- Email: [email protected]
Chitinase from Phaseolus vulgaris leaves
[60]
BEAN L E A F CHITINASE
479
pH. Verticillium chitinase has a sharp pH optimum at 3.7 and is inactive above pH 7.7. The response curves are identical for crude and purified preparations. Temperature. Maximum activity occurs at 51 * but at 65 ° activity is still 40% of maximum. The enzyme is inactivated at 70*. Molecular Weight. In SDS gel electrophoresis two protein bands are observed. The denser band has a molecular weight of 64,000 and the minor band 58,000 by comparison of their mobilities relative to molecular weight markers. Effect of Ions and Inhibitors The effect of heavy metals and biological inhibitors on enzyme activity and substances interfering with N A G determinations is shown in Table II. Standard assay conditions using chitin as substrate at 1.83 mg ml -~ are used but with BSA at 200 pg m1-1 reaction mixture. The effect of substances on NAG determination is studied by substituting NAG at 25 pg ml- ~for enzyme in the reaction mixture. Effects are shown as a percentage of maximum activity in the absence of the test substance.
[60] Chitinase
f r o m P h a s e o l u s vulgaris L e a v e s
By THOMAS BOLLER, ANNETTE GEnRI, FELIX MAUCH, and URS VOGELI It is a surprising fact that some plants contain high activities of chitinase, an enzyme that has no known function in plant metabolism. Plant chitinase was first described from almond emulsin 1 and from bean and other seeds. 2 Abeles et aL3 discovered that chitinase is induced in bean leaves by the plant hormone, ethylene, in parallel with fl-l,3-glucanase. Since chitin and t - 1,3-glucans are important cell wall components of fungi, they have proposed an antifungal function for chitinase and fl-l,3-glucanase. 3 We examined the effect of ethylene on chitinase activity in bean leaves more closely and purified the enzyme in the course of this work. 4 Our L. Zechmeister and G. Toth, Enzymologia 7, 165 (1939). 2 R. F. Powning and H. Irzykiewicz, Comp. Biochem. Physiol. 14, 127 (1965). 3 F. B. Abeles, R. P. Bosshart, L. E. Forrence, and W. H. Habig, Plant Physiol. 47, 129
0970. 4 T. Boller, A. Gehri, F. Mauch, and U. V0geli, Planta 157, 22 (1983).
METHODS IN ENZYMOLOGY, VOL. 161
Copyright© 1988by AcademicPress,Inc. All fightsof reproductionin any form reserved.
480
CHITIN
[60]
purification method is derived from the one used for chitinase from wheat germ. 5,6 It is based on affinity chromatography on chitin and is applicable, with slight modifications, to all chitinases that are readily adsorbed by chitin. Some plant chitinases, for example, the major chitinase from cucumber leaves,7 do not bind well to chitin and cannot be purified by this method. A different method has been used for the purification of chitinase from tomato leaves) Assay M e t h o d
Principle.W e routinely employ the radiochemical chitinase assay9 described elsewhere in this volume, ~° based on the liberation of soluble radioactivity from insoluble [acetyl-3H]chitin.By this method, any possible chitin deacctylase activity would be misjudged as chitinasc. W e have verified that chitin dcacctylasc is not present in ethylene-treated bean leaves (unpublished observations). W e have also used the colorimctric chitinasc assay described elsewhere in this volume.H W e obtain exactly the same resultswith the colorimctric and the radiochemical assay.4,H Reagents [3H]Chitin (6 kBq/mg), 10 mg/ml in 3 m M NaNa. The [3H]chitin is prepared by N-acetylation of chitosan with [3H]acetic anhydride 6,9 (regenerated chitin). From this preparation, colloidal [3H]chitin is prepared according to Berger and Reynolds ~2as described elsewhere in this volume ~3 Sodium phosphate buffer, 0.1 M, pH 6.5 TCA (trichloroacetic acid), 1 M
Procedure. The following are pipetted into an 1.5-ml Eppendorf tube: 50/zl sodium phosphate buffer, 10- 100/zl enzyme, and water to a volume of 150/zl. The reaction is carried out in a shaking water bath at 37 °. It is started by the addition of 100 #1 [3H]chitin and stopped by the addition of 250/tl TCA after 30 min. After centrifugation (1000g for 10 min), the radioactivity of 250/zl of the supernatant is determined. 5 j. Molano, I. Polacheck, A. Duran, and E. Cabib, J. Biol. Chem. 254, 4901 (1979). 6 E. Cabib, this volume [63]. 7 j. p. M~traux and T. Boiler, Physiol. Mol. Plant Pathol. 28, 161 (1986). 8 G. F. Peg~ this volume [61 ]. 9 j. Molano, A. Duran, and E. Cabib, Anal. Biochem. 83, 648 (1977). l0 E. Cabib, this volume [48]. 1, T. Boiler and F. Mauch, this volume [50]. 12 L. R. Berger and D. M. Reynolds, Biochim. Biophys. Acta 29, 522 (1958). 13 K. Shimahara and Y. Takiguchi, this volume [47].
[60]
BEAN LEAF CHITINASE
481
Definition of Unit and Specific Activity. Since formation of reaction product is nonlinearly related to the enzyme concentration, the activity is calculated from a standard curve prepared with a dilution series of the enzyme, as described elsewhere in this volume. ~,~1One unit ofchitinase is defined as the amount that catalyzes the release of soluble chitoohgosaccharides containing 1/~mol of N-acetylgiucosamine residues in 1 min at infinite dilution. Specific activity (units per milligram of protein) is based on protein determinations by the Bradford methodJ 4 Plant Material, E t h y l e n e T r e a t m e n t Primary leaves of Phaseolus vulgaris cv. "Saxa" (bush bean) are employed. The seeds, obtained from Samen Mauser, Ztirich, Switzerland, are steeped for 4 hr in running tap water and then grown on trays containing Vermiculite in a greenhouse with a day/night photoperiod of 16/8 hr and temperatures of 25/20 °. After 12-14 days, when the primary leaves are fully expanded, the whole trays with about 100 seedlings are put into plastic chambers (I00 liters). The chambers are tightly closed. The plants are exposed to an atmosphere containing I0 ppm ethylene by injecting l ml pure ethylene gas into the chamber through an inlet port covered with a gas-tight rubber cap. To obtain maximal induction of chitinase, the plants are incubated for 36-48 hr in ethylene. (Longer treatment results in leaf abscission.) About 70 g primary leaves is obtained from one tray. Purification P r o c e d u r e 4 Unless otherwise specified, all operations are carried out at 0 - 5 °. Step 1: Preparation of the Crude Extract. Leaves from ethylene-treated plants (200 g) are homogenized in 400 ml 0.1 Mcitrate buffer, pH 5.0, in a Sorvall Omnimix homogenizer for 3 × 1 min at top speed. The homogenate is centrifuged (15,000 g, 10 min). The supernatant represents the crude extract. It contains most of the chitinase activity but, at the relatively low pH employed, only about 20% of the total leaf protein. Addition of dithiothreitol (2 m M ) does not interfere but does not increase the yield of chitinase. Step 2: Heat Treatment. The crude extract is incubated at 50 ° for 20 min. After cooling, the precipitate is removed by centrifugation (20,000 g, 10 min). Step 3: Ammonium Sulfate Precipitation. To the supernatant, ammonium sulfate is added to give 60% saturation at 0 °. After an incubation of ,4 M. M. Bradford, Anal Biochern. 72, 248 (1976).
482
cretin
[60]
1 hr with continuous stirring, the precipitate is collected by centrifugation (20,000 g, 20 min) and redissolved in 40 ml 10 m M sodium acetate buffer, pH 5. NaHCO3 is added to give a concentration of 20 mM, and the pH is raised to pH 8.4 with 0.1 N NaOH. Step 4: Chromatography on Regenerated Chitin. A column (8 X 3 cm) is made of regenerated chitin, prepared according to Molano et al. a After equilibration with 20 m M sodium bicarbonate buffer, pH 8.4, the chitinase preparation is put on the column. The absorption of the eluate is monitored at 280 nm. The column is washed with 150 ml 20 m M sodium bicarbonate buffer, pH 8.4, followed by 100 ml 20 m M sodium acetate buffer, pH 5.5. Then, chitinase is eluted with 20 m M acetic acid (pH 3.2). Chitinase activity is eluted in a single peak which coincides with the peak of A2s0. The peak fractions are combined, dialyzed against 10 m M sodium acetate buffer, pH 5.0, and lyophilized. Summary. Table I summarizes the purification protocol. Chitinase is purified about 24-fold, indicating that 4% of the total soluble protein at pH 5 is chitinase. The overall yield is 68%. Properties Purity. The enzyme preparation from the chitin column is homogeneous, as judged by SDS-gel electrophoresis. For a further demonstration of purity, the lyophilized chitinase is taken up in 2 ml 0.05 M sodium phosphate buffer, pH 6.8, containing 0.1 M NaCl, and chromatographed on a 2 × 68 cm column of Sephadex G-75 equilibrated with the same buffer. Chitinase elutes as a single peak with constant specific activity. The position of the peak indicates an apparent molecular weight of 22,000. Obviously, the protein is slightly retarded on the Sephadex column since its molecular weight, estimated with SDS-gel electrophoresis and analytical ultracentrifugation, is about 30,500-32,500 (see below). TABLE I PURIFICATION OF ETHYLENE-INDUCED CHITINASE FROM BEAN LEAVES
Protein (rag)
Chitinase (U)
Specific activity (U/mg)
Yieh
Step
Volume (ml)
Crude extract Heat treatment (NH4)2SO 4 Precipitation Chitin column
500 500 50 20
340 200 154 10
2130 2000 2000 1440
6.3 10.0 13.0 144
100 94 94 68
(%)
[60]
BEAN LEAF CHITINASE
483
Specificity and Mode of Action. Like other plant chitinases,4,9,~5 bean leaf chitinase initially forms chitooligosaccharides of various chain lengths from colloidal or regenerated chitin. 4 After exhaustive digestive of chitin, the main end products are chitobiose and chitotriose; only about 7% of the product is in the form of the monomer, N-acetylglucosamine. Obviously, the exochitinase assay used by Abeles et al., 3 which monitors the production of the monomer only, is unsuitable for bean chitinase.~l Bean leaf chitinase readily depolymerizes glycol chitin; it can be measured viscosimetrically on this basis. 4 The enzyme does not hydrolyze chitosan, glycol chitosan, or p-nitrophenyl-fl-N-acetylglucosaminide; it has no cellulase or r- 1,3-glucanase activity? Bean leaf chitinase releases chitooligosaccharides from purified chitincontaining fungal cell walls. It also attacks bacterial cell walls, acting as a lysozyme.4,~5 Antifungal Activity. Purified bean chitinase is a potent growth inhibitor of Trichoderma viride. 16The smallest concentration that inhibits growth of this fungus is 2/zg m1-1. Physical Properties. The molecular weight of bean leaf chitinase, estimated by SDS-gel electrophoresis, is 32,500. Analytical ultracentrifugation yields a similar molecular weight (30,500), indicating that the enzyme occurs as a monomer in solution. It is a basic protein with an isoelectric point of about 9.4, as determined by chromatofocusing. Incidentally, most plant chitinases are basic proteins, 15 although there are exceptions. 7 Interestingly, the N-terminal primary amino acid sequence of bean leaf chitinase shows a high degree of similarity with WGA, a chitin-binding lectin. 17 The entire amino acid sequence of bean chitinase has been deduced from the nucleotide sequence of a chitinase cDNA clone. TM Effect of pH. Bean leaf chitinase has a broad pH optimum around pH 6.5 for the hydrolysis of colloidal or regenerated chitin; its activity is more than 50% of the maximum between pH 3 and 9. 4,15 In contrast, its pH optimum for the hydrolysis of the bacterial cell wall peptidoglycan is quite narrow, centered at pH 4.8, with less than 50% of the maximal activity above pH 5.25 and below pH 4.15 Effect of Temperature. The enzyme is stable up to 40 ° . It loses about 10% of its activity per hour at 50 ° and about 60%/hr at 60 °. Between 0 and ~5T. Boiler, in "Cellular and Molecular Biology of Plant Stress" (J. L. Key and T. Kosuge, eds.), p. 247. Liss, New York, 1985. 16A. Schlumbaum, F. Mauch, U. VOgeli, and T. Boiler, Nature (London) 324, 365 (1986). 17j. Lucas, A. Henschen, F. Lottspeich, U. Vtgeli, and T. Boiler, FEBSLett. 193, 208 (1985). ,s K. E. Broglie, J. J. Gaynor, and R. M. Broglie, Proc. Natl. Acad. Sci. U.S.A. 83, 6820 (1986).
484
CHITIN
[61]
40 °, its apparent energy of activation, determined from an Arrhenius plot, is 39 kJ mol -~. Stability. Purified bean chitinase is stable for at least 2 years when kept lyophilized or frozen at - 2 0 °. Activators and Inhibitors. Bean leaf chitinase does not require any cofactors. It is not affected by EDTA. The chitin-hydrolyzing activity is inhibited about 50% by 100 m M N-acetylglucosamine and less than 10% by l0 m M histamine. The lysozyme activity is inhibited less than 10% by 100 m M N-acetylglucosamine but more than 90% by 10 m M histamine. Subcellular Localization. In bean leaves, chitinase is located in the central vacuole of the cells. 19 Acknowledgment This work was supported by the Swiss National Science Foundation. t9 T. Boiler and U. V6geli, Plant Physiol. 74, 442 (1984).
[61 ] C h i t i n a s e f r o m T o m a t o
Lycopersicon esculentum
By G. F. PEGG Chitinases are found in a number of higher plants,~-5 including tomato
(Lycopersicon esculentum Mill.). 6,7 Chitin per se is not synthesized by vascular plants, but N-acetylglucosamine as a constituent of other plant polymers, e.g., as a glycoprotein,8,9 is widespread in many spermatophytes, especially in seeds, including tomato?-~2 Sideris et al. ~3have reported the presence of free hexosamine in pineapple but this uncombined form appears to be exceptional. F. B. Abeles, R. P. Bosshart, L. E. Forrence, and W. H. Habig, Plant Physiol. 47, 129 (1970). 2 W. Grassmann, L. Zeehmeister, R. Bender, and G. Toth, Bet. Dtsch. Chem. 67, 1 (1934). 3 j. Molano, I. Potacheek, A. Duran, and E. Cabib, J. Biol. Chem. 254, 4901 (1979). 4 T. Boiler, A. Gehri, F. Mauch, and U. V0geli, Planta 157, 22 (1983). s T. Tsukamoto, D. Koga, A. Ide, T. Ishibashi, M. Hodno-Matsushige, K. Yagishita, and T. I m o t o , Agric. Biol. Chem. 4 8 , 9 3 1 ( 1 9 8 4 ) . 6 G. F. Pegg and J. C. Vessey, Physiol. Plant Pathol. 3, 207 (1982). 7 G. F. Pegg and D. H. Young, Physiol. Plant Pathol. 21, 389 (1982).
METHODS IN ENZYMOLOGY, VOL. 161
Copyright © 1988 by Academic Press, Inc. All fights of reproduction in any form t~erved.
BEAN L E A F CHITINASE
479
pH. Verticillium chitinase has a sharp pH optimum at 3.7 and is inactive above pH 7.7. The response curves are identical for crude and purified preparations. Temperature. Maximum activity occurs at 51 * but at 65 ° activity is still 40% of maximum. The enzyme is inactivated at 70*. Molecular Weight. In SDS gel electrophoresis two protein bands are observed. The denser band has a molecular weight of 64,000 and the minor band 58,000 by comparison of their mobilities relative to molecular weight markers. Effect of Ions and Inhibitors The effect of heavy metals and biological inhibitors on enzyme activity and substances interfering with N A G determinations is shown in Table II. Standard assay conditions using chitin as substrate at 1.83 mg ml -~ are used but with BSA at 200 pg m1-1 reaction mixture. The effect of substances on NAG determination is studied by substituting NAG at 25 pg ml- ~for enzyme in the reaction mixture. Effects are shown as a percentage of maximum activity in the absence of the test substance.
[60] Chitinase
f r o m P h a s e o l u s vulgaris L e a v e s
By THOMAS BOLLER, ANNETTE GEnRI, FELIX MAUCH, and URS VOGELI It is a surprising fact that some plants contain high activities of chitinase, an enzyme that has no known function in plant metabolism. Plant chitinase was first described from almond emulsin 1 and from bean and other seeds. 2 Abeles et aL3 discovered that chitinase is induced in bean leaves by the plant hormone, ethylene, in parallel with fl-l,3-glucanase. Since chitin and t - 1,3-glucans are important cell wall components of fungi, they have proposed an antifungal function for chitinase and fl-l,3-glucanase. 3 We examined the effect of ethylene on chitinase activity in bean leaves more closely and purified the enzyme in the course of this work. 4 Our L. Zechmeister and G. Toth, Enzymologia 7, 165 (1939). 2 R. F. Powning and H. Irzykiewicz, Comp. Biochem. Physiol. 14, 127 (1965). 3 F. B. Abeles, R. P. Bosshart, L. E. Forrence, and W. H. Habig, Plant Physiol. 47, 129
0970. 4 T. Boller, A. Gehri, F. Mauch, and U. V0geli, Planta 157, 22 (1983).
METHODS IN ENZYMOLOGY, VOL. 161
Copyright© 1988by AcademicPress,Inc. All fightsof reproductionin any form reserved.
480
CHITIN
[60]
purification method is derived from the one used for chitinase from wheat germ. 5,6 It is based on affinity chromatography on chitin and is applicable, with slight modifications, to all chitinases that are readily adsorbed by chitin. Some plant chitinases, for example, the major chitinase from cucumber leaves,7 do not bind well to chitin and cannot be purified by this method. A different method has been used for the purification of chitinase from tomato leaves) Assay M e t h o d
Principle.W e routinely employ the radiochemical chitinase assay9 described elsewhere in this volume, ~° based on the liberation of soluble radioactivity from insoluble [acetyl-3H]chitin.By this method, any possible chitin deacctylase activity would be misjudged as chitinasc. W e have verified that chitin dcacctylasc is not present in ethylene-treated bean leaves (unpublished observations). W e have also used the colorimctric chitinasc assay described elsewhere in this volume.H W e obtain exactly the same resultswith the colorimctric and the radiochemical assay.4,H Reagents [3H]Chitin (6 kBq/mg), 10 mg/ml in 3 m M NaNa. The [3H]chitin is prepared by N-acetylation of chitosan with [3H]acetic anhydride 6,9 (regenerated chitin). From this preparation, colloidal [3H]chitin is prepared according to Berger and Reynolds ~2as described elsewhere in this volume ~3 Sodium phosphate buffer, 0.1 M, pH 6.5 TCA (trichloroacetic acid), 1 M
Procedure. The following are pipetted into an 1.5-ml Eppendorf tube: 50/zl sodium phosphate buffer, 10- 100/zl enzyme, and water to a volume of 150/zl. The reaction is carried out in a shaking water bath at 37 °. It is started by the addition of 100 #1 [3H]chitin and stopped by the addition of 250/tl TCA after 30 min. After centrifugation (1000g for 10 min), the radioactivity of 250/zl of the supernatant is determined. 5 j. Molano, I. Polacheck, A. Duran, and E. Cabib, J. Biol. Chem. 254, 4901 (1979). 6 E. Cabib, this volume [63]. 7 j. p. M~traux and T. Boiler, Physiol. Mol. Plant Pathol. 28, 161 (1986). 8 G. F. Peg~ this volume [61 ]. 9 j. Molano, A. Duran, and E. Cabib, Anal. Biochem. 83, 648 (1977). l0 E. Cabib, this volume [48]. 1, T. Boiler and F. Mauch, this volume [50]. 12 L. R. Berger and D. M. Reynolds, Biochim. Biophys. Acta 29, 522 (1958). 13 K. Shimahara and Y. Takiguchi, this volume [47].
[60]
BEAN LEAF CHITINASE
481
Definition of Unit and Specific Activity. Since formation of reaction product is nonlinearly related to the enzyme concentration, the activity is calculated from a standard curve prepared with a dilution series of the enzyme, as described elsewhere in this volume. ~,~1One unit ofchitinase is defined as the amount that catalyzes the release of soluble chitoohgosaccharides containing 1/~mol of N-acetylgiucosamine residues in 1 min at infinite dilution. Specific activity (units per milligram of protein) is based on protein determinations by the Bradford methodJ 4 Plant Material, E t h y l e n e T r e a t m e n t Primary leaves of Phaseolus vulgaris cv. "Saxa" (bush bean) are employed. The seeds, obtained from Samen Mauser, Ztirich, Switzerland, are steeped for 4 hr in running tap water and then grown on trays containing Vermiculite in a greenhouse with a day/night photoperiod of 16/8 hr and temperatures of 25/20 °. After 12-14 days, when the primary leaves are fully expanded, the whole trays with about 100 seedlings are put into plastic chambers (I00 liters). The chambers are tightly closed. The plants are exposed to an atmosphere containing I0 ppm ethylene by injecting l ml pure ethylene gas into the chamber through an inlet port covered with a gas-tight rubber cap. To obtain maximal induction of chitinase, the plants are incubated for 36-48 hr in ethylene. (Longer treatment results in leaf abscission.) About 70 g primary leaves is obtained from one tray. Purification P r o c e d u r e 4 Unless otherwise specified, all operations are carried out at 0 - 5 °. Step 1: Preparation of the Crude Extract. Leaves from ethylene-treated plants (200 g) are homogenized in 400 ml 0.1 Mcitrate buffer, pH 5.0, in a Sorvall Omnimix homogenizer for 3 × 1 min at top speed. The homogenate is centrifuged (15,000 g, 10 min). The supernatant represents the crude extract. It contains most of the chitinase activity but, at the relatively low pH employed, only about 20% of the total leaf protein. Addition of dithiothreitol (2 m M ) does not interfere but does not increase the yield of chitinase. Step 2: Heat Treatment. The crude extract is incubated at 50 ° for 20 min. After cooling, the precipitate is removed by centrifugation (20,000 g, 10 min). Step 3: Ammonium Sulfate Precipitation. To the supernatant, ammonium sulfate is added to give 60% saturation at 0 °. After an incubation of ,4 M. M. Bradford, Anal Biochern. 72, 248 (1976).
482
cretin
[60]
1 hr with continuous stirring, the precipitate is collected by centrifugation (20,000 g, 20 min) and redissolved in 40 ml 10 m M sodium acetate buffer, pH 5. NaHCO3 is added to give a concentration of 20 mM, and the pH is raised to pH 8.4 with 0.1 N NaOH. Step 4: Chromatography on Regenerated Chitin. A column (8 X 3 cm) is made of regenerated chitin, prepared according to Molano et al. a After equilibration with 20 m M sodium bicarbonate buffer, pH 8.4, the chitinase preparation is put on the column. The absorption of the eluate is monitored at 280 nm. The column is washed with 150 ml 20 m M sodium bicarbonate buffer, pH 8.4, followed by 100 ml 20 m M sodium acetate buffer, pH 5.5. Then, chitinase is eluted with 20 m M acetic acid (pH 3.2). Chitinase activity is eluted in a single peak which coincides with the peak of A2s0. The peak fractions are combined, dialyzed against 10 m M sodium acetate buffer, pH 5.0, and lyophilized. Summary. Table I summarizes the purification protocol. Chitinase is purified about 24-fold, indicating that 4% of the total soluble protein at pH 5 is chitinase. The overall yield is 68%. Properties Purity. The enzyme preparation from the chitin column is homogeneous, as judged by SDS-gel electrophoresis. For a further demonstration of purity, the lyophilized chitinase is taken up in 2 ml 0.05 M sodium phosphate buffer, pH 6.8, containing 0.1 M NaCl, and chromatographed on a 2 × 68 cm column of Sephadex G-75 equilibrated with the same buffer. Chitinase elutes as a single peak with constant specific activity. The position of the peak indicates an apparent molecular weight of 22,000. Obviously, the protein is slightly retarded on the Sephadex column since its molecular weight, estimated with SDS-gel electrophoresis and analytical ultracentrifugation, is about 30,500-32,500 (see below). TABLE I PURIFICATION OF ETHYLENE-INDUCED CHITINASE FROM BEAN LEAVES
Protein (rag)
Chitinase (U)
Specific activity (U/mg)
Yieh
Step
Volume (ml)
Crude extract Heat treatment (NH4)2SO 4 Precipitation Chitin column
500 500 50 20
340 200 154 10
2130 2000 2000 1440
6.3 10.0 13.0 144
100 94 94 68
(%)
[60]
BEAN LEAF CHITINASE
483
Specificity and Mode of Action. Like other plant chitinases,4,9,~5 bean leaf chitinase initially forms chitooligosaccharides of various chain lengths from colloidal or regenerated chitin. 4 After exhaustive digestive of chitin, the main end products are chitobiose and chitotriose; only about 7% of the product is in the form of the monomer, N-acetylglucosamine. Obviously, the exochitinase assay used by Abeles et al., 3 which monitors the production of the monomer only, is unsuitable for bean chitinase.~l Bean leaf chitinase readily depolymerizes glycol chitin; it can be measured viscosimetrically on this basis. 4 The enzyme does not hydrolyze chitosan, glycol chitosan, or p-nitrophenyl-fl-N-acetylglucosaminide; it has no cellulase or r- 1,3-glucanase activity? Bean leaf chitinase releases chitooligosaccharides from purified chitincontaining fungal cell walls. It also attacks bacterial cell walls, acting as a lysozyme.4,~5 Antifungal Activity. Purified bean chitinase is a potent growth inhibitor of Trichoderma viride. 16The smallest concentration that inhibits growth of this fungus is 2/zg m1-1. Physical Properties. The molecular weight of bean leaf chitinase, estimated by SDS-gel electrophoresis, is 32,500. Analytical ultracentrifugation yields a similar molecular weight (30,500), indicating that the enzyme occurs as a monomer in solution. It is a basic protein with an isoelectric point of about 9.4, as determined by chromatofocusing. Incidentally, most plant chitinases are basic proteins, 15 although there are exceptions. 7 Interestingly, the N-terminal primary amino acid sequence of bean leaf chitinase shows a high degree of similarity with WGA, a chitin-binding lectin. 17 The entire amino acid sequence of bean chitinase has been deduced from the nucleotide sequence of a chitinase cDNA clone. TM Effect of pH. Bean leaf chitinase has a broad pH optimum around pH 6.5 for the hydrolysis of colloidal or regenerated chitin; its activity is more than 50% of the maximum between pH 3 and 9. 4,15 In contrast, its pH optimum for the hydrolysis of the bacterial cell wall peptidoglycan is quite narrow, centered at pH 4.8, with less than 50% of the maximal activity above pH 5.25 and below pH 4.15 Effect of Temperature. The enzyme is stable up to 40 ° . It loses about 10% of its activity per hour at 50 ° and about 60%/hr at 60 °. Between 0 and ~5T. Boiler, in "Cellular and Molecular Biology of Plant Stress" (J. L. Key and T. Kosuge, eds.), p. 247. Liss, New York, 1985. 16A. Schlumbaum, F. Mauch, U. VOgeli, and T. Boiler, Nature (London) 324, 365 (1986). 17j. Lucas, A. Henschen, F. Lottspeich, U. Vtgeli, and T. Boiler, FEBSLett. 193, 208 (1985). ,s K. E. Broglie, J. J. Gaynor, and R. M. Broglie, Proc. Natl. Acad. Sci. U.S.A. 83, 6820 (1986).
484
CHITIN
[61]
40 °, its apparent energy of activation, determined from an Arrhenius plot, is 39 kJ mol -~. Stability. Purified bean chitinase is stable for at least 2 years when kept lyophilized or frozen at - 2 0 °. Activators and Inhibitors. Bean leaf chitinase does not require any cofactors. It is not affected by EDTA. The chitin-hydrolyzing activity is inhibited about 50% by 100 m M N-acetylglucosamine and less than 10% by l0 m M histamine. The lysozyme activity is inhibited less than 10% by 100 m M N-acetylglucosamine but more than 90% by 10 m M histamine. Subcellular Localization. In bean leaves, chitinase is located in the central vacuole of the cells. 19 Acknowledgment This work was supported by the Swiss National Science Foundation. t9 T. Boiler and U. V6geli, Plant Physiol. 74, 442 (1984).
[61 ] C h i t i n a s e f r o m T o m a t o
Lycopersicon esculentum
By G. F. PEGG Chitinases are found in a number of higher plants,~-5 including tomato
(Lycopersicon esculentum Mill.). 6,7 Chitin per se is not synthesized by vascular plants, but N-acetylglucosamine as a constituent of other plant polymers, e.g., as a glycoprotein,8,9 is widespread in many spermatophytes, especially in seeds, including tomato?-~2 Sideris et al. ~3have reported the presence of free hexosamine in pineapple but this uncombined form appears to be exceptional. F. B. Abeles, R. P. Bosshart, L. E. Forrence, and W. H. Habig, Plant Physiol. 47, 129 (1970). 2 W. Grassmann, L. Zeehmeister, R. Bender, and G. Toth, Bet. Dtsch. Chem. 67, 1 (1934). 3 j. Molano, I. Potacheek, A. Duran, and E. Cabib, J. Biol. Chem. 254, 4901 (1979). 4 T. Boiler, A. Gehri, F. Mauch, and U. V0geli, Planta 157, 22 (1983). s T. Tsukamoto, D. Koga, A. Ide, T. Ishibashi, M. Hodno-Matsushige, K. Yagishita, and T. I m o t o , Agric. Biol. Chem. 4 8 , 9 3 1 ( 1 9 8 4 ) . 6 G. F. Pegg and J. C. Vessey, Physiol. Plant Pathol. 3, 207 (1982). 7 G. F. Pegg and D. H. Young, Physiol. Plant Pathol. 21, 389 (1982).
METHODS IN ENZYMOLOGY, VOL. 161
Copyright © 1988 by Academic Press, Inc. All fights of reproduction in any form t~erved.