Planta (1991)185:344-349
P l a n m 9 Springer-Verlag1991
Metallo-proteinase from the seedlings of kale (Brassica oleracea L. var. sabellica): Preparation, partial characterization and substrate specificity Anna Wilimowska-Pelc 1., Marek DryjafiskP, Tomasz Zal 2, and Tadeusz Wilusz ~ i Institute of Biochemistry, University of Wroctaw, Tamka 2, and 2 Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Czerska 12, PL53114 Wroctaw, Poland Received 25 March; accepted 23 May 1991
Abstract. Metallo-proteinase f r o m 8-d-old seedlings of kale was isolated. The enzyme was extracted with 1% NaC1, concentrated by a m m o n i u m sulfate and finally purified by high-performance liquid chromatography. The isolated enzyme had a molecular weight of 22.4 k D a and showed a m a x i m u m activity at p H 9.0 using casein as a substrate. Proteolytic activity of proteinase was inhibited by chelators. The inhibition by ethylenediaminetetraacetate ( E D T A ) was abolished by some divalent metals ions, especially by Zn 2+. The enzyme showed activity against the synthetic peptides Suc-AlaA l a - P r o - L e u - p N A and Suc-Ala-Ala-Pro-Phe-pNA, and hydrolized the following peptide bonds in the oxidized insulin B-chain: Leu6-Cya7, L e u l 5 Tyrl6, L e u l 7 Vall 8 and Phe25 Tyr26. Key words: B r a s s i c a - Metallo-proteinase - Proteolytic specificity (enzyme)
Introduction Both resting and germinating seeds contain various proteinases and peptidases which are suggested to play a central role in the biochemical mechanism of germination. The best k n o w n proteolytic enzymes present in germinating seeds belong to the class of cysteine proteinases as indicated by their inactivation by thiol-blocking reagents. The presence of enzymes of the cysteine type has been demonstrated in germinating seeds of m u n g bean (Baumgartner and Chrispeels 1977), corn (Abe et al. 1977), pine (Salmia 1981), castor bean (Tully and Beevers 1978) and c o m m o n bean (Boylan and Sussex 1987). * To whom correspondence should be addressed Abbreviations: EDTA=ethylenediaminotetraacetic acid; HPLC =
high-performance liquid chromatography; NEM=N-ethylmaleimide; PCMB=p-mecuribenzoic acid; PMSF=phenylmethylsulfonyl fluoride
In contrast to the extensively studied cysteine proteinase, serine and aspartic proteinases have been isolated only from the resting seeds of some plants. Trypsin-like proteinases have been purified from soybean seeds (Nishikata 1984) and from C u c u r b i t a f i c i f o l i a (Dryjafiski et al. 1990). Aspartic proteinases have been isolated from the resting seeds of rice (Doi et al. 1980), cucumber (Wilimowska-Pelc et al. 1983), buckwheat (Belozersky et al. 1984) and wheat (Belozersky et al. 1989). Metallo-proteinases are c o m m o n enzymes though their presence in plants has seldom been demonstrated. Enzymes belonging to this class are characterized by possessing metal ions, most often Zn 2+, in the active center, and are active at a neutral or slightly alkaline pH. Chelators such as E D T A and 1,10-phenanthroline are reversible inhibitors of metallo-proteinases. So far, there has been only one report of a zinc-containing proteinase in plant material: Voskoboynikova et al. (1989) described a protocol for separating a homogeneous metallo-proteinase from resting buckwheat seeds. In this paper we describe the presence of two caseindigesting proteinases in the seedlings of kale. The activity of both enzymes is inhibited by E D T A and 1,10-phenanthroline. One of the enzymes has been isolated, partially characterized and its proteolytic specificity for the oxidized B-chain of bovine insulin has been determined.
Material and methods Plant mater&l. Seeds of kale (Brasicca oleracea L. var. sabellica)
were supplied by the Plant Seed Corporation in Wroctaw (Poland). Seeds were sterilized by soaking them in 90% acetone for 5 min, then in H202 for 30 min. After that seeds were rinsed with water, sown in a moist bed of filter paper, and allowed to germinate at 2224~ C for 8 d with 10-12 h of light daily. No fungal contamination was observed on the germinating seeds. Assay ofproteolytic activity. Samples of 1 ml of enzyme solution incubated for 30 min at 36~ C with 1 ml of 1% casein (BDH, Poole, Dorset, UK) in 200 mM Tris-HCl buffer, pH 9. The reaction was stopped by adding 3 ml of 5% trichloroacetic acid (TCA). The precipitate was centrifuged off, and absorption at 280 nm was
A. Wilimowska-Pelc et al. : Metallo-proteinase from the seedlings of kale measured in the supernatant. One unit of proteolytic activity was defined as the amount of enzyme which gave an increase of 0.1 absorption unit at 280 nm under the conditions applied.
Inhibitors. The effect of some chemical inhibitors on proteinase activity was established after 15 min preincubation of enzyme with inhibitors. Final concentrations of inhibitors in the assay mixture were 1 m M for phenylmethylsulfonyl fluoride (PMSF), p-mercuribenzoic acid (PCMB), E D T A and cysteine, 3 mM for N-ethylmaleimide (NEM), and 5 mM for 1,10-phenanthroline.
Assay of amidase activity. The activity of the proteinase against Suc-Ala-Ala-Pro-Phe-pNA and Suc-Ala-Ala-Pro-Leu-pNA (Sigma Chemical Co., St.Louis, Mo., USA) was determined in according to Erlanger et al. (1961).
Proteinase purification
345
pancreatic secretory trypsin inhibitor (6.1 kDa) from bovine pancreas, prepared as described by Wilimowska-Pelc et al. (1973), cytochrome c (12.4 kDa), myoglobin (17.6 kDa), chymotrypsinogen A (25 kDa) and egg albumin (45 kDa). The kale proteinase was localized in the eluate by estimation of its proteolytic activity.
The effect ofpH and Zn 2+. The effect of pH and Zn 2+ on enzyme activity was studied using enzyme which had been stored at 5~ C for 30 d at pH 2.0 (0.01 N HCI), pH 4.0 (0.05 M acetate buffer), pH 6.0, 8.0 and 10.0 (0.05 M Tris-HCl buffer). In one of the two series studied samples also contained 0.2 m M ZnSO4. At 3-d intervals aliquots were withdrawn for determination of the residual activity. Determination of pH optimum. The proteinase pH optimum was determinated by hydrolysis of casein in 100 mM Tris-HC1 buffer in the pH range of 6.0-10.5.
The purification procedure was carried out at 5-7 ~ C.
Protein determination. Protein was determinated by the microbiuret method of Goa (1953), and spectrophotometrically at 280 nm.
Extraction. Eight-day-old seedlings were harvested and frozen at
Determination of specificity for oxidized B-chain of bovine insulin.
- 15~ C for 2 h. Each 100-g sample of seedling was homogenized with 150-200 ml of 1% NaC1 solution and extracted for 1 h with constant mechanical stirring. After centrifugation (14000 "9, 20 rain) the supernatant was decanted and adjusted to pH 7.5 with 2 N NaOH. The precipitate formed was centrifuged off.
Ammonium-sulfate precipitation. The supernatant was brought to 90% saturation with ammonium sulfate. After 20 h, sedimented proteins were collected, dissolved in about 15 ml of water and after 2 h clarified by centrifugation at 14 000 - g for 20 rain.
Sephadex G-75 chromatography. The ammonium-sulfate-precipitated protein solution was adjusted to pH 5.9 with 1 M acetic acid and applied to a Sephadex G-75 column (85 cm long, 2 cm i.d. ; Pharmacia, Uppsala, Sweden) equilibrated with 0.1 M acetate buffer pH 5.9. The fractions corresponding to the first and second peak of proteolytic activity were collected separately. Ion-exchange chromatography. The fraction corresponding to the second peak of proteolytic activity was brought to pH 7.0 with 1 N N a O H and applied to a DEAE-Sephadex A-50 column (15 cm long, 2 cm i.d.; Pharmacia) equlibrated with 0.05 M phosphatecitric acid buffer, pH 7.0. The enzyme did not adsorb under the applied conditions and was eluted from the column with starting buffer. Active fractions were pooled and after dialysis against 0.05 M acetate buffer (pH 5.0) subjected to chromatography on SP-Sephadex C-50 (15 cm long, 2 cm i.d. ; Pharmacia) equlibrated with the same buffer. The adsorbed proteins were eluted from the column with a linear concentration gradient of NaCI (0-0.5 M) in starting buffer. Final purification was achieved by means of highperformance liquid chromatography (HPLC) on a Spherogel TSK CM 3 SW column (75 mm long, 7.5 mm i.d. ; Beckman, Palo Alto, Cal., USA) equilibrated with 0.025 M phosphate buffer, pH 6.1. Proteins were eluted with a linear concentration gradient of NaC1 (0 to 0.5 M).
Polyacrylamide gel electrophoresis (PAGE). Proteins were subjected to PAGE using the method of Davis (1964) at pH 8.3 in 7.5% gels. Gels were stained for proteins with 1% Amido black 10B in 7% acetic acid. For detection of proteolytic activity, gels containing 0.1% edestin were used (Leluk et al. 1985); after electrophoresis (4 mA per tube) the gels were soaked in 0.1 M Tris-HCl buffer, pH 9.0, and incubated at room temperature until the transparent zones were visible. Then the gels were stained with 0.1% Amido black in 7% acetic acid. Molecular-weight determination by 9el filtration. A Sephadex G-75 column (80 cm long, 1.5 cm i.d. ; Pharmacia) was equilibrated with 0.05 M Tris-HC1 buffer (pH 7.5) containing 0.5 M NaC1. The following standards at a concentration of 1 mg 9ml-1 were used:
The enzyme (0.1 mol%) was added to 5 mg of insulin dissolved in 0.5 ml of 1% NH4HCO3, pH 7.9. After 24 h incubation at 30 ~ C the sample was frozen, lyophylized, and finally subjected to analysis, Separation of the hydrolysis product was accomplished by HPLC on a Supelcosil LC 18 DB column (250 mm long, 4.6 mm i.d. ; Supelco, Gland, Switzerland) with a linear gradient of acetonitrile (0-50%) in 0.1% trifluoroacetic acid (TFA). The peptides collected were hydrolized (6 N HC1, 110 ~ 24 h) and their amino-acid compositions determined by the method of pre-column derivatization with o-phtaldialdehyde and 2-mercaptoethanol followed by reversed-phase HPLC on Spherisorb ODS II 4 lain (column 60 mm long, 4 mm i.d. ; Knauer, Bad Hamburg, FRG), and fluorescence detection (Reiendra 1987, with a slight modification of the gradient curve).
Results and discussion
Proteolytic activity during kale seed germination. P r o t e o lyric a c t i v i t y w a s n o t d e t e c t a b l e in e x t r a c t s f r o m r e s t i n g seeds u n d e r t h e c o n d i t i o n s used. H o w e v e r , it i n c r e a s e d slowly during germination, displayed a sharp increase between 5 and 8 d and then did not change appreciably d u r i n g t h e n e x t 2 d ( F i g . l ) . T h e s e r e s u l t s a l l o w e d us to
"~ 120 >, 100 .>
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Germination [clays] Fig. 1. Proteolytic activity during kale seed germination. Seedlings or seeds were extracted with 1% NaC1 under the conditions described in Material and methods. Proteolytic activity in crude enzyme extracts was measured using a casein substrate at pH 9
346
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P l a n m 9 Springer-Verlag1991
Metallo-proteinase from the seedlings of kale (Brassica oleracea L. var. sabellica): Preparation, partial characterization and substrate specificity Anna Wilimowska-Pelc 1., Marek DryjafiskP, Tomasz Zal 2, and Tadeusz Wilusz ~ i Institute of Biochemistry, University of Wroctaw, Tamka 2, and 2 Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Czerska 12, PL53114 Wroctaw, Poland Received 25 March; accepted 23 May 1991
Abstract. Metallo-proteinase f r o m 8-d-old seedlings of kale was isolated. The enzyme was extracted with 1% NaC1, concentrated by a m m o n i u m sulfate and finally purified by high-performance liquid chromatography. The isolated enzyme had a molecular weight of 22.4 k D a and showed a m a x i m u m activity at p H 9.0 using casein as a substrate. Proteolytic activity of proteinase was inhibited by chelators. The inhibition by ethylenediaminetetraacetate ( E D T A ) was abolished by some divalent metals ions, especially by Zn 2+. The enzyme showed activity against the synthetic peptides Suc-AlaA l a - P r o - L e u - p N A and Suc-Ala-Ala-Pro-Phe-pNA, and hydrolized the following peptide bonds in the oxidized insulin B-chain: Leu6-Cya7, L e u l 5 Tyrl6, L e u l 7 Vall 8 and Phe25 Tyr26. Key words: B r a s s i c a - Metallo-proteinase - Proteolytic specificity (enzyme)
Introduction Both resting and germinating seeds contain various proteinases and peptidases which are suggested to play a central role in the biochemical mechanism of germination. The best k n o w n proteolytic enzymes present in germinating seeds belong to the class of cysteine proteinases as indicated by their inactivation by thiol-blocking reagents. The presence of enzymes of the cysteine type has been demonstrated in germinating seeds of m u n g bean (Baumgartner and Chrispeels 1977), corn (Abe et al. 1977), pine (Salmia 1981), castor bean (Tully and Beevers 1978) and c o m m o n bean (Boylan and Sussex 1987). * To whom correspondence should be addressed Abbreviations: EDTA=ethylenediaminotetraacetic acid; HPLC =
high-performance liquid chromatography; NEM=N-ethylmaleimide; PCMB=p-mecuribenzoic acid; PMSF=phenylmethylsulfonyl fluoride
In contrast to the extensively studied cysteine proteinase, serine and aspartic proteinases have been isolated only from the resting seeds of some plants. Trypsin-like proteinases have been purified from soybean seeds (Nishikata 1984) and from C u c u r b i t a f i c i f o l i a (Dryjafiski et al. 1990). Aspartic proteinases have been isolated from the resting seeds of rice (Doi et al. 1980), cucumber (Wilimowska-Pelc et al. 1983), buckwheat (Belozersky et al. 1984) and wheat (Belozersky et al. 1989). Metallo-proteinases are c o m m o n enzymes though their presence in plants has seldom been demonstrated. Enzymes belonging to this class are characterized by possessing metal ions, most often Zn 2+, in the active center, and are active at a neutral or slightly alkaline pH. Chelators such as E D T A and 1,10-phenanthroline are reversible inhibitors of metallo-proteinases. So far, there has been only one report of a zinc-containing proteinase in plant material: Voskoboynikova et al. (1989) described a protocol for separating a homogeneous metallo-proteinase from resting buckwheat seeds. In this paper we describe the presence of two caseindigesting proteinases in the seedlings of kale. The activity of both enzymes is inhibited by E D T A and 1,10-phenanthroline. One of the enzymes has been isolated, partially characterized and its proteolytic specificity for the oxidized B-chain of bovine insulin has been determined.
Material and methods Plant mater&l. Seeds of kale (Brasicca oleracea L. var. sabellica)
were supplied by the Plant Seed Corporation in Wroctaw (Poland). Seeds were sterilized by soaking them in 90% acetone for 5 min, then in H202 for 30 min. After that seeds were rinsed with water, sown in a moist bed of filter paper, and allowed to germinate at 2224~ C for 8 d with 10-12 h of light daily. No fungal contamination was observed on the germinating seeds. Assay ofproteolytic activity. Samples of 1 ml of enzyme solution incubated for 30 min at 36~ C with 1 ml of 1% casein (BDH, Poole, Dorset, UK) in 200 mM Tris-HCl buffer, pH 9. The reaction was stopped by adding 3 ml of 5% trichloroacetic acid (TCA). The precipitate was centrifuged off, and absorption at 280 nm was
A. Wilimowska-Pelc et al. : Metallo-proteinase from the seedlings of kale measured in the supernatant. One unit of proteolytic activity was defined as the amount of enzyme which gave an increase of 0.1 absorption unit at 280 nm under the conditions applied.
Inhibitors. The effect of some chemical inhibitors on proteinase activity was established after 15 min preincubation of enzyme with inhibitors. Final concentrations of inhibitors in the assay mixture were 1 m M for phenylmethylsulfonyl fluoride (PMSF), p-mercuribenzoic acid (PCMB), E D T A and cysteine, 3 mM for N-ethylmaleimide (NEM), and 5 mM for 1,10-phenanthroline.
Assay of amidase activity. The activity of the proteinase against Suc-Ala-Ala-Pro-Phe-pNA and Suc-Ala-Ala-Pro-Leu-pNA (Sigma Chemical Co., St.Louis, Mo., USA) was determined in according to Erlanger et al. (1961).
Proteinase purification
345
pancreatic secretory trypsin inhibitor (6.1 kDa) from bovine pancreas, prepared as described by Wilimowska-Pelc et al. (1973), cytochrome c (12.4 kDa), myoglobin (17.6 kDa), chymotrypsinogen A (25 kDa) and egg albumin (45 kDa). The kale proteinase was localized in the eluate by estimation of its proteolytic activity.
The effect ofpH and Zn 2+. The effect of pH and Zn 2+ on enzyme activity was studied using enzyme which had been stored at 5~ C for 30 d at pH 2.0 (0.01 N HCI), pH 4.0 (0.05 M acetate buffer), pH 6.0, 8.0 and 10.0 (0.05 M Tris-HCl buffer). In one of the two series studied samples also contained 0.2 m M ZnSO4. At 3-d intervals aliquots were withdrawn for determination of the residual activity. Determination of pH optimum. The proteinase pH optimum was determinated by hydrolysis of casein in 100 mM Tris-HC1 buffer in the pH range of 6.0-10.5.
The purification procedure was carried out at 5-7 ~ C.
Protein determination. Protein was determinated by the microbiuret method of Goa (1953), and spectrophotometrically at 280 nm.
Extraction. Eight-day-old seedlings were harvested and frozen at
Determination of specificity for oxidized B-chain of bovine insulin.
- 15~ C for 2 h. Each 100-g sample of seedling was homogenized with 150-200 ml of 1% NaC1 solution and extracted for 1 h with constant mechanical stirring. After centrifugation (14000 "9, 20 rain) the supernatant was decanted and adjusted to pH 7.5 with 2 N NaOH. The precipitate formed was centrifuged off.
Ammonium-sulfate precipitation. The supernatant was brought to 90% saturation with ammonium sulfate. After 20 h, sedimented proteins were collected, dissolved in about 15 ml of water and after 2 h clarified by centrifugation at 14 000 - g for 20 rain.
Sephadex G-75 chromatography. The ammonium-sulfate-precipitated protein solution was adjusted to pH 5.9 with 1 M acetic acid and applied to a Sephadex G-75 column (85 cm long, 2 cm i.d. ; Pharmacia, Uppsala, Sweden) equilibrated with 0.1 M acetate buffer pH 5.9. The fractions corresponding to the first and second peak of proteolytic activity were collected separately. Ion-exchange chromatography. The fraction corresponding to the second peak of proteolytic activity was brought to pH 7.0 with 1 N N a O H and applied to a DEAE-Sephadex A-50 column (15 cm long, 2 cm i.d.; Pharmacia) equlibrated with 0.05 M phosphatecitric acid buffer, pH 7.0. The enzyme did not adsorb under the applied conditions and was eluted from the column with starting buffer. Active fractions were pooled and after dialysis against 0.05 M acetate buffer (pH 5.0) subjected to chromatography on SP-Sephadex C-50 (15 cm long, 2 cm i.d. ; Pharmacia) equlibrated with the same buffer. The adsorbed proteins were eluted from the column with a linear concentration gradient of NaCI (0-0.5 M) in starting buffer. Final purification was achieved by means of highperformance liquid chromatography (HPLC) on a Spherogel TSK CM 3 SW column (75 mm long, 7.5 mm i.d. ; Beckman, Palo Alto, Cal., USA) equilibrated with 0.025 M phosphate buffer, pH 6.1. Proteins were eluted with a linear concentration gradient of NaC1 (0 to 0.5 M).
Polyacrylamide gel electrophoresis (PAGE). Proteins were subjected to PAGE using the method of Davis (1964) at pH 8.3 in 7.5% gels. Gels were stained for proteins with 1% Amido black 10B in 7% acetic acid. For detection of proteolytic activity, gels containing 0.1% edestin were used (Leluk et al. 1985); after electrophoresis (4 mA per tube) the gels were soaked in 0.1 M Tris-HCl buffer, pH 9.0, and incubated at room temperature until the transparent zones were visible. Then the gels were stained with 0.1% Amido black in 7% acetic acid. Molecular-weight determination by 9el filtration. A Sephadex G-75 column (80 cm long, 1.5 cm i.d. ; Pharmacia) was equilibrated with 0.05 M Tris-HC1 buffer (pH 7.5) containing 0.5 M NaC1. The following standards at a concentration of 1 mg 9ml-1 were used:
The enzyme (0.1 mol%) was added to 5 mg of insulin dissolved in 0.5 ml of 1% NH4HCO3, pH 7.9. After 24 h incubation at 30 ~ C the sample was frozen, lyophylized, and finally subjected to analysis, Separation of the hydrolysis product was accomplished by HPLC on a Supelcosil LC 18 DB column (250 mm long, 4.6 mm i.d. ; Supelco, Gland, Switzerland) with a linear gradient of acetonitrile (0-50%) in 0.1% trifluoroacetic acid (TFA). The peptides collected were hydrolized (6 N HC1, 110 ~ 24 h) and their amino-acid compositions determined by the method of pre-column derivatization with o-phtaldialdehyde and 2-mercaptoethanol followed by reversed-phase HPLC on Spherisorb ODS II 4 lain (column 60 mm long, 4 mm i.d. ; Knauer, Bad Hamburg, FRG), and fluorescence detection (Reiendra 1987, with a slight modification of the gradient curve).
Results and discussion
Proteolytic activity during kale seed germination. P r o t e o lyric a c t i v i t y w a s n o t d e t e c t a b l e in e x t r a c t s f r o m r e s t i n g seeds u n d e r t h e c o n d i t i o n s used. H o w e v e r , it i n c r e a s e d slowly during germination, displayed a sharp increase between 5 and 8 d and then did not change appreciably d u r i n g t h e n e x t 2 d ( F i g . l ) . T h e s e r e s u l t s a l l o w e d us to
"~ 120 >, 100 .>
$ "6 20
A-
A
It.-
Germination [clays] Fig. 1. Proteolytic activity during kale seed germination. Seedlings or seeds were extracted with 1% NaC1 under the conditions described in Material and methods. Proteolytic activity in crude enzyme extracts was measured using a casein substrate at pH 9
346
A. Wilimowska-Pelc et al. : Metallo-proteinase from the seedlings of kale o
I
4"
4,/.-
"11 "~: 910 2 '9 :_~
o C
~&2-
E e-
@ (NI
"6
,
