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Plant Physiol. (1998) 117: 1363-1371 Characterization of Euphorbia characias Latex Amine Oxidase1
Department of Biochemistry and Human Physiology, University of Cagliari, Cagliari, Italy (A.P., R.M., A.L., B.M., G.F.); Department of Chemistry, Odense University, Odense, Denmark (J.Z.P.); and Department of Experimental Medicine, University of Rome "Tor Vergata," Rome, Italy (A.F.A.)
A copper-containing amine oxidase
from the latex of Euphorbia characias was purified to
homogeneity and the copper-free enzyme obtained by a ligand-exchange
procedure. The interactions of highly purified apo- and holoenzyme with
several substrates, carbonyl reagents, and copper ligands were
investigated by optical spectroscopy under both aerobic and anaerobic
conditions. The extinction coefficients at 278 and 490 nm were
determined as 3.78 × 105 M
Copper-amine oxidases (amine oxygen oxidoreductase deaminating,
copper containing; EC 1.4.3.6) are widespread enzymes oxidizing primary
amines with the formation of the corresponding aldehyde, ammonia, and
hydrogen peroxide:
The catalytic mechanism of amine oxidases has been reported previously
(Medda et al., 1995b An amine oxidase from the latex of Euphorbia characias, a
perennial Mediterranean shrub, has been purified, but only a
preliminary characterization of this enzyme was reported (Rinaldi et
al., 1982
Reagents Putrescine, benzylamine hydrochloride, DABA hydrochloride, kynuramine dihydrobromide, BHY dihydrochloride, 4-hydroxyquinoline, SCA hydrochloride, and diethyldithiocarbamate were purchased from Sigma and used without further purification. TNM was from Aldrich, PHY hydrochloride was from Across (Geel, Belgium) and 4-(dimethylamino)benzaldehyde was from Fluka. All other chemicals were obtained as pure commercial products and used without further purification.Purification of ELAO The purification procedure (Table I) essentially follows the method described elsewhere (Rinaldi et al., 1982 ) with minor modifications as follows.
Steps 1 through 4 The eluate from a DEAE-cellulose column (see Table I) was made 70% saturated with solid ammonium sulfate with constant stirring at 4°C for 30 min and centrifuged at 9000 rpm for 30 min. The precipitate was dissolved in 30 mL of 1 mM KPi buffer, pH 7.0, and dialyzed against 10 L of the same buffer at 4°C for 12 h. The insoluble material was removed by centrifugation.Step 5 The supernatant from fraction 5 was loaded onto a column (2 × 8 cm) of hydroxyapatite equilibrated with 1 mM KPi buffer, pH 7.0. The column was washed with the same buffer until the A280 became 0.01. The ELAO was then
eluted with 50 mM KPi buffer, pH 7.0. The fractions showing
enzymatic activity were pooled.
Step 6 The material resulting from step 6 was loaded onto a column (2 × 10 cm) of -aminohexyl-Sepharose 4B equilibrated with 50 mM KPi buffer, pH 7.0. The column was washed with the same
buffer until the A280 became 0.01. The
ELAO was then eluted with 100 mM KPi buffer, pH 7.0. The
fractions showing enzymatic activity were pooled and concentrated by
filtration under a vacuum.
Copper-Free Enzyme Copper-free ELAO was prepared as follows: ELAO solution (10 mg/mL) was dialyzed against double-distilled water containing 1 mM sodium cyanide at 4°C for 6 h. The dialyzed solution was brought to 10 mM in sodium diethyldithiocarbamate and allowed to stand at 4°C for 6 h, then centrifuged at 105,000g for 2 h. The supernatant was dialyzed against double-distilled water and then centrifuged at 15,000 rpm for 30 min. The pink supernatant was stored at 20°C when not used immediately.
Determination of Metals Copper and manganese contents were determined by atomic absorption using an IL 951 atomic absorption spectrometer Instrumentation Laboratory, Wilmington, DE). The spectral line chosen was 324.7 nm for copper and 285.2 nm for manganese.Protein Concentration Protein concentrations were measured by the Lowry method, as modified by Hartree (1972), with BSA as a standard.
Analytical PAGE and Determination of Relative Molecular Mass Electrophoresis under nondenaturing conditions was performed as described previously (Gabriel, 1971). Continuous SDS-PAGE was carried
out according to the method of Weber and Osborne (1969). Preparation of
protein samples for SDS-PAGE was by two methods. First, the protein
samples were incubated for 48 h at 4°C with 0.2% SDS and 100 mM 2-mercaptoethanol in 100 mM Tris-HCl, pH
8.0. Second, the protein samples were heated at 100°C for 5 min in 10 mM NaPi buffer, pH 7.0, containing 1% SDS and 100 mM 2-mercaptoethanol. No difference was observed with the
two methods. For molecular mass determination the standards used were
myosin (205 kD),  -galactosidase (116 kD), phosphorylase B (97.4 kD),
BSA (66 kD), ovalbumin (43 kD), and carbonic anhydrase (31 kD) as high
standards; and phosphorylase B (97.4 kD), BSA (66 kD), ovalbumin (43 kD), carbonic anhydrase (31 kD), and trypsin inhibitor (20.1 kD) as low
standards.
Spectrophotometric Methods Absorption spectra were recorded at 25°C with a Cary 2300 spectrophotometer (Varian, Victoria, Australia). Anaerobic experiments were made after several cycles of vacuum followed by flushing with O2-free argon at 25°C in a Thunberg-type spectrophotometer cuvette (Vetroscientifica, Rome, Italy) in which anaerobic additions of various reagents could be made through a rubber cap with a syringe. Fluorescence spectra were obtained using a Perkin-Elmer LS-3 spectrofluorimeter.Determination of TPQ Stoichiometry Native ELAO was titrated anaerobically in 100 mM KPi buffer, pH 7.0, at 25°C with putrescine, benzylamine, DABA, and kynuramine as the substrates. The aromatic aldehyde production from benzylamine was measured by the A250 increase using an 250 of 12,800 M 1 cm1
(Neumann et al., 1975). The aldehyde production from DABA was measured by the A350 increase using an
350 of 31,200 M1 cm1
(Ehrlich's reagent; Medda et al., 1995b). The reaction with kynuramine was followed photometrically at 326 and 314 nm (formation of
4-hydroxyquinoline) or at 356 and 255 nm (disappearance of kynuramine;
Padiglia et al., 1997). The amount of reaction product formed was
calculated as the mean of at least three different measurements. ELAO
was also titrated with carbonyl-containing compounds in the presence and absence of oxygen. Titrations were performed by stepwise addition of 5 µL of freshly prepared reagent, and the optical density changes were measured at the appropriate wavelengths (see ``'') on a
Cary 2300 spectrophotometer at 25°C. At each addition the optical
density of the reaction mixture was checked at the appropriate time
(see ``'') and aliquots of the reaction mixture were tested
for activity. The activity of the enzyme was measured polarographically
with an oxygraph (Gilson Medical Electronics, Middleton, WI) at
37°C. The incubation mixture (1 mL) contained 2 µg of purified ELAO in 100 mM KPi buffer, pH 7.0. The reaction was started by
the addition of 50 µL of putrescine solution (17 mM)
after at least 10 min of preincubation.
Carbanion Detection Reaction of TNM with enzyme alone or with enzyme and putrescine as the substrates was monitored by the time-dependent increase in A350 (Medda et al., 1993). The reaction was
initiated by adding the substrates to a reaction mixture (1 mL)
containing TNM and known quantities of ELAO. The rates of nitroform
production reported are initial rates and were determined by the slope
of the tangent to the absorbance versus time curve 6 to 8 s after
addition of the substrate.
Preparation of Genomic DNA from Leaf Tissue Frozen Euphorbia characias leaf tissue was completely powdered using a pestle-and-mortar grinding method. DNA was isolated using a plant DNA-isolation kit (Boehringer Mannheim), according to the manufacturer's instructions.
Cycle Sequencing of Purified Fragment DNA-sequence analysis of fragments was performed with the Digoxigenin Taq DNA Sequencing Kit (Boehringer Mannheim). Forward and reverse nonradioactive digoxigenin-sequencing primers labeled with digoxigenin at the 5 end were synthesized with
the same sequences as the PCR primers. Cycling conditions were the same as those used in PCR amplification. After the sequencing reaction DNA
fragments were separated on the sequencing gel. As they reached the
bottom of the gel, the fragments were transferred directly to a nylon
membrane (Sartorius, Edgewood, NY). The transfer was completed after
approximately 8 to 10 h, and the cross-linking of the transferred
DNA was done using a standard UV-light transilluminator for about 3 min. Colorimetric detection of the blotted sequencing ladders was
performed using a Digoxigenin Nucleic Acid Detection Kit (Boehringer
Mannheim) with additional required reagents. The color precipitate
started to form within a few minutes and the reaction was usually
complete after 16 h. The reaction was stopped by washing the
membrane for 5 min with 10 mM Tris-HCl buffer, pH 8.0, containing 1 mM EDTA.
Molecular Properties The enzyme preparations used in our studies were assayed for copper content by atomic absorption spectrometry. The purified enzyme contained 0.090% (w/w) of copper. On this basis, a minimum Mr of 70,000 can be calculated. Because the crystal structure of PSAO (Kumar et al., 1996) revealed a second site
with a metal tentatively identified as manganese, we looked for this
metal with atomic absorption spectrometry, but none was detected.
Relative Molecular Mass Determination The purified ELAO showed a single symmetrical peak of molecular mass at 145 ± 2 kD in gel-filtration chromatography (results not shown), and a single band in SDS-PAGE (Fig. 1). The molecular mass of the ELAO monomer as determined by SDS-PAGE was found to be 72 ± 1 kD (Fig. 1). These results are in good agreement with the preliminary data reported (Rinaldi et al., 1982).
Reaction of ELAO with TNM ELAO catalyzes the reaction of putrescine with TNM, giving rise to the nitroform anion. No production of nitroform was seen during 2 to 3 min of preincubation of LSAO with 250 µM TNM at 25°C in 10 mM KPi buffer, pH 7.0. Moreover, the rate of nitroform production was negligible in an enzyme-free reaction mixture containing TNM, putrescine, ammonia, hydrogen peroxide, and aldehyde product, indicating that the nitroform generation occurs only in the reaction of TNM with an ELAO-substrate complex. Therefore, no correction of the rates was required. The initial rates for nitroform production were linearly related to the concentration of active sites of ELAO present in each assay (not shown). The amount of nitroform that accumulated in 10 min was 14 to 20 times the content of enzyme active sites, consistent with the requirement of enzyme turnover to release nitroform. The increase at 350 nm was immediate when putrescine was added. A double-reciprocal plot of the data A350 min1/TNM
concentration at saturating concentration of putrescine was linear. The
Km value obtained was 1.25 mM,
whereas the maximal rate of nitroform production was 11.4 nmol
min1.
Spectroscopic Properties and Titration of ELAO In addition to the protein absorbance maximum at 278 nm, the enzyme showed a maximum at 490 nm in the visible region.Reaction of ELAO with Hydrazines Chemical modification of ELAO was made by addition of PHY or BHY to enzyme solutions (1 mL) in 100 mM KPi buffer, pH 7.0, both in air and anaerobically. The addition of PHY or BHY to ELAO was followed by the formation of strong absorption bands at 430 nm (430 = 55,000 M1 cm1) and
380 nm (380 = 60,000 M1 cm1),
respectively. Both derivatives were nonfluorescent. The reaction of
ELAO with either PHY or BHY was irreversible. Formation of the
BHY-modified enzyme was concomitant with the disappearance of the
absorption band at 490 nm, and a clean isosbestic point at 460 nm was
observed upon titration of ELAO with BHY (Fig.
2). Analogous changes could be obtained
upon titration with PHY, with an isosbestic point observed at 514 nm
(data not shown). A titration end point was obtained at a ratio
BHY:ELAO dimer (Fig. 2, inset) or PHY:ELAO dimer (Fig.
3A) of 2. The spectral changes of ELAO with PHY and BHY were also studied as a function of time. The reaction
was very fast for both PHY and BHY, and the end point for maximum
spectral change at high amounts of inhibitor was 10 min.
Reaction of ELAO with SCA The addition of SCA to the incubation mixture was followed by the formation of two absorption bands at 480 nm (480 = 5,000 M1 cm1) and
345 nm (345 = 23,000 M1 cm1).
The formation of these new absorption bands was always concomitant with
the disappearance of the absorption band at 490 nm. The reaction was
very fast and the total time required for maximum spectral change with
increasing amount of SCA was 10 min. Because of the irreversibility of
the reaction, it was possible to titrate ELAO with SCA (Fig. 3B). An
end point at a ratio SCA:ELAO dimer of 2 was always obtained.
Reaction of ELAO with Putrescine Putrescine is the best substrate for ELAO. In 100 mM KPi buffer, pH 7.0, the turnover number was 2300 min1. When putrescine was added to ELAO in
anaerobiosis, the broad absorption band at 490 nm disappeared
instantaneously, indicating the rapid conversion of the TPQ cofactor to
a bleached species, presumably the quinolaldimine. Then the solution
turned yellow as a result of the formation of new absorption bands
centered at 430 and 460 nm, indicative of a free radical intermediate
species generated upon reaction in the absence of oxygen (Finazzi
Agró et al., 1984; Dooley et al., 1987; Medda et al., 1995b).
Precisely 20 nmol of substrate was required to completely reduce 10 nmol of ELAO (results not shown).
Reaction of ELAO with Benzylamine Benzylamine is the best substrate for benzylamine oxidase from pig or beef plasma (Janes and Klinman, 1991; Morpurgo et al., 1991). Also,
ELAO was able to oxidize benzylamine, with a
Km value of 0.4 mM, which is
similar to that found for putrescine (Rinaldi et al., 1982). The
turnover number at saturating benzylamine concentrations was found to
be 10.5 min1. The anaerobic titration of 10 nmol of ELAO with benzylamine required 20 nmol of substrate (not
shown), and the formation of 1.98 ± 0.2 mol of benzaldehyde per
mol of enzyme dimer was directly observable by the increase in
A250.
Oxidation of DABA DABA was a poor substrate for ELAO. The turnover number with DABA was 0.028 min1, a small value compared with the
corresponding number of 2300 min1 found for
putrescine. However, when DABA was added to ELAO in anaerobiosis, the
broad absorption band at 490 nm disappeared instantaneously. Together
with the formation of the quinolaldimine, a new band centered at 400 nm
appeared. This band was assigned to the protonated tautomeric form of
the quinolaldimine, as reported for DABA oxidation by LSAO (Medda et
al., 1995b). Under anaerobic conditions this species decayed slowly, in
parallel with formation of the yellow radical intermediate (Fig.
4). These spectral changes occurred
simultaneously with the liberation of 1.98 ± 0.2 mol of the
corresponding aldehyde directly observable by the increase in
A350. In the presence of air, the radical
species reacted rapidly with oxygen to restore the oxidized enzyme and
to liberate ammonia and hydrogen peroxide (data not shown).
Oxidation of Kynuramine Kynuramine is an endogenous amine that is formed from tryptamine through indole 2,3-dioxygenase and formamidase, or from decarboxylation of kynurenine (Johnson and Clarke, 1981). Kynuramine is an excellent substrate used in assays for the measurement of both types A and B of
monoamine oxidase activity. In fact, this compound is converted by
monoamine oxidase to an aldehyde that spontaneously rearranges to
4-hydroxyquinoline, which fluoresces strongly in alkaline solutions (Morinan and Garratt, 1985). Kynuramine is also a substrate for pig
plasma copper amine oxidase and its fluorescence was used to probe the
catalytic site of this enzyme (Massey and Churchich, 1977). However,
kynuramine behaves as a competitive inhibitor of the SCA-sensitive
copper amine oxidases (Elliott et al., 1988). ELAO was able to oxidize
kynuramine to 4-hydroxyquinoline. At pH 7.0, in 100 mM KPi
buffer, the turnover number for kynuramine was 0.24 min1. At the same pH value the substrate
inhibition was apparent at lower concentrations than when the substrate
was putrescine (2 mM for kynuramine and 17 mM
for putrescine; data not shown).
The Radical Species
The Effect of Cyanide Cyanide is known to trap the semiquinone form by stabilizing Cu(I), and the yield of the radical increases in Arthrobacter P1 and porcine kidney amine oxidases by addition of cyanide (McGuirl et al., 1997). When cyanide (2 mM) was added to anaerobically reduced ELAO using
benzylamine as the substrate, the spectrum of the radical form showed a
70% increase in intensity.
The Effect of Azide Azide is known to bind to Cu(II), generating a Cu(II)-N3 complex that absorbs
at 400 nm. In 100 mM KPi buffer, pH 7.0, and at 20°C, the
Keq of the Cu(II)-azide complex for
oxidized ELAO was determined to be 34 ± 5 M1. When ELAO was first
reduced by 2 mM benzylamine to form a mixture of
Cu(I)-semiquinolamine and Cu(II)-aminoresorcinol, and then azide was
added to the sample, the semiquinolamine radical spectrum decreased,
probably because of conversion to the aminoresorcinol form (results not
shown). However, in ELAO, as in other amine oxidases (McGuirl et al.,
1997), the reduction of TPQ cofactor by the substrate had no effect on
the magnitude of Keq for azide binding to
Cu2+.
Temperature Effect The Cu(I)-semiquinolamine radical form of ELAO was monitored as a function of temperature. As reported for the pea enzyme (Turowski et al., 1993), the concentration of the yellow species increased with the
temperature in the range 10°C to 30°C and was constant from 30°C
to 40°C, the highest temperature investigated (results not shown).
Reaction of Copper-Free ELAO Copper could be removed from native ELAO by treatment with cyanide and diethyldithiocarbamate (see ``Materials and Methods''). The residual copper, measured by atomic absorption spectroscopy, was 0.2 ± 0.2% of the original content. Copper-free ELAO showed a broad absorption peak at 480 nm (Fig. 6, inset), shifted toward shorter wavelengths with respect to the native enzyme, but with similar intensity (480 = 6000 M1 cm1).
The addition of DABA to the copper-free enzyme caused the formation of
the peak at 400 nm, which later decayed in parallel with the release of
aldehyde. Within the experimental error the absorption changes observed
at 350 and 400 nm were identical to those seen for the holoenzyme in
anaerobiosis. However, the complete absence of bands at 434 and 464 nm
showed that no formation of the free radical intermediate occurred
(results not shown).
PCR Amplification and Sequencing Reactions Alignment of amino acid sequences of all known amine oxidases shows that the homology is in the range of 20% to 99% and that the important residues determining the enzyme activity are conserved. The homology is high for amine oxidases obtained from related organisms, as for LSAO and PSAO (>92%), but becomes very low when comparing plant and mammal enzymes (25%; Frébort and Adachi, 1995). Comparison
of the amino acid sequence around the TPQ cofactor in several amine
oxidases obtained from either cDNA or direct sequencing of the protein
shows a much higher homology for the C than for the N terminus. TPQ
bound in the typical sequence NyD/E is located somewhere in the middle.
The motif HXH (X = Q, D, or T), found in the amine oxidases
located at the positions around 40 to 50 residues from the TPQ position
toward the C terminus, is conserved in all amine oxidases (with a
single exception, the amine oxidase from Aspergillus niger).
Moreover, two conserved histidyl residues were found in all enzymes 30 residues from TPQ, one toward the N terminus and one near the C
terminus. It was shown that three histidyl residues, two of them in the
HXH motif and one at the C terminus, are ligands of copper. The DNA
sequence of the ELAO active site and the amino acid sequence shown in
Figure 7 are very similar to the
corresponding sequences of PSAO and LSAO, in which the position of TPQ
has been demonstrated to be the Tyr located between an Asn and an Asp.
Therefore, we propose that in ELAO this tyrosinyl residue can be
modified to TPQ in the mature protein.
ELAO has been prepared to homogeneity by a new method of
purification. Highly purified ELAO has a specific activity of 34 µmol
substrate min
* Corresponding author; e-mail florisg{at}vaxca1.unica.it; fax 39-070-675-4524. Received February 5, 1998;
accepted May 12, 1998.
Abbreviations:
BHY, benzylhydrazine.
DABA, p-(dimethylamino)benzylamine.
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  M. G. Kocsis and A. D. Hanson Biochemical Evidence for Two Novel Enzymes in the Biosynthesis of 3-Dimethylsulfoniopropionate in Spartina alterniflora Plant Physiology, July 1, 2000; 123(3): 1153 - 1162. [Abstract] [Full Text]
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Abstract
Introduction
X174
HaeIII digest as a molecular mass marker (Sigma). In a large
number of reactions a clear band was observed that corresponded
approximately to a 200-bp product; this product was purified from 1%
agarose gel using the QIAquick gel-extraction kit protocol (Qiagen,
Chatsworth, CA).
) and 5-µL portions of the reaction mixture were taken
simultaneously for activity assay (
).
), and 350 nm (
-adrenoceptor inhibitory action of kynuramine.
Eur J Pharmacol
72:
351-356