PURIFICATION OF AMINE OXIDASE FROM BEEF PLASMA* · PURIFICATION OF AMINE OXIDASE FROM BEEF PLASMA* BY CELIA WHITE TABOR, HERBERT TABOR, AND SANFORD M. ROSENTHAL (From the Nalional

PURIFICATION OF AMINE OXIDASE FROM BEEF PLASMA*

BY CELIA WHITE TABOR, HERBERT TABOR, AND SANFORD M. ROSENTHAL

(From the Nalional Institute of Arthritis and Metabolic Diseases, National Institutes of Health, United States Public Health Service, Bethesda, Maryland)

(Received for publication, December 14, 1953)

Numerous investigators have studied monoamine oxidase since it was first described (2) in 1928. The literature has recently been reviewed by Blaschko (3) and Zeller (4). Monoamine oxidase has usually been considered as a single enzyme (5), but some authors (Werle and Roewer (6) and Alles and Heegaard (7)) have presented evidence indicating that these preparations contained more than one type of amine oxidase activity. All of these studies, however, have been carried out with relatively crude preparations, since monoamine oxidase is associated with particulate material and has resisted efforts to purify it. Although many tissues were found to contain this activity, liver preparations have been most commonly used. Amine oxidase activity has been reported to be present in dog blood by Werle and Roewer (6), but has usually been considered to be absent in the blood of other species (3). Although Hirsch (8) reported the oxidation of spermine and spermidine by beef and sheep serum, no significant activity Tvas observed with those monoamines and diamines tested.

In the present work a soluble amine oxidase has been purified 150- to 200- fold from steer plasma; the enzyme oxidatively deaminates a variety of amines with the stoichiometric formation of the corresponding aldehydes, ammonia, and hydrogen peroxide. The substrate and inhibitor specific- ities of this enzyme differ markedly from those described for the liver preparations.

Materials

The various amines and aldehydes were obtained from commercial sources. Sperm&e hydrochloride was obtained from Hoffmann-La Roche and Company and L. Light and Company, Ltd. A sample of synthetic spermidine phosphate was kindly supplied by Dr. H. Schultz of the Univer- sity of Miami. Recrystallized pyridoxamine and pyridoxamine phosphate were kindly supplied by Dr. E. Peterson and Dr. A. Meister. Crystalline

* A preliminary report on this work was presented at the meeting of the American Society for Pharmacology and Experimental Therapeutics, New Haven, September 7, 1953 (1).

645

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646 PURIFICATION OF AMINE OXIDASE

catalase was obtained from the Worthington Biochemical Corporation and freed from ammonium sulfate by dialysis against 0.002 M phosphate buffer, pH 7.2. Calcium phosphate gel (9), aged 4 to 10 months, contained 14 mg. of dry weight per ml. Saturated ammonium sulfate solutions were equi- librated at room temperature (25-30”) and contained approximately 53 gm. of ammonium sulfate per 100 ml. of solution (approximately 4 M).

Methods

Manometric experiments were carried out in a conventional Warburg ap- paratus at 37.3”; the gas phase was air. Spectrophotometric measurements were made in a Beckman model DU spectrophotometer (with photomul- tiplier attachment) at 30” in cuvettes with a 1 cm. light path. Amine oxidase activity was measured as described below. Calalase activity was measured according to Sumner and Somers (5). Ammonia was determined calorimetrically with Nessler’s solution after distillation in Conway dishes, with saturated sodium borate for alkalization (10). Sperm&e and spermidine were determined by an unpublished method of Rosenthal; the details of this procedure (involving coupling with diazotized p-nitroaniline) will be published separately. Protein was determined by measuring the absorption at 280 rnp (11). Conductivity determinations were made with a Barnstead purity meter PM-2 and were used to check the salt concentration in all ammonium sulfate fractionations; the solutions were diluted 1: 50,000 in water, and the conductivity readings were compared with those of similar dilutions of a standard ammonium sulfate solution (12).

Results

Enzyme Assay. Manometric Units-The incubation mixture contained 0.2 ml. of 0.2 M potassium phosphate buffer (pH 7.2), 0.05 unit of catalase, and enzyme in a volume of 2.4 ml. After equilibration, 6 PM (0.1 ml.) of spermine hydrochloride (adjusted to approximately pH 7) were added from the side arm. The initial rate of oxygen consumption was measured and expressed as microliters of oxygen consumed per 10 minute period. 1 unit of enz,yme activity was defined as the amount of enzyme catalyzing the consumption of 1 ~1. of oxygen per 10 minutes. Usually the assays were carried out with 20 to 30 units of enzyme per Warburg flask. The specific activity was defined as units of enzyme per mg. of protein. Mano- metric units are used in this paper unless otherwise indicated.

Spectrophotometric Units-Although the manometric assay was used in following the purification reported in Table I, a spectrophotometric assay was used in other experiments and found to be more convenient. The spectrophotometric assay was based on the difference between the molar extinction coefficients of benzylamine and benzaldehyde at 250 mp (Fig.


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C. W. TABOR, H. TABOR, AND S. M. ROSENTHAL 647

1). The incubation mixtures contained 200 PM of potassium phosphate buffer (pH 7.2), 10 PM of benzylamine, and enzyme in a total volume of 3 ml. 1 spectrophotometric unit was defined as the amount of enzyme catalyzing an increase of 0.001 per minute in the optical density reading at 250 w. 1 manometric unit was approximately equivalent to 10 spectrophotometric units. The proportionality of this assay to enzyme concentration is shown in Fig. 2. Other amines, such as furfurylamine and p-(aminomethyl)sulfanilamide (homosulfanilamide) could also be used for this pur-

O1’lr~~’ 240 260 280 300

WAVE LENGTH 230 250 270 290

( MILLIMICRONS)

FIG. 1. Molar extinction coefficients of furfural and benzaldehyde compared with the corresponding amines. Optical density measurements were carried out on 5 X 10e5 M solutions of furfural and benzaldehyde and on 1OP M solutions of furfurylamine and benzylamine. All reagents were redistilled prior to use. Dilutions were made immediately before the spectrophotometric reading to avoid autoxidation.

w tFURFURYLAMINE\- i -Ll

pose (Figs. 1, 2, and 5). Both the manometric and the spectrophotometric assays gave essentially the same results when used to follow the purification of the plasma enzyme.

Benzylamine and homosulfanilamide were also degraded by liver amine oxidase (Z-4, 7), and in other experiments we have found the spectrophotometric method also applicable for studies with liver preparations. Crude calf liver homogenates contained 6 spectrophotometric units per mg. of protein, as compared with 2.2 units per mg. in titrated beef plasma. In comparable manometric experiments with the liver preparations, benzylamine was rapidly attacked; essentially no oxygen consumption was found, however, with spermine as the substrate.

Enzyme PuriJication (Table I). Step A-Steer blood, collected at the


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slaughter-house, was immediately treated with Q volume of a citrate solution, which contained 8 gm. of citric acid and 26.7 gm. of sodium citrate- 5.5H20 per liter (13). The titrated blood was centrifuged at 2000 r.p.m. for 30 minutes, and the plasma was collected. In other preparations serum was used instead of plasma, with essentially the same results. All subsequent steps were carried out at O-3”.

Step B-To 13,600 ml. of plasma, 7300 ml. of saturated ammonium sulfate were added (final concentration approximately 1.4 M); the solution

I I I I I I I

BENZYLAMI N E > FURFURYLAM INE HOMOS~LFANILAMIDE

: ,,L 1250 1 X275 1x250 I .3 -I

.I

.05

5’ ‘IO 0 ’ 5 . IO 0” 5 ‘IO TIME - ( MINUTES)

FIG. 2. Proportionality of enzyme activity to enzyme concentration. The incubation mixtures contained 200 PM of potassium phosphate buffer (pH 7.2), enzyme (indicated by the numerals on the chart in ml.), and 10 pM of substrate in a final

volume of 3.0 ml. The incubations were carried out at 30”. The enzyme solution (Step D) contained 1.1 mg. of protein per ml.

was cooled to O-3”, and the precipitate was removed by filtration through a fluted filter paper. To the filtrate 13,100 ml. of saturated ammonium sulfate were added (final concentration approximately 2.4 M). The precipitate was collected by filtration, and dissolved in water (final volume 3100 ml.).

This solution was then refractionated with ammonium sulfate. Con- ductivity measurements indicated that the solution was already 26 per cent saturated (approximately 1 M), and 1000 ml. of saturated ammonium sulfate were added to achieve a final ammonium sulfate concentration of 1.8 M. The precipitate was discarded after filtration, and 2000 ml. of saturated ammonium sulfate were added to give a final concentration of


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approximately 2.5 M. The precipitate was dissolved in 900 ml. of water. The solution was dialyzed against three changes of 0.01 M sodium acetate (total of 18 liters) to reduce the ammonium sulfate concentration to <0.02 M. Since inadequate dialysis caused poorer fractionation in the next steps, the efficiency of the dialysis was checked by conductivity measurements.

Step C-The dialyzed solution, divided into 50 ml. portions, was heated rapidly (1 to 2 minutes) to 65” with stirring and maintained at this temperature for 10 minutes. Usually no precipitate developed, and the solution was immediately cooled to 0”.

Portions (400 ml.) were then fractionated with ethanol at 0” after the addition of 40 ml. of 0.1 M MnC$. to each. With mechanical stirring 200 ml. of cooled (- 10”) 25 per cent ethanol were added over a period of 5 to

TABLE I

PuriJication of Amine Oxidase from Steer Plasma

step

A. Original. ........................ B. Ammonium sulfate ............... C. AlcoholtS. ...................... D. Calcium phosphate?. .............

Total volume -

ml.

Total units* Specific activity

13,600 180,000 0.22 1,440 108,000 2.3

117 42,000 16 1,639 35,000 33

* Manometric units = microliters of 02 per 10 minutes. t As indicated in the text, these steps were carried out on aliquots; the data given

here represent the total units obtained for all the aliquots. $ Including heating step.

10 minutes, while keeping the temperature of the solution at 0”. The precipitate was collected by centrifugation for 15 minutes at 4000 X g in a re- frigerated centrifuge (Precipitate I). In a similar manner additional precipitates were collected after the following successive additions’ of alcohol: 100 ml. of 25 per cent ethanol (Precipitate II), 100 ml. of 25 per cent ethanol and 20 ml. of absolute ethanol (Precipitate III), 100 ml. of absolute ethanol (Precipitate IV), 80 ml. of absolute ethanol (Precipitate V), and 110 ml. of absolute ethanol (Precipitate VI). Each precipitate was dissolved in approximately 35 ml. of cold water. The fractions showing the highest specific activities (usually Precipitates IV + V) were then combined.

r Although most of the inert protein was precipitated in the early fractions, at- tempts to shorten the above procedure by precipitating all these early fractions together gave poorer results, owing to coprecipitation of the enzyme activity with the bulky precipitate.


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Portions (57 ml.) of these combined alcohol fractions were refractionated with ethanol at 0” after the addition of 5.7 ml. of 0.1 M MnC&. Precipi- tates were collected after the following successive additions of cooled absolute ethanol: 3.0 ml. (Precipitate I), 2.0 ml. (Precipitate II), 1.8 ml. (Pre-

TABLE II

Relative Rates of Oxidation of Various Substrates by Plasma Amine Oxidase*

The incubation mixtures contained 0.2 ml. of 0.2 M potassium phosphate buffer, pH 7.2, 25 to 50 units of amine oxidase, catalase, and 6 pM of substrate in a total volume of 2.5 ml. KOH was present in the center well. The initial rates are expressed as the oxygen consumption (microliters) in 10 minutes per 10 manometric units of enzyme (i.e., spermine rate = IO).

Relative rate Relative rate

Substrate Crudet P ‘urifiedj Substrate Crudet Purifiedt

Monoamines Ethyl- Propyl- Butyl- Amyl- Hexyl- Heptyl- octy1- Decyl- Dodecyl- Octadecyl- Benzyl- Phenylethyl- Furfuryl- N-Methylbenzyl-

amine Trimethyleneimine Tyramine Mescaline

6.8

8.1

5

=G 2.0 3.6 3.3 5.3 5.9 3.1 3.3 4.4 f

4.8 2.3 3.6 f

Monoamines-continued Tryptamine Epinephrinejj Norepinephrine Serotonin Agmatine Homosulfanilamide

Diamines Ethylene- Trimethylene- Tetramethylene- Pentamethylene- Hexamethylene- Decamethylene- Histamine

Polyamines Spermine Spermidine

Amino acid Lysine

f f f f f

5.1

4.2

f f f f f

4.0 f

f 0.8 1.0

10 10

10 5.7

f

* The oxidative deamination of most of these amines in liver preparations has been studied by numerous investigators. These results are summarized in the re- views by Zeller (4) and Blaschko (3).

t Citrated steer plasma. $ Step D, purified 175 times. 3 f indicates 0 to 0.1. Essentially the same results were found with these sub-

strates when tested with a 5-fold increase in substrate concentration. At the con- clusion of the experimental period, 6 PM of spermine were added; there was an immediate oxygen consumption, indicating that the enzyme was still active.

jj Essentially no oxygen consumption (above the blank) was found at pH 7.7 or at pH 6.1, as well as at pH 7.2, or when 30 PM of substrate were used instead of 6 pM. The same results were obtained when the epinephrine incubation mixture contained 10 pM of ethylenediaminetetraacetate in order to reduce the blank.


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cipitate III), 2.5 ml. (Precipitate IV), and 2.5 ml. (Precipitate V). The various precipitates were each dissolved in approximately 20 ml. of cold water and assayed. The fractions showing the highest specific activities (usually Precipitates III + IV) were used for Step D.

Step D-6 ml. portions of the enzyme solution from Step C were treated with 21 ml. of calcium phosphate gel. The gel was collected by centrifugation and eluted with 42 ml. of 0.01 M KzHP04.

Most of the studies were carried out with enzyme fractions from Step C or D. Frequently, material carried through Step C was lyophilized and

c SPERMINE

6.0

E 4 4.0 - a

4" I= z - 2.0-

0- 6.0 7.0 0.0

0

i\

0

A

0

I I 7.0 0.0

PH PH

FIG. 3. Effect of pH on enzyme activity with spermine and with benzylamine as substrates. The incubation mixtures contained 40 pM of buffer, 6 PM of substrate, catalase, and enzyme (Step D) in a final volume of 2.5 ml. The initial rates of oxygen consumption were measured and expressed as microliters of oxygen per minute per ml. of enzyme. 0, potassium phosphate buffer; a, tris(hydroxymethyl)amino- methane; l , barbital buffer.

BENZYLAMINE

2.0

1.0

stored as the dry powder. Although the enzyme preparations (at all stages) were stable at ice box temperatures, prolonged storage was usually carried out at -15” to -20”. Essentially no loss of activity occurred during the period of observation (1 to 4 months). No activity was sedimented when the enzyme (Step C) was centrifuged at 100,000 X g for 1 hour in the Spinco preparative centrifuge.2

Substrate SpeciJicity-The relative rates of oxidation of various substrates at pH 7.2 are listed in Table II. As indicated in this table, the rates of oxidation, relative to spermine, of the various aliphatic amines and of benzylamine with the purified preparations were similar to those observed with the crude preparations.

* We wish to thank Dr. H. Saroff and Mr. E. Adamik for the use of this centrifuge.


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The relative rates of oxidation reported in Table II were only valid for the conditions listed; i.e., pH = 7.2 and substrate concentration = 6 PM per 2.5 ml. Since at this concentration the enzyme was not saturated with all of the substrates studied, different ratios were found at other substrate levels. Changes in pH, likewise, affected the ratios found. For example, the rate for spermine at pH 6.2, the pH optimum for spermine oxidation, was approximately 1.5 times greater than the value found at pH

I I I I I I B r CATALASE ADDED

ADDITIONAL 0 20 40 60 80 100 120 ‘ok ‘“““‘:‘:““‘ol ,

0 30 60 90 120 150 180

.

TIME (MINUTES) TIME (MINUTES)

FIG. 4. Effect of catalase on benzylamine and spermine oxidation. In the experiment in A both incubation mixtures contained 0.2 ml. of 0.2 M potassium phosphate buffer (pH 7.2), 35 units of amine oxidase (Step D), and 6 ,UM of benzylamine in a total volume of 2.5 ml. Incubation II also contained 1.0 unit of catalase. Subsequently 1.0 unit of catalase and 6 pM of benzylamine were added to each flask (indicated at the arrows). In B the experiment was essentially the same except for 3 PM of sper- ~mne msceaa 01 oenzylamme.

7.2 (Table II, Fig. 3). With spermidine, on the other hand, the rate at,

pH 7.2 was 3 times greater than the rate at pH 6.0. With similar concentrations of benzylamine the maximal rate was observed at pH 7.5 (Fig. 3). Although we have not extended these pH studies to other amines, similar differences in pH optima with a wide variety of substrates had previously been reported by Alles and Heegaard (7), using liver amine oxidase preparations. The influence of substrate concentrations on these pH effects has not been evaluated.

Stoichiometry. Monoamines-Previous work demonstrated that liver monoamine oxidase catalyzes the oxidation of tyramine and other monoamines to ammonia and the corresponding aldehydes. Although these


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preparations still contained catalase, indirect evidence demonstrated the formation of HzOz (2-4, 14-16). Hz02 has also been shown during the oxidation of phenylethylamine by plant preparations (17).

The data in Fig. 4 and Table III indicate that a similar pathway occurs with the substrates degraded by the plasma enzyme. In the absence of catalase, the oxidation of monoamines proceeded in accordance with the equation R-CH2NH2 + 02 + Hz0 -+ R-CHO + NH3 + HsOZ. In the presence of catalase, Hz02 --+ Hz0 + 402. In Fig. 4, A, it is shown that the oxidation of each micromole of benzylamine in the presence of catalase consumed 1 microatom of oxygen. In the absence of catalase 2

TABLE III

Stoichiometry of Oxygen Consumption and Ammonia Production

Conditions essentially as in Table II.

Substrate

PM

n-Heptylamine ........................ 6 Benzylamine. ......................... 10 Homosulfanilamide ................... 6 Decamethylenediamine. .............. 6 Spermine ............................ 6 Spermidine ............................ 6

02 consumption NHx production

microatoms IrM

7 7.4 9.3 9.5 6.1 6.4

11.5 13.3 11.7 12.1*

5.2 5.0*

* All the ammonia values were measured by nesslerization after diffusion in Conway vessels from a borate buffer. Consequently, the presence of other material, which would react like ammonia in this assay, has not been excluded; this may be particularly true with spermine and spermidine, for which there is some preliminary evidence that an unstable aminoaldehyde may be one of the products of the reaction (unpublished evidence based on XE64-hydrogen form chromatography).

microatoms of oxygen mere consumed; the formation of hydrogen peroxide was shown by the evolution of the calculated amount of oxygen upon the subsequent addition of catalase. 0.95 pM of ammonia was produced for each micromole of benzylamine oxidized. No CO2 was produced. Essen- tially the same oxygen and ammonia stoichiometry was observed with the other monoamines tested (Table III).

The formation of an aldehyde as a product of amine oxidase action was most conveniently shown with benzylamine, homosulfanilamide, and furfurylamine, since the corresponding aldehydes (Fig. 1) have a relatively high absorption in the ultraviolet. After oxidative deamination of these substrates by the enzyme, the spectral readings indicate over 90 per cent conversion to the corresponding aldehydes (Fig. 5).

Diamines-The only diamine oxidized in the series tested was deca-


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methylenediamine. For each micromole of the substrate oxidized by the enzyme in the presence of catalase, approximately 2 microatoms of oxygen were consumed and 2 micromoles of ammonia were produced.

I I I I 1 I I I I I I I 230 250 270 230 250 270 240 260 280

WAVE LENGTH ( MILLIMICRONS )

FIG. 5. Aldehydes produced by the action of amine oxidase on benzylamine, p-(aminomethyl)benzenesulfonamide (homosulfanilamide), and furfurylamine. The reaction mixtures (volume 2.5 ml.) contained 88 units of enzyme (specific activity, 33 units per mg.), 40 PM of phosphate buffer (pH 7.2), 0.05 unit of catalase, and either 6 PM of benxylamine (A), 5.4 .UM of homosulfanilamide (B), or 6.4 PM of furfurylamine (C). These were then shaken in Warburg vessels at 37.2” until the reaction was more than 80 per cent completed. The various solutions were diluted 1:25 with water, and the optical densities were read immediately in view of the well known instability of benzaldehyde. The oxygen consumption values were 5.2 microatoms for benzylamine (30 minutes), 5 microatoms for homosulfanilamide (20 minutes), and 5.7 microatoms for furfurylamine (22 minutes). The optical densities have been corrected for the small blank of a control mixture without substrate. The readings indicate the presence of 4.8 PM of benzaldehyde in the benzylamine experiment and 6.9 pM of furfural in the furfurylamine experiment.

Polyamines-1 microatom of oxygen was consumed and 1 pM of ammonia was produced per micromole of spermidine oxidized in the presence of catalase (Table III). 2 microatoms of oxygen were consumed and 2 PM

of ammonia were produced per micromole of spermine oxidized in the presence of catalase (Fig. 4, B). In the absence of catalase the oxygen consumption was doubled; subsequent addition of catdase released approximately 1 PM of oxygen, indicating the presence of HzOz. No CO2 was


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produced. No spermine disappeared in a helium atmosphere. Hirsch (8) has previously reported ammonia production during spermine oxidation by crude sheep serum.

No increase in the rate of oxygen consumption was observed when spermine (6 PM) and spermidine (6 pM) were both added together, although

SUBSTRATE SUBSTRATE CONCENTRATION CONCENTRATION

pM/ml. pM/ml.

25

--Y-e 6 I S

FIG. 6. A and B, effect of substrate concentration on the rate of oxidation of benzylamine and homosulfanilamide. The incubation mixtures contained 1 ml. of 0.2 M phosphate buffer (pH 7.2), enzyme (Step D), and substrate in a final volume of 3.0 ml. The initial rates of aldehyde formation were measured in the spectrophotometer at 250 rnp and are plotted against the substrate concentration. C, inhibitory effect of spermine on homosulfanilamide oxidation. The conditions were the same as those in B, except for the addition of spermine hydrochloride; the amount of added spermine is indicated by the numerals on the chart in micromoles per ml. (final concentration). The reciprocals of the velocity (spectrophotometric units) and the substrate concentration (micromoles per ml.) are plotted in the usual Line- weaver-Burk (20) representation.

the final oxygen uptake reached the value expected for the oxidation of both substrates. This observation favors the postulation that spermine and spermidine are oxidized by the same enzyme.

Preliminary evidence has been obtained (unpublished experiments) for the formation of spermidine as an intermediate in spermine oxidation. When the reaction was interrupted after only 50 per cent of the expected oxygen uptake had occurred, chromatographic analysis (Amberlite XE64) indicated the appearance of both an aldehyde component and spermidine. The latter accounted for approximately one-half of the degraded spermine;


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it was isolated as spermidine hydrochloride3 and identified by infrared spectroscopy and x-ray diffraction analysis. When the oxygen uptake was permitted to reach completion, spermidine was no longer found. The

TABLE IV

Effect of Various Inhibitors on Spermine Oxidation

The incubation mixtures contained 0.2 ml. of 0.2 M potassium phosphate buffer, pH 7.2, catalase, purified amine oxidase (50 to 100 manometric units), and inhibitor in a volume of 2.4 ml. After the preincubation period 6 PM of spermine (0.1 ml.) were added from the side arm.

Substance

Ephedrine Dibenamine 53 98 00

Quinacrine 63 Benzedrine 68 Urethane Trimethylenedi- 60

amine Ethylenediamine- Trimethyleneimine 53

tetraacetate Pyridoxamine* 00 (31) Isoniazid 00 (63)

00 (80) Pyridoxamine 0

phosphate Iproniazid Phenobarbital 0

Pyribenzamine 91 Benadryl 78 Octyl alcohol 0 Hydroxylamine 00 Semicarbazide 00 Cyanide 00

- * Inhibition of spermine oxidation by pyridoxamine was only transient; for ex-

ample, in the experiment with 5 X 10e3 M pyridoxamine, oxygen consumption began 15 minutes after the spermine was added. The figures in parentheses represent the per cent inhibition during this later period.

t Per flask.

70 4 x IO-3 10 4 x 10-a

2 x 10-z 4 x 10-2

36 4 x 10-a 10 4 x 10-Z

10 4 X 10-S

65 1 X 10-S 1 X IO-4

1 X 10-z 2 x 10-Z

65 2 X 1O-4 8 x 10-4

1 X 10-s 2 x 10-a

8 '3 2 c L3

- w

cent

0 0

82 92

0 0

109 2 X 10-b 2 x 10-4

2 x 10-a 70 2 x 10-3

1152 X 1O-3 36 2 X IO-3

0

36 97 98 98 18 86 97 95

36 2 X 10-3 30 5 x 10-4

2 x 10-3

5 ‘X 10-3 36 I X 10-3

75 2 x 10-s 109 2 x IO-3

60 2 X 10-S 127 0.1 ml.t

454 x 10-4 454 x 10-3

60 1 x 10-a

-

Substance

8 3 r: e’cf COIWS- 0.0 tration SE ela 4 -

Inhibition

per cent

possibility that a labile intermediate closely related to spermidine is con- verted into spermidine during isolation procedures has not been excluded.

3 We wish to thank Dr. H. Bauer for his assistance in this isolation, Dr. E. Horning for the infra-red analysis, and Mr. William C. White for the x-ray diffraction analysis.


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Further details on the intermediary products of spermine metabolism in vitro and in vivo will be published separately.

Evidence for the formation of one or more aldehydes during the above reactions was obtained by adding a saturated solution of 2,4-dinitrophenylhydrazine in 2 N HCl to the trichloroacetic acid filtrate of the spermine incubation mixture. Subsequent alkalization with 5 N NaOH resulted in the purple color characteristic of dinitrophenylhydrazones (18). In other

TABLE V

Effect of Preincubation on Isoniazid Inhibition of Amine Oxidase

The incubation mixture contained 1 ml. of 0.2 M potassium phosphate buffer, pH 7.2, isoniazid, 12 spectrophotometric units of amine oxidase, and water in a volume of 2.94 ml. After the preincubation period indicated at 30”, 0.06 ml. of 0.1 M benxylamine sulfate was added, and the reaction was followed at 250 rnp in the spectrophotometer for 10 minutes.

Concentration of isoniazid Time of preincubation Inhibition’

Y

2 x 10-4

2 x 10-s

2 x 10-e

min. per cent

1 41 15 89 37 98

1 0 1 6

22 40 60 64

109 88 135 81

1 0 105 15

* A manometric experiment was carried out with a final concentration of 1 X 10-d M isoniazid and 6 PM of benzylamine as the substrate. There was essentially no inhibition without a preincubation period; after 60 minutes of preincubation at 37”, 93 per cent inhibition was observed in the rate of oxygen uptake. Similar results were obtained with iproniazid.

experiments, with a more concentrated incubation mixture, a crystalline reddish brown precipitate was obtained upon the addition of the 2,4- dinitrophenylhydrazine; although this is presumably a dinitrophenylhy- drazone, more definitive identification has not been carried out. The formation of an aldehyde from spermidine has been shown by Silverman and Evans (19) during the oxidation of spermidine by lyophilized Pseudo- monas pyocyanea.

Kinetic Studies-The effect of substrate concentration on the reaction rates with benzylamine and homosulfanilamide is shown in Fig. 6. The Michaelis constants, as calculated by the method of Lineweaver and Burk (20), were 1.6 X 1O-3 and 2 X 10h3 M, respectively.


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The affinity of spermine for the enzyme could not be measured directly, as the enzyme was completely saturated in manometric experiments containing 3 PM per 2.5 ml. volume. An indirect measure of this affinity was obtained by measuring the effect of spermine as an inhibitor of homosulfanilamide oxidation (Fig. 6, C). K1 (the enzyme-inhibitor dissociation constant) was calculated as 8 X lO+ M.

200

180 CYANIDE CONCENTRATION

160 q = = 1.0x10-3M

3140

Fi g 120

k x 100

2 o 80 0 0” 60

40

0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 MINUTES

FIG. 7. Effect of cyanide on benzylamine oxidation. The incubation mixtures contained 100 pM of phosphate buffer (pH 7.2), enzyme (Step D), and sodium cyanide in a total volume of 2.44 ml. An amount of acetic acid, equivalent to the sodium cyanide concentration, was included in the incubation mixture. After a 15 minute preincubation at 37.2”, 6 pM of benzylamine (0.06 ml.) were added from the side arm. At the arrows, an additional 6 pM of benzylamine were added to each flask.

Inhibition Studies-The effects of various inhibitors on spermine oxidation are summarized in Table IV. Table V summarizes the inhibition studies with isonicotinoylhydrazine (isoniazid), demonstrating that the inhibitory effect was not immediate, but required incubation of the enzyme and the inhibitor. A similar effect of preincubation was found with l-iso- nicotinoyl-2-isopropylhydrazine (iproniazid).

The oxidation of spermine by the purified amine oxidase preparations was inhibited by cyanide, hydroxylamine, and semicarbazide, thus con- firming the findings of Hirsch (8) with sheep serum. Comparable inhibition was also observed in the oxidation of benzylamine. Although a lag in cyanide inhibition was noted with low cyanide concentrations (Fig. 7),


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longer preincubation periods (100 minutes) did not affect the results obtained. This was in contrast to the isoniazid inhibition reported above.

The inhibition of spermine oxidation by quinacrine observed in these experiments is similar to the inhibitory effects of quinacrine reported by Silverman and Evans (19) during spermidine oxidation by lyophilized P. pyocyanea.

DISCUSSION

Although the relationship between the plasma enzyme and the liver monoamine oxidase is still unclear, various differences and similarities in the behavior of the two enzymes have been noted in this work. These are somewhat difficult to evaluate at present, since the liver enzyme is particulate and resists purificationP Most striking are the differences in substrate and inhibitor specificity. Our preparations show strong activity against spermine, relatively weak activity against tyramine, and essentially no activity against tryptamine and epinephrine. This is in contrast to the usual liver preparations (2-4), which show considerable activity against the latter substrates and, as reported above, essentially no activity against spermine . Werle and Roewer (6) have recently presented evidence that in rabbit liver butylamine and tyramine are oxidized by separate amine oxidases. The inactivity against long chain monoamines and short chain diamines, as well as the active oxidation of the one long chain diamine tested, is in agreement with the findings of Blaschko and Duthie (22) and Blaschko and Hawkins (23) for liver monoamine oxidase.

Zeller et al. (24) have recently reported that liver and brain monoamine oxidases are inhibited by iproniazid, but are resistant to isoniazid. The plasma enzyme, on the other hand, is inhibited by both iproniazid and isoniazid.

The plasma preparations are inhibited by 10e3 M KCN. Liver monoamine oxidase preparations, on the other hand, are very resistant to cyanide inhibition (24, 14-16). The inhibition of the enzyme by carbonyl reagents (Table IV) suggests the possibility that a carbonyl group is present as an active center on the enzyme or a coenzyme (possibly pyridoxal phosphate).

In general, the data indicate that in beef plasma preparations one enzyme is involved in the oxidation of spermine, benzylamine, and the aliphatic amines. For example, the relative reaction rates with different substrates with the preparation purified 175 times are similar to those found with the crude plasma. Further support is offered by various competitive experiments, as well as similar behavior towards KCN and other inhibitors.

4 Barsky, Berman, and Zeller (21) have recently reported the preparation of a non-sedimentable amine oxidase from hog liver mitochondria by combined treat- ment with desoxycholic acid and sonic oscillation.


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However, unequivocal proof for a single enzyme would require further purification of the enzyme, as well as some independent determination of the K, for spermine.

Spermine was not only very actively degraded, but also had a very high affinity for the enzyme. These findings may be of particular relevance to the physiologic significance of this enzyme, in view of the recent studies on the nephrotoxic action of spermine (Rosenthal et al. (25, 26)), as well as its relationship to bacterial metabolism (19, 27-30). Our purified amine oxidase preparations, when added to culture media containing spermine, have the same inhibitory effect on the growth of Mycobacterium tuberculosis (BCG strain) as the preparations of Hirsch and Dubos (27, 28). (Un- published experiments of Dr. Y. T. Chang.)

SUMMARY

1. A soluble amine oxidase from beef plasma, which oxidizes various amines with the stoichiometric formation of the corresponding aldehyde, HzOz, and NH3, has been purified 150- to 200-fold.

2. The substrates most rapidly attacked are spermine, spermidine, benzylamine, homosulfanilamide, furfurylamine, and various aliphatic amines. Essentially no activity is observed towards epinephrine.

3. This preparation differs in substrate and inhibitor specificity from the particulate monoamine oxidase described in liver.

4. A convenient spectrophotometric assay for amine oxidase is described, which depends on the change in the absorption spectrum when benzylamine is oxidized to benzaldehyde.

BIBLIOGRAPHY

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M. RosenthalCelia White Tabor, Herbert Tabor and Sanford

FROM BEEF PLASMAPURIFICATION OF AMINE OXIDASE

1954, 208:645-662.J. Biol. Chem.

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PURIFICATION OF AMINE OXIDASE FROM BEEF PLASMA* · PURIFICATION OF AMINE OXIDASE FROM BEEF PLASMA* BY CELIA WHITE TABOR, HERBERT TABOR, AND SANFORD M. ROSENTHAL (From the Nalional