- Email: [email protected]
[53] Polyamine oxidase (oat seedlings)
[53]
POLYAMINEOXIDASE(OAT SEEDLINGS)
311
vitro. With crude peroxisomal extracts the oxidation of spermidine is not as strictly dependent on the presence of benzaldehyde as with the purified enzyme.
[53] P o l y a m i n e O x i d a s e (Oat S e e d l i n g s )
By TERENCE A. SMITH NH2CHzCH2CH2NHCH2CH2CHzCH2NHz + 0 2 Sperm[dine
H
C -CH~ NH2CH2CH2CH2NHz + N~/ [ + HzO 2 C -CH2 Hz Diam inopropane
A' -Pyrroline
NH2CHzCHzCH2NHCHzCHzCHzCHaNI'ICHzCH2CHzNH2 + 0 z Spermine
H
H
/C = C NHzCHzCH2CH2NI"I2 + NH2CH2CHzCHzN I C-C Hz Hz Diaminopropane 1 -(3-Aminopropyl) pyrroline 1
+ H20 z
Amine oxidases and amine dehydrogenases are found in various microorganisms. 2-4 In the higher plants, amine oxidases that effect the oxidation of polyamines by the above mechanisms have been found only in the Gramineae (the grasses)5-13; they have not yet been detected in animals. TM 1 The structure is s h o w n as a derivative of A2-pyrroline, although the position of the double bond is not yet established. 2 R. H. W e a v e r and E. J. Herbst, J. Biol. Chem. 231, 647 (1958). 3 K. Isobe, Y. Tani, and H. Y a m a d a , Agric. Biol. Chem. 45, 727 (1981). 4 C. W. Tabor and P. D. Kellogg, J. Biol. Chem. 245, 5424 (1970). 5 T. A. Smith, Biochem. Biophys. Res. Commun. 41, 1452 (1970). 6 T. A. Smith, Phytochemistry 11, 899 (1972). 7 T. A. Smith, Phytochemistry 13, 1075 (1974). 8 T. A. Smith and D. A. Bickley, Phytochemistry 13, 2437 (1974). 9 T. A. Smith, Phytochemistry 15, 633 (1976). ~0 T. A. Smith, Phytochemistry 16, 1647 (1977). u y . Suzuki and E. Hirasawa, Phytochemistry 12, 2863 (1973). i2 E. Hirasawa and Y. Suzuki, Phytochemistry 14, 99 (1975). 13 y . Suzuki and H. Yanagisawa, Plant Cell Physiol. 21, 1085 (1980). 14 See, however, this volume ~52] for a polyamine oxidase in animal tissues that converts spermidine and acetylspermidine to putrescine.
METHODS IN ENZYMOLOGY, VOL. 94
Copyright © 1983by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181994-9
312
AMINE OXIDASES AND DEHYDROGENASES
[53]
Preliminary work with oats, barley, and maize had indicated that the enzymes are associated with a particulate fraction, 6,9,11 though a more recent study, supporting an earlier observation, 8 demonstrates that this is an artifact of extraction and that this enzyme normally occurs in the cell wall. 15 The enzyme is particularly active in vascular tissue. 5,~5 Assay Methods There are several methods for the determination of this enzyme. Perhaps the least ambiguous depends on the estimation of oxygen consumption using a Clark electrode. 8 However, measurement of the hydrogen peroxide by a coupled peroxidase system7 is more sensitive, owing to the greater baseline stability. 8
Reagents Tris buffer, 0.1 M, pH 6.5 (correct at 30°), air equilibrated Guaiacol, 25 mM Peroxidase, Sigma, type II (200 purpurogallin units/mg), 1 mg/ml Spermidine, 25 mM Procedure. Buffer (2 ml), guaiacol (0.1 ml), peroxidase (0.1 ml), and the oat leaf polyamine oxidase preparation (0.2 ml) are placed in optically matched cylindrical spectrophotometer cells (1.2 × 12.5 cm). After preincubation for 2 rain in a water bath at 30° the tube is transferred to a spectrophotometer cuvette holder, also thermostatted at 30°. Spermidine (0.1 ml) is added, and the increase in absorbance at 470 nm is determined. The spectrophotometer output is fed into a logarithmic recorder (fullscale deflection = 0.2 absorbance unit). Activity is determined on extrapolation of the curve to zero time. Activity declines rapidly during the assay, especially with spermine as substrate, l0 Spermidine is therefore to be preferred in the assay. The use of fluorogenic peroxidase substrates 16 should allow a considerable increase in sensitivity. Purification Procedure 9
Material. Oats (Arena sativa L., cv 'Black Supreme') are grown in the dark for 3 weeks at 22° in sand culture in a medium containing 2 mM K2SO4, 3 mM MgSO4, 8 mM CaCI2, 4 mM NaHzPO4, and 12 mM NaNO3 together with Fe (as FeEDTA) 5.6 ppm, Mn 0.6 ppm, Cu 0.06 ppm, Zn 0.07 ppm, Mo 0.05 ppm, and B 0.7 ppm. 15 R. K a u r - S a w h n e y , H. E. Flores, and A. W. Galston, Plant Physiol. 68, 494 (1981). 16 K. Zaitsu and Y. Ohkura, Anal. Biochem. 109, 109 (1980).
[53]
POLYAMINE OXIDASE (OAT SEEDLINGS)
313
TABLE I PURIFICATION OF POLYAMINE OXIDASE FROM OAT LEAVES9
Step Aqueous extractiona pH 4 precipitation Acetone precipitation Gel filtration
Total Volume protein (ml) (mg) 550 22 2.4 66
800 34 2.9 0.22
Total activity (nkat? 5600 4740 4580 1340
Specific activity (nkat/mg Purification Yield protein) (fold) (%) 6.8 140 1620 6020
1 20 240 890
(100) 85 82 24
a The extract was prepared from 150 g of 3-week-old dark-grown shoots. b The enzyme was assayed by the peroxidase-guaiacol method at pH 6 with spermine as substrate.
Extraction. The shoots are blended in 4 volumes of cold water, the macerate is squeezed through muslin, and the residue is reextracted with 2 volumes of cold water. p H 4 Precipitation. The combined substrates are adjusted to pH 4 with saturated citric acid. The precipitate is collected by centrifuging at 2500 g for 15 min and extracted in 0.1 M pH 6 citrate buffer containing 1 M NaC1. On recentrifuging at 3000 g for 15 min the precipitate is discarded. Acetone Precipitation. The supernatant is cooled to 0 °, and 1 volume of acetone at - 1 5 ° is added. The precipitate collected by centrifugation is extracted with 1 M NaCI in 0.1 M p H 6 citrate buffer; on recentrifugation, the precipitate is discarded. Gel Filtration. The supernatant is applied to a column of Sephadex G-100 (3.5 × 87 cm) equilibrated with 1 M NaC1 in 0.1 M pH 6 citrate buffer. Fractions (8 ml each at 24 ml/hr) eluted in the same buffer are collected; the active band is pooled and concentrated by Millipore filtration (UM-10 filter). On polyacrylamide gel electrophoresis the final product migrates as a single band at pH 4.3 toward the anode, and enzyme activity is coincident with the major protein band.l° A typical purification is summarized in Table I. Properties of the Oat L e a f E n z y m e
Activators and Inhibitors. Oxidation of the polyamines is strongly inhibited by NHz(CHzh0NH(CH2)3NH2, a spermidine homolog. 1° The fungicide, guazatine, [NH2C(-m-NH)NH(CH2)8]2NH (KenoGard AB, Stockholm, Sweden), is also a powerful inhibitor (Ki ca 10 -8 M). The
314
[54]
AMINE OXIDASES AND DEHYDROGENASES T A B L E II COMPARISON OF THE PROPERTIES OF OAT, BARLEY, AND MAIZE SEEDLING POLYAMINE OXIDASES
Property Molecular weight Relative activity, spermine vs spermidine" pH Optimum for spermidine pH Optimum for spermine Cofactors Maximum specific activity (nkat/mg protein) a
Oatsg.to
BarleyT.9
80,000 1
85,000 14
6.5 6.5 ND C 6020
Maize H t3 65,000 0.7
8.0 b 4.8 ND --
6.3 5.5 FAD 476
Activity at the pH optimum for the respective substrate.
b The enzyme is unstable at this pH. " ND, Not detected.
enzyme is activated by the chlorides of Li +, Na +, K +, and Rb + at 1 M in order of increasing effectiveness. 17 The Km values for spermidine and spermine, respectively, are 8 × 10 -6 M and 2 × 10 -6 M in air-saturated buffer. The Km values for oxygen are 1.83 x 10 -4 M and 0 8 5 x 10 -4 M with spermidine and spermine, respectively, as substrates.10 For the barley enzyme the Km for oxygen is relatively high, and oxygen in air-saturated buffer is limiting. 8 Properties of the polyamine oxidases from oat, barley, and maize seedlings are compared in Table II. Stability. In intact oat leaves, loss of activity at - 15° is 5% per week; for the purified enzyme, loss of activity at - 1 5 ° is 10% per week. 1° 17 T. A. Smith and P. E. Gay, unpublished (1981).
[54] P u r i f i c a t i o n o f B o v i n e P l a s m a A m i n e O x i d a s e
By B.
MONDOVI,
P.
TURINI, O. BEFANI,
and S. SABATINI
Bovine plasma amine oxidase is a CuE+-containing enzyme that catalyzes the oxidation of primary amines I as follows: R - - C H 2 - - N H 3 + + 02 + H20 --~ R C H O + H,,O2 + NH4 ~
E. A. Zeller, Helv. Chim. Acta 21, 880 (1938).
METHODS IN ENZYMOLOGY, VOL. 94
Copyright© 1983by AcademicPress, Inc. All rightsof reproductionin any formreserved. ISBN 0-12-181994-9
POLYAMINEOXIDASE(OAT SEEDLINGS)
311
vitro. With crude peroxisomal extracts the oxidation of spermidine is not as strictly dependent on the presence of benzaldehyde as with the purified enzyme.
[53] P o l y a m i n e O x i d a s e (Oat S e e d l i n g s )
By TERENCE A. SMITH NH2CHzCH2CH2NHCH2CH2CHzCH2NHz + 0 2 Sperm[dine
H
C -CH~ NH2CH2CH2CH2NHz + N~/ [ + HzO 2 C -CH2 Hz Diam inopropane
A' -Pyrroline
NH2CHzCHzCH2NHCHzCHzCHzCHaNI'ICHzCH2CHzNH2 + 0 z Spermine
H
H
/C = C NHzCHzCH2CH2NI"I2 + NH2CH2CHzCHzN I C-C Hz Hz Diaminopropane 1 -(3-Aminopropyl) pyrroline 1
+ H20 z
Amine oxidases and amine dehydrogenases are found in various microorganisms. 2-4 In the higher plants, amine oxidases that effect the oxidation of polyamines by the above mechanisms have been found only in the Gramineae (the grasses)5-13; they have not yet been detected in animals. TM 1 The structure is s h o w n as a derivative of A2-pyrroline, although the position of the double bond is not yet established. 2 R. H. W e a v e r and E. J. Herbst, J. Biol. Chem. 231, 647 (1958). 3 K. Isobe, Y. Tani, and H. Y a m a d a , Agric. Biol. Chem. 45, 727 (1981). 4 C. W. Tabor and P. D. Kellogg, J. Biol. Chem. 245, 5424 (1970). 5 T. A. Smith, Biochem. Biophys. Res. Commun. 41, 1452 (1970). 6 T. A. Smith, Phytochemistry 11, 899 (1972). 7 T. A. Smith, Phytochemistry 13, 1075 (1974). 8 T. A. Smith and D. A. Bickley, Phytochemistry 13, 2437 (1974). 9 T. A. Smith, Phytochemistry 15, 633 (1976). ~0 T. A. Smith, Phytochemistry 16, 1647 (1977). u y . Suzuki and E. Hirasawa, Phytochemistry 12, 2863 (1973). i2 E. Hirasawa and Y. Suzuki, Phytochemistry 14, 99 (1975). 13 y . Suzuki and H. Yanagisawa, Plant Cell Physiol. 21, 1085 (1980). 14 See, however, this volume ~52] for a polyamine oxidase in animal tissues that converts spermidine and acetylspermidine to putrescine.
METHODS IN ENZYMOLOGY, VOL. 94
Copyright © 1983by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181994-9
312
AMINE OXIDASES AND DEHYDROGENASES
[53]
Preliminary work with oats, barley, and maize had indicated that the enzymes are associated with a particulate fraction, 6,9,11 though a more recent study, supporting an earlier observation, 8 demonstrates that this is an artifact of extraction and that this enzyme normally occurs in the cell wall. 15 The enzyme is particularly active in vascular tissue. 5,~5 Assay Methods There are several methods for the determination of this enzyme. Perhaps the least ambiguous depends on the estimation of oxygen consumption using a Clark electrode. 8 However, measurement of the hydrogen peroxide by a coupled peroxidase system7 is more sensitive, owing to the greater baseline stability. 8
Reagents Tris buffer, 0.1 M, pH 6.5 (correct at 30°), air equilibrated Guaiacol, 25 mM Peroxidase, Sigma, type II (200 purpurogallin units/mg), 1 mg/ml Spermidine, 25 mM Procedure. Buffer (2 ml), guaiacol (0.1 ml), peroxidase (0.1 ml), and the oat leaf polyamine oxidase preparation (0.2 ml) are placed in optically matched cylindrical spectrophotometer cells (1.2 × 12.5 cm). After preincubation for 2 rain in a water bath at 30° the tube is transferred to a spectrophotometer cuvette holder, also thermostatted at 30°. Spermidine (0.1 ml) is added, and the increase in absorbance at 470 nm is determined. The spectrophotometer output is fed into a logarithmic recorder (fullscale deflection = 0.2 absorbance unit). Activity is determined on extrapolation of the curve to zero time. Activity declines rapidly during the assay, especially with spermine as substrate, l0 Spermidine is therefore to be preferred in the assay. The use of fluorogenic peroxidase substrates 16 should allow a considerable increase in sensitivity. Purification Procedure 9
Material. Oats (Arena sativa L., cv 'Black Supreme') are grown in the dark for 3 weeks at 22° in sand culture in a medium containing 2 mM K2SO4, 3 mM MgSO4, 8 mM CaCI2, 4 mM NaHzPO4, and 12 mM NaNO3 together with Fe (as FeEDTA) 5.6 ppm, Mn 0.6 ppm, Cu 0.06 ppm, Zn 0.07 ppm, Mo 0.05 ppm, and B 0.7 ppm. 15 R. K a u r - S a w h n e y , H. E. Flores, and A. W. Galston, Plant Physiol. 68, 494 (1981). 16 K. Zaitsu and Y. Ohkura, Anal. Biochem. 109, 109 (1980).
[53]
POLYAMINE OXIDASE (OAT SEEDLINGS)
313
TABLE I PURIFICATION OF POLYAMINE OXIDASE FROM OAT LEAVES9
Step Aqueous extractiona pH 4 precipitation Acetone precipitation Gel filtration
Total Volume protein (ml) (mg) 550 22 2.4 66
800 34 2.9 0.22
Total activity (nkat? 5600 4740 4580 1340
Specific activity (nkat/mg Purification Yield protein) (fold) (%) 6.8 140 1620 6020
1 20 240 890
(100) 85 82 24
a The extract was prepared from 150 g of 3-week-old dark-grown shoots. b The enzyme was assayed by the peroxidase-guaiacol method at pH 6 with spermine as substrate.
Extraction. The shoots are blended in 4 volumes of cold water, the macerate is squeezed through muslin, and the residue is reextracted with 2 volumes of cold water. p H 4 Precipitation. The combined substrates are adjusted to pH 4 with saturated citric acid. The precipitate is collected by centrifuging at 2500 g for 15 min and extracted in 0.1 M pH 6 citrate buffer containing 1 M NaC1. On recentrifuging at 3000 g for 15 min the precipitate is discarded. Acetone Precipitation. The supernatant is cooled to 0 °, and 1 volume of acetone at - 1 5 ° is added. The precipitate collected by centrifugation is extracted with 1 M NaCI in 0.1 M p H 6 citrate buffer; on recentrifugation, the precipitate is discarded. Gel Filtration. The supernatant is applied to a column of Sephadex G-100 (3.5 × 87 cm) equilibrated with 1 M NaC1 in 0.1 M pH 6 citrate buffer. Fractions (8 ml each at 24 ml/hr) eluted in the same buffer are collected; the active band is pooled and concentrated by Millipore filtration (UM-10 filter). On polyacrylamide gel electrophoresis the final product migrates as a single band at pH 4.3 toward the anode, and enzyme activity is coincident with the major protein band.l° A typical purification is summarized in Table I. Properties of the Oat L e a f E n z y m e
Activators and Inhibitors. Oxidation of the polyamines is strongly inhibited by NHz(CHzh0NH(CH2)3NH2, a spermidine homolog. 1° The fungicide, guazatine, [NH2C(-m-NH)NH(CH2)8]2NH (KenoGard AB, Stockholm, Sweden), is also a powerful inhibitor (Ki ca 10 -8 M). The
314
[54]
AMINE OXIDASES AND DEHYDROGENASES T A B L E II COMPARISON OF THE PROPERTIES OF OAT, BARLEY, AND MAIZE SEEDLING POLYAMINE OXIDASES
Property Molecular weight Relative activity, spermine vs spermidine" pH Optimum for spermidine pH Optimum for spermine Cofactors Maximum specific activity (nkat/mg protein) a
Oatsg.to
BarleyT.9
80,000 1
85,000 14
6.5 6.5 ND C 6020
Maize H t3 65,000 0.7
8.0 b 4.8 ND --
6.3 5.5 FAD 476
Activity at the pH optimum for the respective substrate.
b The enzyme is unstable at this pH. " ND, Not detected.
enzyme is activated by the chlorides of Li +, Na +, K +, and Rb + at 1 M in order of increasing effectiveness. 17 The Km values for spermidine and spermine, respectively, are 8 × 10 -6 M and 2 × 10 -6 M in air-saturated buffer. The Km values for oxygen are 1.83 x 10 -4 M and 0 8 5 x 10 -4 M with spermidine and spermine, respectively, as substrates.10 For the barley enzyme the Km for oxygen is relatively high, and oxygen in air-saturated buffer is limiting. 8 Properties of the polyamine oxidases from oat, barley, and maize seedlings are compared in Table II. Stability. In intact oat leaves, loss of activity at - 15° is 5% per week; for the purified enzyme, loss of activity at - 1 5 ° is 10% per week. 1° 17 T. A. Smith and P. E. Gay, unpublished (1981).
[54] P u r i f i c a t i o n o f B o v i n e P l a s m a A m i n e O x i d a s e
By B.
MONDOVI,
P.
TURINI, O. BEFANI,
and S. SABATINI
Bovine plasma amine oxidase is a CuE+-containing enzyme that catalyzes the oxidation of primary amines I as follows: R - - C H 2 - - N H 3 + + 02 + H20 --~ R C H O + H,,O2 + NH4 ~
E. A. Zeller, Helv. Chim. Acta 21, 880 (1938).
METHODS IN ENZYMOLOGY, VOL. 94
Copyright© 1983by AcademicPress, Inc. All rightsof reproductionin any formreserved. ISBN 0-12-181994-9