BIOCHEMICAL SOCIETY TRANSACTIONS
74
in the biosynthesis of lignin (Stafford, 1974), in which hydroxylation of p-coumaric acid to caffeic acid is an important step, we have studied hydroxylation of pcoumaric acid by the peroxidase-dihydroxyfumarate system. Table 1 shows that this hydroxylation is completely inhibited by catalase. It is also stopped by superoxide dismutase and by the metal ions Cu2+ and Mn2+, which can react with the superoxide radical (Oz-) under these conditions (Rabani et al., 1973; Lumsden & Hall, 1975). Oxidation of dihydroxyfumarate in these reaction mixtures was severely inhibited by catalase (in agreement with the results of Swedin & Theorell, 1940), but only slightly inhibited by Cuz+ or superoxide dismutase and actually stimulated by Mn2+. It is therefore clear that, except for catalase, inhibition of hydroxylation by these reagents could not be attributed to an inhibition of dihydroxyfumarate oxidation. Scavengers of the hydroxyl radical ('OH), such as formate, mannitol, ethanol and Tris (Fridovich, 1974; Paschen & Weser, 1975) also inhibited hydroxylation much more effectively than they inhibited dihydroxyfumarate oxidation (Table 1 ) . The initial stage of the oxidation of dihydroxyfumarate in this system is a simple peroxidase reaction which requires H20z and so is inhibited by catalase (Yamazaki & Piette, 1963). The effects of superoxide dismutase and Cu2+ indicate that 02'-also plays a small role in this oxidation. This 02'-appears to be essential for the hydroxylation of p-coumaric acid ;hence the complete inhibition by superoxide to give Mn3+(eqn. 1): dismutase, Mn2+and Cu2+.Mn2+ reacts with 4'MnZ+ Oz'- 2H+ .+ Mn3+ H20z (1) Mn3+ increases the rate of dihydroxyfumarate oxidation but probably changes the mechanism of this reaction (see Yamazaki & Piette, 1963). Oz'- breaks down to give H202(eqn. 2 ) which can react with a further molecule of Oz'- to give 'OH (Fridovich, 1974: eqn. 3) 02*-+02*-+2H+ + HzOz+Oz (2) HzOz 0 2 ' - + 0 2 OH- 'OH (3) 'OH readily hydroxylates aromatic compounds such as p-coumaric acid (4hydroxycinnamic acid). The inhibition of hydroxylation in this system by scavengers of 'OH (Table 1) suggests that formation of *OH from 0 2 ' - is responsible for at least part of the caffeic acid formation. Buhler, D. R. & Mason, H. S. (1961) Arch. Biochem. Biophys. 92,424437 Chance, B. (1952) J. Biol. Chem. 197, 577-589 Fridovich, I. (1974) Adv. Enzymol. Relat. Areas Mol. Biol. 41, 35-97 Halliwell, B. (1975) FEBS Lett. 56, 34-38 Lumsden, J. & Hall, D. 0. (1975) Biochem. Biophys. Res. Commun. 64, 595-602 McCord, J. M. & Fridovich, I. (1969) J. Biol. Chem. 244,6049-6055 Paschen, W.& Weser, U. (1975) Hoppe-Seyler's Z. Physiol. Chern. 356,727-737 Rabani, J., mug-Roth, D. & Lilie, J. (1973) J. Phys. Chem. 77, 1169-1175 Stafford, H. A. (1974) Annu. Rev. Plant Physiol. 25,459486 Swedin, B. & Theorell, H. (1940) Nature (London) 14!5,71-72 Yamazaki, I. & Piette, L. H. (1963) Biochim. Biophys. Acta 7 7 , 4 7 4
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Polyamine Oxidase from the Leaves of Cereals TERENCE A. SMITH Long Ashton Research Station (University of Bristol), Long Ashton, Bristol BS18 9AF, U.K. and WILLIAM L. HAYGARTH and JANET F. WILLIAMS* School of Biological Sciences, University of Bath, Claverton Down, Bath BA2 7 A Y, U.K.
The polyamines spermine (NH2[CHz13NH[CH214NH[CH2]3NH2) and spermidine [NH2(CH2)3NH(CHz)rNH2] have been reported in many animals, plants and micro* Present address: School of Education, University of Exeter, Exeter, Devon, U.K. 1976
e
74
in the biosynthesis of lignin (Stafford, 1974), in which hydroxylation of p-coumaric acid to caffeic acid is an important step, we have studied hydroxylation of pcoumaric acid by the peroxidase-dihydroxyfumarate system. Table 1 shows that this hydroxylation is completely inhibited by catalase. It is also stopped by superoxide dismutase and by the metal ions Cu2+ and Mn2+, which can react with the superoxide radical (Oz-) under these conditions (Rabani et al., 1973; Lumsden & Hall, 1975). Oxidation of dihydroxyfumarate in these reaction mixtures was severely inhibited by catalase (in agreement with the results of Swedin & Theorell, 1940), but only slightly inhibited by Cuz+ or superoxide dismutase and actually stimulated by Mn2+. It is therefore clear that, except for catalase, inhibition of hydroxylation by these reagents could not be attributed to an inhibition of dihydroxyfumarate oxidation. Scavengers of the hydroxyl radical ('OH), such as formate, mannitol, ethanol and Tris (Fridovich, 1974; Paschen & Weser, 1975) also inhibited hydroxylation much more effectively than they inhibited dihydroxyfumarate oxidation (Table 1 ) . The initial stage of the oxidation of dihydroxyfumarate in this system is a simple peroxidase reaction which requires H20z and so is inhibited by catalase (Yamazaki & Piette, 1963). The effects of superoxide dismutase and Cu2+ indicate that 02'-also plays a small role in this oxidation. This 02'-appears to be essential for the hydroxylation of p-coumaric acid ;hence the complete inhibition by superoxide to give Mn3+(eqn. 1): dismutase, Mn2+and Cu2+.Mn2+ reacts with 4'MnZ+ Oz'- 2H+ .+ Mn3+ H20z (1) Mn3+ increases the rate of dihydroxyfumarate oxidation but probably changes the mechanism of this reaction (see Yamazaki & Piette, 1963). Oz'- breaks down to give H202(eqn. 2 ) which can react with a further molecule of Oz'- to give 'OH (Fridovich, 1974: eqn. 3) 02*-+02*-+2H+ + HzOz+Oz (2) HzOz 0 2 ' - + 0 2 OH- 'OH (3) 'OH readily hydroxylates aromatic compounds such as p-coumaric acid (4hydroxycinnamic acid). The inhibition of hydroxylation in this system by scavengers of 'OH (Table 1) suggests that formation of *OH from 0 2 ' - is responsible for at least part of the caffeic acid formation. Buhler, D. R. & Mason, H. S. (1961) Arch. Biochem. Biophys. 92,424437 Chance, B. (1952) J. Biol. Chem. 197, 577-589 Fridovich, I. (1974) Adv. Enzymol. Relat. Areas Mol. Biol. 41, 35-97 Halliwell, B. (1975) FEBS Lett. 56, 34-38 Lumsden, J. & Hall, D. 0. (1975) Biochem. Biophys. Res. Commun. 64, 595-602 McCord, J. M. & Fridovich, I. (1969) J. Biol. Chem. 244,6049-6055 Paschen, W.& Weser, U. (1975) Hoppe-Seyler's Z. Physiol. Chern. 356,727-737 Rabani, J., mug-Roth, D. & Lilie, J. (1973) J. Phys. Chem. 77, 1169-1175 Stafford, H. A. (1974) Annu. Rev. Plant Physiol. 25,459486 Swedin, B. & Theorell, H. (1940) Nature (London) 14!5,71-72 Yamazaki, I. & Piette, L. H. (1963) Biochim. Biophys. Acta 7 7 , 4 7 4
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+
+
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Polyamine Oxidase from the Leaves of Cereals TERENCE A. SMITH Long Ashton Research Station (University of Bristol), Long Ashton, Bristol BS18 9AF, U.K. and WILLIAM L. HAYGARTH and JANET F. WILLIAMS* School of Biological Sciences, University of Bath, Claverton Down, Bath BA2 7 A Y, U.K.
The polyamines spermine (NH2[CHz13NH[CH214NH[CH2]3NH2) and spermidine [NH2(CH2)3NH(CHz)rNH2] have been reported in many animals, plants and micro* Present address: School of Education, University of Exeter, Exeter, Devon, U.K. 1976
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