The Mechanism of Inhibition of Pig-Plasma Benzylamine Oxidase by the Copper-Chelating Reagent Cuprizone

Eur. J. Biochem. 48, 229 -236 (1974)

The Mechanism of Inhibition of Pig-Plasma Benzylamine Oxidase by the Copper-Chelating Reagent Cuprizone Anders LINDSTROM and Gosta PETTERSSON Avdelningen for Biokemi, Kemicentrum, Lunds Universitet

(Received March 3O/July 8, 1974)

1. Treatment of pig-plasma benzylamine oxidase with the copper-chelating reagent cuprizone leads to the reversible formation of an enzyme . cuprizone complex with a dissociation constant of about 3 mM, followed by an irreversible conversion of this complex into an enzyme species exhibiting a new absorption band centered around 350 nm. The latter enzyme species shows no reactivity towards substrate or carbonyl reagents such as phenylhydrazine. One mole cuprizone per mole protein is sufficient to complete the chromophoric changes and to inactive the enzyme completely.

2. Cuprizone reacts with free pyridoxal phosphate under formation of a reaction product showing maximum absorption at 350 nm in the visible region, and competes with phenylhydrazine for the protein-bound pyridoxal phosphate in benzylamine oxidase.

3. Cuprizone does not extract any significant amounts of copper from benzylamine oxidase, but affects electron paramagnetic resonance spectra of the enzyme and its phenylhydrazone derivative in a way suggesting a direct interaction with at least one of the two copper ions present in the protein. The latter interaction does not appear to be identical with those involved in the formation of the inactive enzyme species absorbing at 350 nm.

4. It is concluded that the inhibitory action of cuprizone cannot be attributed to its copper- chelating properties, but reflects an interaction with enzyme-bound pyridoxal phosphate through a mechanism analogous to that governing Schiff-base or hydrazone formation between enzyme and substrates or hydrazine derivatives.

Pig-plasma benzylamine oxidase, similarly to other plasma amine oxidases, contains firmly protein-bound copper and pyridoxal phosphate [l]. It appears well established that the function of pyridoxal phosphate in these enzymes is closely related to its ability to react with the amine substrates under Schiff-base formation [2,3], but the catalytic role of copper remains obscure. Evidence that copper is essential for enzyme activity has been obtained by the preparation of inactive copper-free proteins from pig [4] and beef [ 5 ] plasma amine oxidase, the activity of which could be partially restored by cupric copper. On the other hand, there is no evidence as to whether copper functions as a structural element or participates more directly in the mechanism of action of plasma amine oxidases.

Abbreviation. EPR, electron paramagnetic resonance. Enzyme. Benzylamine oxidase, monoamine oxidase or

monoamine : 0, oxidoreductase (deaminating) (EC 1.4.3.4).

Examination of the effect on enzyme activity of ligand-binding to copper while still being protein bound has provided valuable information with bearing on the mechanistic function of the metal in several copper-containing oxidases [6]. Although there is no direct evidence for the binding of external ligands to copper in amine oxidases from various sources, certain reagents known to have a high affinity for copper have been reported to inhibit such enzymes under much milder conditions than those required for copper extraction, cuprizone being the most efficient inhibitor of this category [l]. It was, therefore, considered of interest to examine the interaction between pig-plasma benzylamine oxidase and the latter copper-chelating reagent in detail by kinetic and spectrometric techniques. The data reported in the present investigation strongly indicate that cuprizone does bind to copper in benzylamine oxidase, but also provide evidence that the inhibitory effect of

Eur. J. Biochem. 48 (1974)

230 Inhibition of Benzylamine Oxidase by Cuprizone

cuprizone is due to interaction with enzyme-bound pyridoxal phosphate rather than with enzyme-bound copper.

EXPERIMENTAL PROCEDURE

Ma ter ials

The preparation of homogeneous benzylamine oxidase from pig plasma and methods for determination of protein concentration and assay of enzyme activity have been described previously [7]. Enzyme preparations used in the present investigation were at least 90 %pure according to specific activity determinations. The phenylhydrazone derivative of the enzyme was prepared by titration with phenylhydrazine [8], excess of the carbonyl reagent being removed by dialysis. Other chemicals used were commercial samples of highest available purity. Cuprizone (biscyclohexanone oxalyldihydrazone) was obtained from BDH Chemicals (Poole).

Methods

Unless otherwise stated, experiments reported in the present investigation were carried out at 25 "C in 0.1 M phosphate buffer, pH 7.0, saturated with air (0.25 f 0.005 mM oxygen). In some experiments the oxygen concentration was varied between 0.03 and 1.1 mM by the techniques described elsewhere [S], oxygen concentrations being determined polarographi- cally with a Clark electrode combined to an Esch- weiler Combi-Analysator U.

Optical absorption spectra were recorded with a Zeiss PM QII spectrophotometer. The time-course of the reaction between enzyme and cuprizone was followed at 350 nm using an Amico-Morrow stopped- flow system described in detail elsewhere [S]. Recorded transmittances were routinely converted into absorb- ances, and estimates of apparent first-order rate constants for the 350nm absorbance changes were determined by iterative non-linear regression analysis [9], preliminary parameter estimates being obtained by conventional graphical analysis. Second-order rate constants for the reaction between cuprizone and free pyridoxal phosphate were determined spectrophotometrically at 350 nm by techniques analogous to those described for the reaction between pyridoxal phosphate and various hydrazines [3].

Electron paramagnetic resonance spectra were recorded with a Varian E-3 spectrometer at 77 K and 9.13 GHz, using about 0.1 mM enzyme. Copper determinations were performed with a Unicam SP90 atomic absorption spectrophotometer.

RESULTS

Effect of Cuprizone on Absorption Spectra of Benzylamine Oxidase

Absorption spectra in the visible region recorded at different times after mixing pig-plasma benzylamine oxidase with a 3-fold excess of cuprizone are shown in Fig. 1, from which it can be seen that the interaction between enzyme and cuprizone leads to a slow decolorization of the enzymatic 470 nm chromophore and a simultaneous appearance of a new absorption band centered around 350 nm. No tran- sient or final absorbance changes due to the appearance or disappearance of other absorption bands than those at 350 nm and 470 nm could be detected. In particular, no significant increase in absorbance at 600 nm was observed even after an incubation time of 24 h. Since the free copper . cuprizone complex exhibits a strong absorption band centered around 600 nm [lo], the latter observation excludes that the above chromophoric changes are due to the removal of copper from the protein.

The spectral changes induced by the addition of cuprizone to benzylamine oxidase were found to be irreversible by dialysis. Determinations of the copper content of the enzyme after treatment with 5 mM cuprizone and subsequent dialysis, or after dialysis for 24 h against buffer solutions containing 5 mM cuprizone, established that no significant amounts of copper were extracted from the protein under such conditions (Table 1).

Kinetics of the Reaction between Cuprizone and Benzylamine Oxidase

Decolorization of the 470 nm chromophore in benzylamine oxidase by cuprizone was found to parallel the appearance of the 350nm absorption band over a wide range of initial enzyme and cuprizone concentrations tested. Detailed kinetic studies of the process responsible for these spectral changes were carried out at 350 nm, since the amplitude of the absorbance change at this wavelength greatly exceeded that observed at 470 nm (Fig. 1).

A typical oscilloscope trace for the 350 nm absorbance changes observed on mixing benzylamine oxidase with an excess of cuprizone is shown in Fig. 2. The same final absorbance level was reached at all cuprizone concentrations tested, and no lag phase could be detected at the early stage of the process. The appearance of the 350 nm chromophore was found to conform to apparent first-order kinetics for at least 90% of the reaction, and the linear double- reciprocal plot in Fig. 3 shows that apparent first-


A. Lindstrom and G. Pettersson 23 1

0.:

0.L

: 0.: m .D Lo D 4

0.

0.

I

Time ( m i n )

I I I

Wavelength (nrn)

Fig. 1. Effect of‘ cuprizone on the visible absorption spectrum of’ henzjdamine oxidase. Spectra recorded at different times after mixing 15 pM enzyme with 50 pM cuprizone in 0.1 M phosphate buffer, pH 7,25 ‘C

Table 1 . Copper content of benzjdamine oxidase after various treatments Data refer to two different enzyme preparations (I and 11). Dialysis and incubations were performed using 0.1 M phosphate buffer, pH 7, and copper was determined by atomic absorption spectrophotometry

Treatment Copper content of

I I I1 I1

moljmol protein

Dialysis for 24 h at 4 “C 2.04 2.05 1.92 1.94

Incubation with 5 mM cuprizone for 4 h at 25 “C followed by dialysis for 24 h at 4 :’C

Dialysis for 24 h at 4 “C against buffer containing 5 mM cuprizone - - 1.87 2.99

2.14 2.28 - -

order rate constants (kapp) for the process vary hyperbolically with the concentration of cuprizone.

These observations are consistent with a kinetic scheme in which enzyme (E) and cuprizone (I) pre- equilibrate rapidly to form an intermediate (El) which


0 10 20 30 40 Time (s)

Fig. 2. Time course qf the 350 nrn absorbance changes observed on mixing benzylamine oxidase with cuprizone. 3.1 pM enzyme and 1 mM cuprizone in 0.1 M phosphate buffer, pH 7, 25 “C

I . /

I I I I 1 2 3 4

1 / [ Cu pr izone] (mM-’) Fig. 3. Variation with cuprizone concentration of the apparent first-order rate constant for the reaction between henzylamine oxidase and cuprizone. Conditions as in Fig. 2

is converted into the spectrally modified enzyme species (E*) in an irreversible first-order reaction :

K1 k 2

E + I * E I - - t E*. (1)

Scheme (1) predicts that the apparent first-order rate constant for the absorbance changes observed is given by

where K1 stands for the dissociation constant of the enzyme . cuprizone complex EI. The parameter estimates obtained on fitting Eqn (2) to the data in Fig. 3 were Kl = 2.7 (i 0.5) mM and k2 = 0.8 (f 0.2) s-’.


Table 2. Time-dependence of’ the decrease in enzyme activity during the reaction between henzy lamine oxidase and cuprizone 5.8 pM enzyme was reacted with 25 pM cuprizone at 25 “C in 0.1 M phosphate buffer, pH 7, formation of the enzyme species E* exhibiting the 350 nm absorption band being followed spectrophotometrically. Samples withdrawn at 2-min intervals were tested for enzyme activity using 2 mM benzylamine, sufficient to prevent further reaction between enzyme and cuprizone

~

Reaction time Inhibition Formation of E*

min ”/,

2 55 50 4 80 85 6 95 90 8 95 95

30 100 100

Effect of Cuprizone on the Catalytic Activity of Bemylamine Oxidme

The inhibition of benzylamine oxidase by cuprizone has previously been reported to be time- dependent [8], and a more detailed examination of this time-dependence showed that the decrease in enzyme activity parallels the appearance of the 350 nm chromophore (Table 2). The activity of the inhibited enzyme could not be restored by dialysis. Titration of the enzyme with cuprizone established that one mole inhibitor per mole protein is sufficient to complete the 350 nm absorbance changes and to inactivate the enzyme completely (Fig. 4).

While variation of the oxygen concentration from 0.03 to 1.0 mM had no effect on the reaction between benzylamine oxidase and cuprizone, preincubation of the enzyme with benzylamine drastically decreased the rate of appearance of the 350 nm chromophore. The 350 nm absorbance changes observed in the presence of substrate did not strictly conform to first- order kinetics, for which reason no detailed kinetic analysis of these data was attempted. Substrate protection of the enzyme towards inactivation by cuprizone is, however, competitive with the inhibitor in the sense that half-times for the formation of the spectrally modified enzyme species E* steadily increase with increasing substrate concentrations and decrease with increasing concentrations of cuprizone (Table 3).

Effect of Cuprizone on the Reaction between Benzylamine Oxiduse and Phenylhydrazine

Benzylamine oxidase is known to react irreversibly with various hydrazines under formation of inactive complexes which most likely represent hydrazone derivatives of the protein-bound pyridoxal phosphate

[Cuprizone] (pM)

Fig. 4. Titrution of benzylamine oxidase with cuprizone. Enzyme activity (+) and 350 nm absorbance changes (0) were determined 10 min after each addition of cuprizone to 30 pM enzyme in 0.1 M phosphate buffer, pH 7, 25 “C

Table 3. Effect of’ substrate on the reaction between benzyl- m i n e oxidase and cuprizone The table lists the time (s) required for 50% conversion of benzylamine oxidase into the spectrally modified species after mixing cuprizone with 6.4 pM enzyme preincubated for 1 min with different concentrations of benzylamine. 0.1 M phosphate buffer, pH 7, 25 “C

Concn of benzylamine

Time for 50 ”/, conversion with

2 mM cuprizone 4 mM cuprizone

0 20

100 500

2500

2.0 1.4 5.2 3.3

22 14 64 35

180 95

[3]. The phenylhydrazone derivative of the enzyme exhibits a narrow absorption band centered around 430 nm [3,7], which is sufficiently well separated from the 350nm absorption band of the enzyme . cuprizone product E* to permit independent assays to be made of the simultaneous interactions between enzyme and the inhibitors phenylhydrazine and cuprizone.

Pilot experiments established that cuprizone (5mM) has no effect on the absorption spectrum of the phenylhydrazone derivative (E-Phz) of benzylamine oxidase. The other way round, formation of the enzyme species E* was found to result in a total loss of enzyme reactivity towards phenylhydrazine.


A. Lindstrom and G. Pettersson 233

These observations might indicate that cuprizone (I) competes with phenylhydrazine (Phz) for the protein- bound pyridoxal phosphate according to a kinetic scheme such as

I

E E I E m

K1 k 2

E-Phz

which predicts that (cf. [ 3 ] )

where cE denotes the total concentration of enzyme and [E-Phz], stands for the final concentration of E-Phz. Rearrangement of Eqn (4) yields

The validity of Scheme ( 3 ) was tested by spectro- photometric determination of the amount of phenylhydrazone formed after completed reaction of benzylamine oxidase with 5 mM cuprizone and different concentrations of phenylhydrazine, the two inhibitors being added simultaneously to the enzyme by the use of stopped-flow techniques. The results obtained are shown in Fig. 5 , from which it can be seen that experimental points in a plot of cE/[E-Phz], vs l/[Phz] fall well along the straight line calculated from Eqn ( 5 ) using k3 = 10.5 mM-' x s-' [3] and the above estimates X, = 2.7 mM and kz = 0.8 x sC1.

The pilot experiments mentioned above justify the assumption that there is no interaction between phenylhydrazine and the enzyme species E", but do not exclude that phenylhydrazine reacts with the enzyme . cuprizone complex EI. If the reaction

(6) k3 EI + Phz --+ EI-Phz

is added to Scheme ( 3 ) and the complex EI-Phz is assumed to exhibit an absorption similar to that of E-Phz, however, experimental points in Fig. 5 would be expected to fall along a straight line with the slope k2[I]/k3(Kl + [I]), indicated by the dashed line in Fig. 5 . The experimental data are, obviously, in- consistent with the latter idea and strongly suggest that the kinetically established reversible binding of cuprizone to benzylamine oxidase is sufficient to prevent formation of the phenylhydrazone derivative of the enzyme.

4 1 0

0 5 10 15 20 1 / \Phenylhydrazine] (rnM-')

Fig. 5. Plot according to Eqn ( 5 ) o j data obtained for the simultaneous reaction of benzylamine oxidase with cuprizone and phenylhydrazine. 4 pM enzyme, 5 mM cuprizone, and 0.05 - 4 mM phenylhydrazine in 0.1 M phosphate buffer, pH 7, 25 "C. The full line indicates the relationship expected according to Scheme (I), the dashed line that corresponding to an alternate kinetic model discussed in the text

Wavelength ( n m )

Fig. 6. EJfect of cuprizone on the visible absorption spectrum of pyridoxal phosphate. Spectra recorded immediately (A) and 3 h (B) after the addition of 0.4 mM cuprizone to 0.1 mM pyridoxal phosphate in 0.1 M phosphate buffer, p H 7, 25 "C

Reaction between Cuprizone and Free Pyridoxal Phosphate

Fig. 6 shows that the addition of an excess of cuprizone to a phosphate buffer solution of pyridoxal phosphate leads to the appearance of a new absorption band centered around 350 nm, indicating the formation of a reaction product with an absorption maxi-

Eur. J . Biochem. 48 (1 974)


I I I I I

I i n II

I \ \ \ I 1 >

I

I

V I I I I L

2600 2800 3000 3200 34c Magnet ic f i e l d (gauss)

Fig. 7. Effect of cuprizone on EPR spectra of benzylamine oxidase. Spectra were recorded at 77 K after preincubation of 85 pM enzyme with 0 (A), 0.1 (B), 0.2 (C), 0.4 (D), and 1.25 mM (E) cuprizone for 5 min at 25 "C in 0.1 M phosphate buffer, pH 7 . Spectrum F was obtained for the phenylhydrazone derivative of the enzyme in the presence of 0.4 mM cuprizone. Microwave frequency 9132 MHz

mum agreeing with that of the spectrally modified enzyme species E" formed from benzylamine oxidase and cuprizone. While no attempts were made to determine the structure of this product, kinetic studies indicated that it is formed through a second-order interaction between cuprizone and pyridoxal phosphate. The second-order rate constant calculated for the reaction in 0.1 M phosphate buffer at pH 7 and 25 "C was 1.0 sC1 xM-' , to be compared with rate constants of 10- 100 s-' x M-' for the reaction between pyridoxal phosphate and various hydrazines under the same conditions [3].

Effect of Cuprizone on Electron- Paramagnetic- Resonance (EPR) Spectra q j Benzylumine Oxidase

Fig. 7A shows the low-temperature EPR spectrum at 9.13 GHz of native benzylamine oxidase, and Fig. 7 B the spectrum recorded after preincubation

of the enzyme with a slight excess of cuprizone for 5 min at 25 "C. Since the latter treatment is sufficient to ensure at least 90% conversion of the enzyme into the species exhibiting the 350 nm absorption band, it may be concluded that formation of this species per sr does not result in any extensive EPR spectral changes. The presence of increasing concentrations of free cuprizone, however, leads to a gradual change of the EPR spectrum of the protein towards that obtained with 1.25 mM cuprizone (Fig. 7C-E), in which positions of the hyperfine lines at about 2630 and 2940 gauss are significantly shifted and a line at 3060 gauss appears in the main-peak region. No further spectral changes were observed on raising the cuprizone concentration from 1.25 to 5.0 mM.

It has previously been reported that hydrazone formation between phenylhydrazine and protein- bound pyridoxal phosphate in benzylamine oxidase has no significant effect on the EPR spectrum of the enzyme [3]. This was confirmed in the present investigation, and Fig. 7F shows that the addition of cuprizone to the phenylhydrazone derivative of the protein results in EPR spectral changes closely similar to those observed with native enzyme. The latter result definitely excludes that the pronounced effect of cuprizone on the EPR spectrum of benzylamine oxidase can be attributed to the irreversible process leading to the 350 nm chromophoric changes.

DISCUSSION

Cuprizone or biscyclohexanone oxalyldihydrazone has been frequently used for analytical purposes, since it forms a strong and intensely coloured complex with cupric ions in solution [lo]. This copper-chelating capacity of the reagent has also motivated its use in investigations of various copper-containing proteins, and the observation that it functions as a strong inhibitor of plasma amine oxidase from various sources has been taken to indicate that copper is essential for the catalytic activity of the latter enzyme systems [l]. Less attention has been paid to the fact that cuprizone is a hydrazone derivative, and hence might undergo a transimination reaction with protein- bound pyridoxal phosphate in these enzymes. The occurrence of transimination reactions involving pyridoxal phosphate has been well established in model systems [ll], and the reaction product formed between cuprizone and free pyridoxal phosphate (Fig. 6) might be obtained through such a mechanism. While elucidation of the detailed nature of this product is of minor importance for the purpose of the present investigation, it is of great interest to note that cuprizone does interact with free pyridoxal phosphate,


A. Lindstrom and G. Pettersson 235

and that this interaction leads to the formation of a product exhibiting an optical absorption band centered at the same wavelength (350 nm) as that of the inactive enzymatic species formed after treatment of pig-plasma benzylamine oxidase with cuprizone (Fig. 1).

Even though absorption coefficients for the two absorption bands are significantly different, the latter observation strongly suggests that the concomitant inhibition of enzyme activity by cuprizone is due to an interaction with protein-bound pyridoxal phosphate, presumably involving the carbonyl function of the prosthetic group. Evidence in support of this idea is given by the lack of effect of cuprizone on the absorption spectrum of the phenylhydrazone derivative of the enzyme, the loss of reactivity of the protein towards carbonyl reagents after cuprizone treatment, and the apparent irreversibility of the inhibitory action of cuprizone. Furthermore, the observation that one mole cuprizone per mole protein is sufficient to complete the 350 nm chromophoric changes and to inactivate the enzyme completely (Fig. 4) is consistent with the reported content of reactive pyridoxal phosphate in the protein [7], and the kinetic experiments showing that cuprizone competes with phenylhydrazine for enzyme-bound pyridoxal phosphate appear to definitely establish that the inactive enzyme species exhibiting the 350 nm absorption band is formed through an interaction between cuprizone and the prosthetic group of the protein.

Although it should be emphasized that the present interpretation of the kinetic data in Fig. 3 is not unique, Scheme (1) offers a plausible explanation for the saturation kinetics observed in assuming that the irreversible interaction between cuprizone and pyridoxal phosphate is preceded by a reversible formation of an enzyme . cuprizone complex with a dissociation constant of about 3 mM. This raises the question of whether or not enzyme-bound copper is involved in the corresponding binding of cuprizone to the protein. Benzylamine oxidase contains two cupric ions per enzyme molecule [7,12], and the EPR spectral data in Fig. 7 show that cuprizone has a pronounced effect on the binding situation of at least one of these paramagnetic ions under conditions where no copper is extracted from the protein (Table 1). Since the EPR spectral changes observed cannot be attributed to the interaction between cuprizone and enzyme-bound pyridoxal phosphate, it seems most likely that they arise from a direct binding of cuprizone to copper in the protein. The apparent heterogeneity of the spectrum in Fig. 7E might indicate that only one of the copper ions is involved in this interaction, but no firm conclusion can be drawn in this respect as the two copper ions may give rise to slightly different

overlapping EPR signals that are differently affected by ligand substitutions. For similar reasons, no precise quantitative evaluation of the EPR-detectable binding of cuprizone can be made at present. Nevertheless, it is obvious from the data in Fig. 7 that the kinetically detected reversible binding of cuprizone to the enzyme is considerably weaker than the interaction leading to the EPR spectral changes, the latter being indicative of complex formation corresponding to a dissociation constant of about 0.2mM rather than 3mM. This observation per se does not exclude that the two interactions take place at the same site, since the affinity of protein-bound copper for cuprizone might increase upon the reaction between cuprizone and pyridoxal phosphate in the protein. Recent studies on ligand-binding to copper in benzylamine oxidase, however, render the latter possibility less likely in showing that the affinity of protein-bound copper for azide is not significantly affected by formation of the phenylhydrazone derivative of the enzyme [ 141.

The present investigation, therefore, gives no support whatsoever to the idea that the inhibitory action of cuprizone is related to its copper-chelating properties, but strongly indicates that cuprizone reacts with the enzyme-bound pyridoxal phosphate by a mechanism closely similar to that governing the corresponding reactions involving substrate [8] or substrate analogues such as hydrazine derivatives [3]. In the latter reactions, the actual steps of Schiff-base and hydrazone formation, respectively, appear to be too rapid to permit the kinetic detection of inter- mediately appearing enzymatic species, although Michaelis-complex formation prior to the interaction with pyridoxal phosphate can be inferred from indirect evidence [3,13]. According to the present results, cuprizone reacts 10- 100 times slower than hydrazines with free pyridoxal phosphate, and might hence be expected to exhibit a corresponding comparatively low reactivity towards enzyme-bound pyridoxal phosphate, This might explain the kinetic significance of the reversible process of complex formation in the reaction between enzyme and cuprizone which, in fact, may be taken as supporting evidence that similar complexes do appear as intermediates in the reactions involving substrates and hydrazines. The observation that, according to the present interpretation of the kinetic data, formation of the reversible complex between enzyme and cuprizone is sufficient to prevent phenylhydrazine from reacting with the enzyme- bound pyridoxal phosphate lends further support to this idea.

The present results thus appear to give a satis- factory description of the mechanism of action of cuprizone as an inhibitor of pig-plasma benzylamine oxidase. They also strongly indicate that external

Eur. J . Biochem. 48 (1974)

236 A. Lindstrom and G. Pettersson: Inhibition of Benzylamine Oxidase by Cuprizone

ligands such as cuprizone may interact with the protein-bound copper in this enzyme, and that such interactions not necessarily have any evident effect on enzyme activity. Further investigations will, therefore, be directed towards the process of ligand-binding to copper in benzylamine oxidase and its effect on the spectral and catalytic properties of the enzyme.

This investigation was supported by grants from the Swedish Natural Science Research Council.

REFERENCES I . Kapeller-Adler, R. (1970) Amine Oxiduses and Methods

for Their Study, pp. 124- 134, Wiley, London. 2. Buffoni, F. (1968) in Pyridoxal Catalysis: Enzymes and

ModelSystems (Snell, E. E., Braunstein, A. E., Severin, E. S. & Torchinsky, Y. M., eds) pp. 363-374, Inter- science, New York.

3. Lindstrom, A, , Olsson, B. & Pettersson, G. (1974) Eur. J . Biochem. 42. 177 - 182.

4. Buffoni, F. (1966) Pharmacol. Rev. 18, 1163- 1199. 5. Yamada, H.&Yasunobu, K. T. (1962) J . Biol. Chem. 237,

6. Malkin, R. & Malmstrom, B. G. (1970) Adv. Enzynzol.

7. Lindstrom, A. & Pettersson, G. (1973) Eur. J . Biochem.

8. Lindstrom, A., Olsson, B. & Pettersson, G. (1973) Eur.

9. Pettersson, G. & Pettersson, I. (1970) Acta Chem. Scand.

10. Freegarde, M. & Jones, B. (1959) Anulyst, 84, 736-719. 11. Jencks, W. P. & Cordes, E. (1963) in Chemical and Bio-

logical Aspects of Pyridoxal Catalysis (Snell, E. E., Fasella, P. M., Braunstein, A. & Rossi Fanelli, A,, eds) pp. 57- 67, Pergamon Press, London.

12. Buffoni, F., Della Corte, L. & Knowles, P. F. (1968) Bio- diem. J . 106, 575- 576.

13. I.indstrom, A,, Olsson, B. & Pettersson, G. (1974) Eur. J . Biochem. 42, 377- 381.

14. Lindstrom, A,, Olsson, B. & Pettersson, G. (1974) Eur. J . Biochem. 48,237 - 243.

3077- 3083.

33, 177-242.

34, 564- 568.

J . Biochem. 35, 70-77.

24, 1275- 1286.

A. Lindstrom and G. Pettersson, Avdelningen for Biokemi, Kemicentrum, Lunds Universitet, Box 740, S-220 07, Lund 7, Sweden


Documents

The Mechanism of Inhibition of Pig-Plasma Benzylamine Oxidase by the Copper-Chelating Reagent Cuprizone