato, H. Tanaka, and M. Misawa
215
Table 1. Schematic diagram of isolation procedures of proteinase inhibitors from S . japonica cultured cells
Table 2. Typicalpurijicationoffive inhibitorsfrom S . japonica cultured cells Inhibitory activity was determined for trypsin using Bz-DL-Arg-ONan as the substrate Preparation
(1) 80 % methanol
(2) concentrate Duolite A-7 chromatography (1) adjust to pH 7.0 with 0.1 N NaOH (2) lyophilize (3) dissolve in 0.05 M Tris-HCI buffer (pH 8.2) containing 0.02 M CaCl, Affinity chromatography on trypsin-Sepharose 4B (1) 0.05 M Tris-HC1 buffer (pH 8.2) containing 1 M NaCl (2) deionized water (3) 0.01 N HC1 I
(rejected)
(rejected)
1.5
Total activity
Specific activity
mg
IU
mIU/mg
89 700 7 120 6 780 115 6.3 1.1 4.1 14.6 22.2
735 3280 2217 6459 4397 718 4932 5 358 10856
8.19 46 1 332 6.46 x 10" 6.98 x 105 7.10 x lo6 1.20 x lo6 3.67 x 105 4.89 x 105
histidine, two threonines, four serines, four glycines and two phenylalanines but no tyrosine or tryptophan.
1
I
Extract Diaion HP-20 Duolite A-7 Trypsin-Sepharose Bio-Gel P-4 1.1 1.2 1.3 1.4
Solid weight
0.01 N HC1 (2) lyophilize (3) dissolve in deionized water
Molecular Weight
A typical plot for molecular weight determination is given for 1.4 in Fig.3. Linear extrapolation of the plotted points to infinite dilution allowed one to estiI ____ I I mate the molecular weight of 5870 for 1.4. Further1.1 1.2 1.3 1.4 1.5 more, the good linearity of logf(fringe displacement) Bio-gel P-4 chromatography
I
Table 3. Amino acid composition of four proteinase inhibitors from S . japonica cultured cells Amino acids
Lysine Histidine Arginine Aspartic acid Threonine Serine Gutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine
Composition
Number of residues
1.1
1.3
1.4
1.5
1.1
1.3
1.4
1.5
2.75 1.65 5.03 8.80 3.39 3.29 5.60 3.16 5.81 5.17 1.97 1.00 0.01 3.74 < 0.01 2.07 1.52
3.03 1.01 1.97 4.90 1.61 3.22 2.31 < 0.01 3.28 1.97 < 0.01 1.oo 2.67 < 0.01 < 0.01 < 0.01 1.42
4.65 1.00 3.41 7.01 2.20 4.48 3.10 < 0.01 4.20 5.05 6.90 < 0.01 2.50 < 0.01 < 3.65 < 0.01 1.65
3.93 1.00 < 0.01 5.98 1.85 4.08 2.30 1.48 4.18 2.79 < 0.01 < 0.01 < 0.01 2.65 < 0.01 < 0.01 1.76
3 2 5 9 4 3 6 3 6 5 2 1 0 4 0 2 2
3 1 2 2 4 3 0 4 2 0 1 3 0 0 0 2
5 1 3 7 2 5 3 0 4 5 7
4 1 0 6 2 4 3 2 4 3 0 0 0 3 0 0 2
6283
3963
5
0 3 0 4 0 2
Total residues Molecular weight" a
6076
3704b
Molecular weights were calculated from amino acid composition by comparing with the data of sedimentation equilibrium. Molecular weight of 1.5 were calculated from amino acid composition by assuming one histidine residue/molecule.
Eur. J. Biochem. 55 (1975)
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Proteinase Inhibitors from S . japonica Cultured Cells
-0.55
._
-
c
a
-Lo ._ -D
Broad-Specificity Proteinase Inhibitors in Scopolia japonica (Solanaceae) Cultured Cells Isolation, Physicochemical Properties, and Inhibition Kinetics Kuniaki SAKATO, Hozumi TANAKA, and Masanaru MISAWA Tokyo Research Laboratory, Kyowa Hakko Kogyo Co., Ltd., Tokyo (Received October 11, 1974 / March 10, 1975)
Proteinase inhibitors were isolated from Scopoliu juponicu cultured cells. Isolation procedures involve concentration by a hydrophobic resin of Diaion HP-20, decolorization by Duolite A-7, affinity chromatography on trypsin-Sepharose, and Bio-Gel P-4 chromatography. It was found that the proteinase inhibitors from S.juponicu cells are a mixture of at least five components. For the inhibitory components except one, amino acid analyses, measurements of sedimentation equilibrium and optical rotatory dispersion (ORD) were carried out. The inhibitors were shown to be the polypeptides with molecular weights in the range of approximately 4000 to 6000. In addition, one of them was found to have approximately 15% a-helical conformation by the MoffittYang analysis of ORD data. The inhibitors were found to have potent inhibitory activity for trypsin, chymotrypsin, plasmin, kallikrein and pepsin but not for papain with synthetic and natural substrates. These inhibitors formed stable complexes with trypsin and chymotrypsin in an equimolar ratio, and their inhibitory mechanisms for both enzymes were of non-competitive type.
sin-kallikrein inhibitor of Kunitz [3] and 4-aminomethylcyclohexane carboxylic acid [4] have reached to the stage of clinical uses. An assay method for inhibition of plasmin was designed in our laboratory for screening a useful plasmin inhibitor from natural sources [ 5 ] . This method has the following advantages : (a) inhibitory activities for plasmin and thrombin can be estimated simultaneously; (b) the crude material can be assayed in small quantities. By this method, the screening of new inhibitors for plasmin were extensively performed with plant calli, microbial cultures and synthetic compounds. In the screening, aqueous extracts from several calli were found to inhibit plasmin activity [5,6]. An extract from Scopolia japonicu (family Solanaceae) showed the strongest inhibition among Abbreviations. Bz-nL-Arg-ONan, a-N-benzoyl-DL-arginine-p- them. The cultural conditions for cell growth and nitroanilide; Bz-L-Arg-ONan, a-N-benzoyl-L-arginine-p-nitroanilide; Glt-Phe-ONan, a-N-glutaryl-L-phenylalanyl-p-nitroanilide; accumulation of the inhibitor were examined in detail with S.juponicu cell suspensions [ 5 ] .As a result, Bz-Arg-OEt, u-N-benzoyl-L-arginine ethyl ester; Ac-Phe-Tyr(I,), a-N-acetyl-~-phenylalanyl-~-3,5-diiodotyrosine ; ORD, optical rotaS.juponicu cultured cells were found to accumulate tory dispersion. higher amounts of the inhibitor than the intact plant. Enzyrnes(CBN Recommendations 1972).Trypsin (EC 3.4.21.4); Cell cultures of higher plants have been of much chymotrypsin (EC 3.4.21.1): plasmin (EC 3.4.21.7): kallikrein interest for the biosynthetic potential of various (EC 3.4.ii.8); pepsin (EC 3.4.23.1); papain (EC 3.4.22.2); thrombin metabolites because of the possibility for the produc(EC 3.4.21.5).
In living organisms, the action of proteolytic enzymes is controlled by many ingenius ways. One of control mechanisms is to use the proteinase inhibitor, although very little has been known about its physiological functions. Proteinase inhibitors occur naturally as polypeptides, proteins, glycoproteins or polysaccharides [l]. More recently they have been isolated from not only plants and animals but also microorganisms [2]. This type of control has made possible the therapeutic application of the pathophysiological processes due to the blood coagulation, the fibrinolysis and the liberation of kinins [l]. Many proteinase inhibitors have been found in natural sources and also have been synthesized for clinical uses [ 2 ] .As the result, a basic bovine pancreatic tryp-
Eur. J. Biochem. 55 (1975)
212
tion of useful constituents. However, very few has been reported on the production of important metabolites by plant cell cultures. This may be ascribed mainly to the fact that the accumulation of metabolites, especially secondary metabolites such as alkaloids, decreases remarkably in dedifferentiated plants. We have induced a variety of plant calli and have examined the production of important metabolites by their cells in cultures. These results will be reviewed elsewhere [6]. The present paper deals with the isolation procedures and physicochemical properties of the proteinase inhibitor from S. japonica cultured cells together with their inhibitory activities for several proteolytic enzymes.
Proteinase Inhibitors from S . .japonir-a Cultured Cells
[8] containing 1.O mg/l of 2,4-dichlorophenoxyacetic acid, 0.02 mg/l of kinetin and 3 % sucrose, using a gas bubble fermentor (20 1) with an aeration of 10 l/min at 28 “C [ 5 ] . After 14 days of culture, 5.8 kg of fresh cells was harvested. Extraction Fresh cells (5.8 kg) were homogenized with 11.6 1 of deionized water in a homogenizer for approximately 10 min. The suspension was heated in boiling water for approximately 10 min. This treatment enhanced remarkably the inhibitory activity for plasmin. Then, the suspension was filtered through a Miracloth (Chicopee Manufacturing Co., U.S.A.) to remove insoluble materials.
EXPERIMENTAL PROCEDURE Muteriuh Porcine trypsin (grade V) was obtained from MilesSeravac Co., {J.S.A. As trypsin substrates, a-N-benzoyl-DL-arginine-p-nitroanilide (Bz-DL-Arg-ONan) (Bz-L-Argand a-N-benzoyl-L-arginine-p-nitroanilide ONan) were obtained from Merck AG, Germany and the Protein Research Foundation, Japan, respectively. Bovine x-chymotrypsin was obtained from Worthington Biochemicals, Corporation, U.S.A. As an a-chymotrypsin substrate, a-N-glutaryl-L-phenylalanine-p-nitroanilide (Glt-Phe-ONan) was obtained from Merck AG, Germany. Plasminogen was prepared from human plasma fraction I11 according to the method of Kline [7]. Streptokinase, obtained from Lederle Laboratories, U.S.A., was used for activation of plasminogen. As plasmin substrates, Bz-DL-ArgONan and casein “Hammersten” (Merck AG) were used. Porcine pancreas kallikrein for clinical use is the product of Bayer Co., Germany, and a-N-benzoylL-arginine ethyl ester (Bz-Arg-OEt) from Tokyo Kasei Kogyo Co., Japan was used as its substrate. Porcine pepsin was obtained from Pentex Biochemicals, U.S.A., and its activity was determined with a-Nacetyl-~-phenylalanyl-~-3,5-diiodotyrosine [Ac-PheTyr(I,)] from Merck AG. Papain was purchased from Worthington Biochemicals Corporation, U.S.A., and was employed as a substrate. A basic bovine pancreatic trypsin-kallikrein inhibitor, which is available commercially as Trasylol, was obtained from Bayer Co., Germany. All other chemicals used were of reagent grade. Suspension Cultures A cell line derived from the stem of S . japonica was incubated in 17 1 of the Murashige and Skoog’ medium
Hydrophobic Resin Chromatography Inhibitory substances in the filtrate were concentrated with the hydrophobic resin, Diaion HP-20 (Mitsubishi Chemical Industries Ltd., Japan). Inhibitor solutions were passed through a column (2 1) of Diaion HP-20 which was washed with deionized water. After washing the column with deionized water, the inhibitor was eluted with 80% methanol. The eluate was evaporated to dryness at 40 “C and was dissolved in a small volume of water. Duolite A-7 Chromatography Inhibitor solution was discharged by passing through a column of Duolite A-7 C1- (Chemical Process Co., U.S.A.). The effluent was neutralized immediately with 0.1 N NaOH for prevention against inactivation of the inhibitor by acid. Affinity Chromatography on Trypsin-Sepharose Inhibitors were separated completely from impurities with an affinity chromatography on trypsinSepharose which was prepared by the method of Axen and Ernback [9]. Sepharose 4B activated by cyanogen bromide was obtained from Pharmacia, Sweden. Trypsin-Sepharose was packed into a column (2.0 x 20.0 cm) in 0.05 M Tris-HC1 buffer (pH 8.6) containing 0.02 M CaC1,. Approximately 2 g of crude inhibitor was dissolved in 100 ml of the same buffer and insoluble materials were removed by centrifugation. The supernatant was applied to the column and washed thoroughly with 0.05 M Tris-HC1 buffer (pH 8.6) containing 1 M NaCl and then with deionized water. The inhibitor was eluted rapidly with 0.01 N HC1 and the eluate was neutralized immediately. Eur. J. Biochem. 55 (1975)
213
K. Sakato, H. Tanaka, and M. Misawa
Bio-Gel P-4 Chromatography The inhibitor separated by affinity chromatography was fractionated into five components by the chromatography on Bio-Gel P-4 (Bio-Rad Laboratories, U.S.A.) equilibrated with deionized water. Each fraction was further purified by rechromatography on the same column (2.5 x 100 cm) and lyophilized. Amino Acid Analysis Approximately 2 m g of each component was hydrolyzed with 6 N HC1 for 24 h at 110 "C in an evacuated, sealed tube. Analyses were performed in a JEOL model JLC-5AH amino acid analyzer (Japan Electron Optics Laboratories Co., Japan) with a JEOL model DK digital integrator. Tryptophan was determined spectrophotometrically by the method of Beaven and Holiday [lo]. Sedimentation Equilibrium Molecular weight of purified inhibitor was determined by sedimentation equilibrium measurements in a Hitachi model UCA-1 ultracentrifuge (Hitachi Ltd, Japan) at a rotor speed of 19780rev./min and 20 "C according to the short-column method of van Holde and Baldwin [11,12]. Increase of the protein concentrations was determined with the Rayleigh interference optical system. The inhibitor solutions with three different concentrations were centrifuged simultaneously with a six-channel centerpiece. Inhibitor concentration prior to centrifugation was determined with a double-sector synthetic boundary cell. Measurements were made in 0.05 M Tris-HC1 buffer (pH 8.6) containing 0.5 M NaCl. Inhibitor solutions could not be equilibrated against the same buffer by dialysis because of their low molecular weights. Accordingly, inhibitor solutions were almost equilibrated by passing through a column of Sephadex G-25 pre-equilibrated with the buffer used. Density measurements could not be made due to shortage of samples. We used therefore the partial specific volume V calculated from amino acid composition (Table 3). The V values for components 1.1,1.3,1.4 and 1.5 were 0.699, 0.703, 0.695 and 0.712 ml/g, respectively [13]. Optical Rotatory Dispersion Optical rotatory dispersion (ORD) was measured for each component in the wavelength range from 240 to 500 nm with a Jasco model ORD/UV-5 spectropolarimeter (Japan Spectroscopic Co., Japan). The ORD data were expressed in terms of [m'],the reduced Eur. J. Biochem. 55 (1975)
mean residue rotation. The ORD data were analyzed in terms of the two-term equation proposed by Moffitt and Yang [14]. Determinat ion of Enzyme Activity and Inhibitory Activity Hydrolytic activity of trypsin was determined with Bz-Arg-ONan as a substrate at 25 "C in 0.2 M triethanolamine buffer (pH 7.8) containing 0.01 M CaC1, [15,16]. One inhibitor milliunit (1 mIU) corresponds to a reduction of 1 pmol of hydrolysis of Bz-Arg-ONan per min and also is equivalent to a decrease of absorbance at 405nm, 0.00332 per min. For the assay of inhibition, trypsin was pre-incubated with the inhibitor in the period from 1 to 30 min. The level of inhibition was independent of the pre-incubation time. Hydrolytic activity of chymotrypsin was determined with Glt-Phe-ONan as a substrate at 25 "C in 0.2 M triethanolamine buffer (pH 7.8) containing 0.02 M CaC12 [17]. One inhibitor milliunit (1 mIU) corresponds to a reduction of 1 pmol of hydrolysis Glt-Phe-ONan per min and also to a decrease of absorbance at 405 nm, 0.00332 per min. The level of inhibition was independent of the pre-incubation time of chymotrypsin and inhibitor in the period from 1 to 30 min. Plasmin activity was measured spectrophotometrically by a similar procedure to the measurement of trypsin activity using Bz-Arg-ONan as a substrate in 0.02 M triethanolamine buffer (pH 8.2) at 25 "C [17]. Plasminogen was activated by incubating with streptokinase (5000 units/ml) at 25 "C for 5 min. Hydrolytic activity of plasmin was also determined with casein as a substrate in 0.02 M phosphate buffer (pH 7.4) at 25 "C [18]. For the assay of inhibition, plasmin was pre-incubated with the inhibitor for 5 min at 25 "C before addition of the substrate. Esterolytic activity of kallikrein was determined colorimetrically with Bz-Arg-OEt as a substrate by using Roberts' modification of the Hestrin method in 0.05 M triethanolamine buffer (pH 8.2) at 25 "C [18]. The inhibitor was pre-incubated with kallikrein ( 5 KU) for 5 min at 25 "C before addition of Bz-Arg-OEt. Hydrolytic activity of pepsin was determined colorimetrically with the ninhydrin method for diiodotyrosine formed in the hydrolysis of Ac-Phe-Tyr(1,) at 25 "C [20]. Inhibitory activity was estimated after pepsin and inhibitor were incubated for 5 min. Hydrolytic activity of papain was determined spectrometrically by hydrolysis of Bz-Arg-ONan in 0.05 M Tris-HC1 buffer (pH 7.5) containing 0.05 M cysteine and 0.002 M EDTA at 25 "C [21]. The procedure used is similar to that for measurement of trypsin activity.
214
In the inhibition spectrum for various proteinases, inhibitory activity of each inhibitory component was estimated as the inhibitor amount (pg/ml) required for 50 inhibition of each proteinase activity. In the kinetic experiments for inhibitions of trypsin and chymotrypsin, the initial rates of hydrolyses of Bz-L-Arg-ONan and Glt-Phe-ONan were assessed by increases in absorbance at 405 nm with a Hitachi model 139 spectrophotometer (Hitachi Ltd, Japan), in which the temperature of the cell could be kept constant to 95 & 0.1 "C with the use of the thermomodule. The inhibition constant Ki was determined by the Lineweaver-Burk method.
RESULTS Isolation und Purification of Inhibitors
Inhibitors in the extract of S. japonica cultured cells were adsorbed on a column of Diaion HP-20 and eluted with 80:, methanol. The active eluate was evaporated to dryness, dissolved in small amount of deionized water and passed through a column of Duolite A-7 C1- . The effluent was lyophilized to a yellow powder. It was further purified with the affinity chromatography on trypsin-Sepharose followed by elution with 0.01 N HCl (Fig. 1). The eluate was lyophilized and a white powder with higher inhibitory activity was obtained. The inhibitor was separated into five components, designated as 1.1 to 1.5, by chromatography on Bio-Gel P-4 equilibrated with deionized water (Fig. 2). In the gel filtration with 0.05 M ammonium formate as eluant, this separation into each component did not occur on the same column. Individual components obtained were further purified by rechromatography on the same column. Separation into five components was shown also by ion-exchange chromatography on SP-Sephadex C-25 (Pharmacia, Sweden), in which the inhibitor components were eluted with both linear gradients of pH and ionic strength using two buffers, 0.01 M NaH,PO, and 0.01 M Na,HPO, containing 0.1 M NaCl. A schematic diagram of the whole procedure of isolation and purification is described in detail in Table 1. The yield and inhibitory activity for trypsin at each step of isolation are shown typically in Table 2. In the table, it must be noted that the total activity increases in the course of purification but only in the step of Duolite A-7 the total and specific activity decreases to a large extent. The former is considered due to the fact that the colored materials exist more or less in active fractions in the steps prior to Bio-Gel P-4 chromatography and therefore reduce the inhibitory potency for trypsin. The latter can be ascribed clearly to the pH stability of the inhibitor. The inhibitory
Proteinase Inhibitors from S . japonica Cultured Cells
I
i -I I 1M NaCl
Sample
water
0.01 N HCI
Elution
Fig. 1. Purification of the proteinase inhibitor .from S . japonica cultured cells by affinity chromatography oti a tryysin-Sepharose column. The column was equilibrated with 0.05 M Tris-HCI buffer (pH 8.6) containing 0.02 M CaCI,. Approximately 2 g of the crude inhibitor was dissolved in the same buffer and the solution was centrifuged at 31 000 x g for 20 min. The supernatant was applied to the column. After washing the column with 0.05 M Tris-HCI buffer (pH 8.6) containing 1 M NaCI. the inhibitor (horizontal bar) was eluted with 0.01 N HCI. The solid line represents the absorbancy at 280 nm
g o.lol N
I
1.1
I .3
1.2
I .4
1.5
Fraction number
Fig. 2. Separation oftheproteinuse inhibitorfroin S. japonica cultured cells into its components by Bio-Gel P-4 chromatography. The column was equilibrated with deionized water adjusted to pH 7.0 and the elution was run at a flow rate of 50 mljh. Components separated are shown by horizontal bars and by absorbances at 780 nm
activity for plasmin was remarkably stable in the range of pH 4 to 9.6. The pH value of the effluent from Duolite A-7 fell to approximately 2, and the inhibitor was considered to be inactivated by acid. Amino Acid Composition
Four inhibitor components except 1.2 were determined their amino acid composition (Table 3). The absence of tryptophan was confirmed by the spectrophotometric procedure of Beaven and Holiday [lo]. As seen in Table 3, the contents of aspartic acid (or asparagine), serine, glutamic acid and glycine exist at relatively higher level but those of aromatic amino acids are lower as a whole. In addition, there are some similarities in amino acid composition among three components except 1.1; each component contains one Eur. J . Biochem. 55 (1975)
K. Sakato, H. Tanaka, and M. Misawa
215
Table 1. Schematic diagram of isolation procedures of proteinase inhibitors from S . japonica cultured cells
Table 2. Typicalpurijicationoffive inhibitorsfrom S . japonica cultured cells Inhibitory activity was determined for trypsin using Bz-DL-Arg-ONan as the substrate Preparation
(1) 80 % methanol
(2) concentrate Duolite A-7 chromatography (1) adjust to pH 7.0 with 0.1 N NaOH (2) lyophilize (3) dissolve in 0.05 M Tris-HCI buffer (pH 8.2) containing 0.02 M CaCl, Affinity chromatography on trypsin-Sepharose 4B (1) 0.05 M Tris-HC1 buffer (pH 8.2) containing 1 M NaCl (2) deionized water (3) 0.01 N HC1 I
(rejected)
(rejected)
1.5
Total activity
Specific activity
mg
IU
mIU/mg
89 700 7 120 6 780 115 6.3 1.1 4.1 14.6 22.2
735 3280 2217 6459 4397 718 4932 5 358 10856
8.19 46 1 332 6.46 x 10" 6.98 x 105 7.10 x lo6 1.20 x lo6 3.67 x 105 4.89 x 105
histidine, two threonines, four serines, four glycines and two phenylalanines but no tyrosine or tryptophan.
1
I
Extract Diaion HP-20 Duolite A-7 Trypsin-Sepharose Bio-Gel P-4 1.1 1.2 1.3 1.4
Solid weight
0.01 N HC1 (2) lyophilize (3) dissolve in deionized water
Molecular Weight
A typical plot for molecular weight determination is given for 1.4 in Fig.3. Linear extrapolation of the plotted points to infinite dilution allowed one to estiI ____ I I mate the molecular weight of 5870 for 1.4. Further1.1 1.2 1.3 1.4 1.5 more, the good linearity of logf(fringe displacement) Bio-gel P-4 chromatography
I
Table 3. Amino acid composition of four proteinase inhibitors from S . japonica cultured cells Amino acids
Lysine Histidine Arginine Aspartic acid Threonine Serine Gutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine
Composition
Number of residues
1.1
1.3
1.4
1.5
1.1
1.3
1.4
1.5
2.75 1.65 5.03 8.80 3.39 3.29 5.60 3.16 5.81 5.17 1.97 1.00 0.01 3.74 < 0.01 2.07 1.52
3.03 1.01 1.97 4.90 1.61 3.22 2.31 < 0.01 3.28 1.97 < 0.01 1.oo 2.67 < 0.01 < 0.01 < 0.01 1.42
4.65 1.00 3.41 7.01 2.20 4.48 3.10 < 0.01 4.20 5.05 6.90 < 0.01 2.50 < 0.01 < 3.65 < 0.01 1.65
3.93 1.00 < 0.01 5.98 1.85 4.08 2.30 1.48 4.18 2.79 < 0.01 < 0.01 < 0.01 2.65 < 0.01 < 0.01 1.76
3 2 5 9 4 3 6 3 6 5 2 1 0 4 0 2 2
3 1 2 2 4 3 0 4 2 0 1 3 0 0 0 2
5 1 3 7 2 5 3 0 4 5 7
4 1 0 6 2 4 3 2 4 3 0 0 0 3 0 0 2
6283
3963
5
0 3 0 4 0 2
Total residues Molecular weight" a
6076
3704b
Molecular weights were calculated from amino acid composition by comparing with the data of sedimentation equilibrium. Molecular weight of 1.5 were calculated from amino acid composition by assuming one histidine residue/molecule.
Eur. J. Biochem. 55 (1975)
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Proteinase Inhibitors from S . japonica Cultured Cells
-0.55
._
-
c
a
-Lo ._ -D
