Pergamon 0031-9422(94)00612-1 Phytochemistry, Vol. 38, No. 1, pp. 23 25, 1995 Copyright © 1995 Elsevi©r Science Ltd Printed in Great Britain. All rights reserved 0031-9422/95 $9.50 + 0.00 AMINO OXIDASE FROM TRIGONELLA FOENUM-GRAECUM SEEDLINGS LENKA LUHOVA, LUDI~KSLAViK, IVO FRI~BORT,* MARTIN HALATAt and PAVEL PE(~ Department of Analytical and Organic Chemistry, Faculty of Science, Palack~ University, TL Svobody 8, 771 46 Olomouc, Czech Republic; *Laboratory of Applied Microbiology, Faculty of Agriculture, Yamaguchi University, 1677-1 Yoshida, 753 Yamaguchi, Japan; tMunicipal Hygienic Station, Wolkerova 6, 772 00 Olomouc, Czech Republic (Received in revisedform 27 June 1994) Key Word Index--Trigonellafoenum-graecum; Fabaceae; fenugreek; amine oxidase; protein purifica- tion; substrate specificity and inhibitors. Al~trmct--Amine oxidase was isolated from eight-day-old Tri#onella foenum-graecum seedlings grown in the dark, using rivanol lactate and ammonium sulphate fractionations, heat denaturation and hydroxylapatite chromato- graphy. The yellow enzyme is a monomer M, of 80 000 with pI 6.8, Area x at 280 nm and a shoulder at 415 nm. Substrate specificity and inhibitor interactions were similar to those of pea amine oxidase. The N-terminal of the enzyme was blocked and no reaction was observed with antibody against amine oxidase either from Escherichia coli or Micrococcus luteus. INTRODUCTION Amine oxidase (EC 1.4.3.6) [amine: 0 2 oxidoreductase (deaminating, copper containing)], has been found in microorganisms, and various plants and animals [1]. The enzyme is widespread in the Fabaceae. Amine oxidase catalyses the oxidative deamination of amines, producing the corresponding aldehyde, H20 2 and ammonia. Cop- per and a carbonyl cofactor, recently identified as the quinone 6-hydroxydopa (topa) [2, 3], mediate substrate reaction following a ping-pong mechanism [4]. In plants, amine oxidase acts in various physiological processes in connection with polyamine degradation [5]. We have now isolated and characterized amine oxidase from Tri- gonella foenum-graecum (fenugreek). RESULTS AND DISCUSSION Amine oxidase (T-AO) activity was followed in extracts from fenugreek grown in continuous light or in the dark during the first 16 days. Enzyme activity appeared soon after seed swelling and rose sharply until six-eight days of cultivation, mainly in the cotyledon. Higher activity was found in etiolated plants than in those cultivated in light. For amine oxidase purification, eight-day-old etiolated germinating seeds were found to be optimum. The purification procedure is based on the method applied for pea amine oxidase (P-AO) [6]; rivanol lactate and ammonium sulphate fractionation, heat denatur- ation of inert proteins and hydroxylapatite chromato- graphy. The summary of the purification procedure is presented in Table 1. SDS-PAGE showed a single band corresponding to M, 80 000 and native PAGE showed an :~Author to whom correspondence should be addressed. 23 activity band corresponding to a single protein band for purified T-AO. Since gel HPLC on TSK gel showed a single peak corresponding also to M, of 80000, the monomeric structure of T-AO was confirmed. SDS- PAGE purified enzyme was subjected to Edman degrada- tion after blotting to PVDF membrane, but the N- terminal sequencing of the enzyme was not possible probably due to blocking. The purified enzyme was yellow having Amazat 280 nm, and a shoulder at 415 nm. Copper content was ca 1 atom mole 1. The enzyme was active toward 1,4-diaminobutane (putrescine) with Km 0.33 mM and 1,5-diaminopentane (cadaverine) with Km 0.11 mM. The activity of the enzyme was measured over a pH range of 4-8; the optimum was ca 7. T-AO is inhibited, as in other Fabaceae amine oxi- dases, by substrate analogues (1,5-diamino-3-pentanone, E- and Z-l,4-diamino-2-butene), Cu chelating agents (o- phenanthroline, 2,2'-bipyridyl), carbonyl reagents (amino- guanidine, phenylhydrazine) and some alkaloids (quini- dine and L-lobeline). Inhibition by E- and Z-1,4-diamino- 2-butene was determined as inhibition by an excess of the substrate. Surprisingly, spectral changes of amine oxi- dases giving a new Amax at 420-440 nm due to the formation of a Schiffbase with the carbonyl cofactor were not observed in the reaction of T-AO with phenylhydra- zine or p-nitrophenylhydrazine, although they showed total inhibition. Also, inhibition was relatively slow com- pared with P-AO, especially when p-nitrophenylhydra- zine was used. Since phenylhydrazine can also inhibit flavine containing bovine kidney mitochondrial amine oxidase (EC 1.4.3.4) I-7] bleaching Area x at 455, pargyline as a typical irreversible inhibitor of flavine containing amine oxidase was tested. Pargyline causes a new Amax at 410 nm with flavine inhibiting bovine kidney mitochon-