Enzymic oxidation of epinephrine to adrenochrome by the salivary gland

BIOCHIMICA ET BIOPHYSICAACTA

247

BBA 65017

ENZYMIC OXIDATION OF E P I N E P H R I N E TO ADRENOCHROME BY THE SALIVARY GLAND JULIUS AXELROD Laboratory of Clinical Science, National Institute of Mental Health, National Institutes of Health, Bethesda, Md. (U.S.A.)

(Received November Igth, 1963)

SUMMARY An enzyme in the cat salivary gland that oxidizes epinephrine to adrenochrome is described. Enzyme activity is measured by trapping the unstable adrenochrome with fl-phenylisopropylphenylhydrazine. Several catecholamines were found to serve as substrates for this enzyme. Ascorbic acid, GSH and diethyldithiocarbamate inhibit, and monophenols increase enzyme activity. INTRODUCTION In a study on the catechol 0-methyltransferase activity of the salivary gland, it was observed that this tissue metabolized [3H]epinephrine to form two metabolic products one of which was identified as metanephrine 1. When a monoamine oxidase inhibitor fl-phenylisopropylhydrazine was added to the soluble supernatant fraction of the salivary gland and then incubated with [ZH]epinephrine a large amount ofa metabolite was formed which was extractable into organic solvents. This report describes the identity of the metabolic product and the properties of the enzyme that forms it. METHODS AND MATERIALS [7-3H]Epinephrine • HC1, and [7-SH]norepinephrine .HC1 were obtained from New England Nuclear Co., [SH]dopamine .HC1 from Tracerlab, and [14C]serotonin from Chicago Nuclear. Adrenochrome was generously supplied by R. A. HEACOCK and fl-phenylisopropylhydrazine and its derivatives was kindly donated by Lakeside Laboratories. Assay for the enzymic formation of adrenochrome

Enzyme activity was determined by incubating enzyme preparation with [3H]epinephrine and fl-phenylisopropylhydrazine in a IS-ml glass-stoppered centrifuge tube. The resulting radioactive hydrazone of adrenochrome is extracted into a mixture of toluene and isoamyl alcohol at pH I0 and the radioactivity in the extract measured after the addition of phosphor. The enzymic oxidation of dopamine, norepinephrine and serotonin, were examined in a similar manner. Bioehim. Biophys. Acta, 85 (1964) 247-254

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J. AXELROD

A typical i n c u b a t i o n consisted of 25/A enzyme preparation, Ioo/,moles t)hosphate buffer (pH 7.o), 25 #moles MgClz, 2 m/tmoles [all]epinephrine, o.5 ffmole fl-phenylisopropylhydrazine. After 15 rain i n c u b a t i o n at 37 °, o.5 ml ofo.5 M borate buffer (pH IO) a n d 6 ml t o l u e n e - i s o a m y l alcohol (3:2, v/v) were added to the i n c u b a t i o n mixture. After shaking for 5 min a n d centrifllging, a 4-ml aliquot of the extract was transferred to a vial c o n t a i n i n g I ml ethanol and IO ml phosphor a n d the r a d i o a c t i v i t v measured in a scintillation spectrometer. A control i n c u b a t i o n in which the enzyme was heated a t I O 0 ° for 3 m i n was carried t h r o u g h the above procedure at tile same time t(, correct for the small a m o u n t s of IaH]epinephrine that is extracted as well as oxidized n . n enzymically. U n d e r the conditions described above the [aH]adrenoehromc hydraz,n(,. was q u a n t i t a t i v e l y extracted. The hydrazone ()f adrenochrolne was prepared by mixing 5 nlg adrenochrolne with Ioo mg fl-phenylisopropylhydrazine in o. 5 ml o.t M phosphate buffer (pH 7.o). After i n c u b a t i n g for 5 rain at 37 ° the yellow precipitate was washed with ice-cold buffer (pH 7.o). RESULTS

Enzymic formation of adrenochrome by cat-salivary-gland extracts Cats were killed b y a n overdose of n e m b u t a l a n d the parotid gland was removed, chilled a n d homogenized with IO volumes of cold distilled water. After centrifugation at I5 ooo >< ;,, an aliquot of the soluble s u p e r n a t a n t fraction was i n c u b a t e d with [:~H]epinephrine a n d fi-phenylisopropylhydrazine. After 3 ° rain i n c u b a t i o n the m i x t u r e was extracted into a toluene isoamyl alcohol m i x t u r e a n d the r a d i o a c t i v i t y measured in the extract. U n d e r these conditions a radioactive p r o d u c t was formed t h a t was extracted into the above solvent m i x t u r e (Table I). I n the absence of flp h e n y l i s o p r o p y l h y d r a z i n e , negligible a m o u n t s of the metabolite were found. A smal a m o u n t of radioactive material was also formed nonenzymically. The n o n e n z y m i c oxidation of epinephrine was suppressed when Mg 2: was added to the i n c u b a t i o n mixture. Mg 2+ also s t i m u l a t e d the enzymic formation of the epinephrine metabolite. TABLE l E N Z Y M I C Y O R M A T I O N OF A D R E N O C H R O M E

BY CAT SALIVARY GLAND

Soluble supernatant fraction obtained from 5 mg of cat parotid gland was incubated at 37° with o.i ml o. 5 M phosphate buffer (pH 7.o), 25 F1 o. 5 M MgCI~, 2 m/~moles [3H]epinephrine (65 ooo counts/rain), o. 5/~mole fl-phenylisopropylhydrazine in a final volume of 25o/~1. After 15 rain, radioactive adrenochrome hydrazone was measnred in the incubation mixture. All values were corrected for small amounts of unchanged [all]epinephrine (goo counts/min) extracted by this procedure. Conditions

Complete system Heated enzyme Mg~+ omitted Heated enzyme, Mg2+ omitted fl-Phenylisopropylhydrazine omitted

Counts]mit~

A drenochrome formed/g tissue ( ml~moles )

28 55o 840 i6 ooo 2 9oo

19o 5 98 18

86o

5

Biochim. Biophys. dcta, 85 (r964) 247-254

OXIDATION OF EPINEPHRINE BY SALIVARY-GLAND ENZYME

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The requirement for a hydrazine suggested the formation of a hydrazone presumably that of adrenochrome. The toluene-isoamyl alcohol extract was reduced to a small volume in vacuo and chromatographed (ascending) in three solvent systems : butanolacetic acid-water (4: I : I, v/v), isopropanol-ammonia-water (8 : I : I, v/v), sec.-butanolpyridine-acetic acid-water (4°: 1:4: IO, v/v). A single radioactive peak was found that had the same RE value as synthetic adrenochrome hydrazone in all chromatographic systems. The enzymically formed adrenochrome hydrazone also had the same partition coefficient as the synthetic compound after distribution between toluene-isoamyl alcohol (3:2, v/v) and various buffers. These observations were taken as evidence for the presence of an enzyme in the salivary gland that oxidizes [SH]epinephrine to [aH~adrenochrome. The unstable adrenochrome is then trapped to form a stable radioactive hydrazone with fl-phenylisopropylhydrazine. The later compound can then be separated from the unreacted [aHlepinephrine by extraction in toluene-isoamyl alcohol. A number of hydrazine derivatives were examined for their ability to trap the enzymically formed adrenochrome. Those hydrazines with a phenylisopropyl sidechain were the best trapping agents (Table II). The hydrazines that react with adrenochrome are also potent monoamine oxidase (EC 1.4.3.4) inhibitors. However the capacity to inhibit monoamine oxidase was unrelated to their ability to form adrenochrome hydrazones since other monoamine oxidase inhibitors such as ipronazid, which has a hydrazine group that is blocked, or MO 91 I, which does not have a hydrazine group, were unable to trap adrenochrome. T A B L E II STRUCTURAL

REQUIREMENTS

FOR THE ADRENOCHROME

TRAPPING

AGENT

Soluble s u p e r n a t a n t fraction obtained from 5 m g of cat parotid was incubated with o.I ml 0.5 M p h o s p h a t e buffer (pH 7.o), 25/zl 0. 5 M MgCI,, 0. 3/*mole agent, i m/~mole [3H]epinephrine in a final volume of o. 5 ml. After 15 mill the incubation m i x t u r e was extracted for [3H]hydrazone as described under METHODS. Relative activity (%)

A gent

fl-Phenylisopropylhydrazine I-(3'-Chloro) phenyl-2 -hydrazinopropane a-Methyl-r-(4' methoxy) phenylethylhydrazine 4-Phenyl-2 -hydrazinobutane 2 -Phenylethylhydrazine Iproniazid N-Methyl-N-benzylpropylamine (MO 911) Phenylisopropylamine (amphetamine) Phenylhydrazine

I oo 81 76 28 8 2 i o o

Purification of the epinephrine oxidizing enzyme Submaxillary and parotid glands obtained from six male cats were homogenized with 5 volumes of ice-cold water using a mortor and pestle and washed sea sand. All subsequent procedures were carried out at 0-4 °. After centrifugation for I h at 8o ooo × g, 21. 9 g of solid ammonium sulfate was added to 7 ° m l of the supernatant Biochim. Biophys. Mcta, 85 (1964) 247-254

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J. AXELROD

fraction (o-5o% saturation). The precipitate was discarded after centrifllgation mid 9.6 g a m m o n i u m sulfate was added to the s u p e r n a t a n t solution (5o 7 ° 0 / s a t u r a t i o n ) . After centrifugation the precipitate was t a k e n up in 13 ml water (5.8 mg p r . t e i n / m l ) a n d dialyzed for I8 h against o.ooi M buffer (pH 7.0). The enzyme preparation was adjusted to p H 5.3 with I N acetic acid a n d i o ml calcium phosphate gel (I~ mg solids/ml) were slowly added. The suspension was centriiuged a n d the gel eluted with 5 ml o.I M phosphate buffer (pH 7.o). A b o u t six-fold purification of the enzyme was o b t a i n e d using the above procedure with a b o u t a 15 ~V~yield (Tat)le I I i). The enzyme was found to be stable up to four weeks of storage at lO ° a n d then lost a c t i v i t y after this time. TABI,E IIt PURIFICATION

i unit

OF

EPINEPHRINE

OXIDIZING

ENZYMF

IN

CAT

SALIVARY

GLAND

1 m/anole adrenoehrome hydrazone formed in I5 nfiu. Purification

step

Units/rag :#rolein

Soluble supernatant fraction 5° 75 °0 ammonimn sulfate Calcium phosphate gel adsorption an(] elution

2. t 4.o ~2.2

l'olal lcn~l~

io ro 33° t4(~

Properties of the enzyme I n phosphate buffers the partially purified epinephrine oxidizing enzyme had an o p t i m a l a c t i v i t y at p H 7.0. The enzyme was f o u n d to be almost entirely i n a c t i v a t e d when i n c u b a t e d at 37 ° for 15 rain. The Km for the enzyme was found to be 3" IO-5 M. The a b i l i t y of the partially purified enzyme to oxidize other catecholamines was e x a m i n e d (Table IV). E p i n e p h r i n e was the best substrate b u t other catecholamines such as norepinephrine a n d d o p a m i n e could be oxidized. A small a m o u n t of serotonin appeared to be metabolized b y the enzyme a n d the r e s u l t a n t metabc)lite reacted with fi-phenylisopropylhydrazine, TABLE IV SUBSTRATE

SPECIFICITV

Partially purified enzyme (57/~g protein) was incubated with o.1 ml 0. 5 M phosphate buffer (pH 7.o), 25 pl o. 5 M MgCI=, 4 mpmoles radioactive substrates, o. 3 t~mole fl-phenylisopropylhydrazine in a final volume of 25o kd. After I5 min the radioactive metabolites were measured as described under METHODS. 5 ubstrate

Epinephrine Norepinephrine Dopamine Serotonin

Relative activity ( oo )

l oo 32 -,l 2

Biochim. Biophys. Acta, 85 (1904) 247-254

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TABLE V INHIBITION

AND

ACTIVATION

OF EPINEPHRINE

OXIDIZING

ENZYME

Partially purified enzyme (88/,g protein) obtained f r o m cat salivary gland was i n c u b a t e d with 2 m p m o l e s [*H]epinephrine, I . i o - S M inhibitor or activator, o . 5 p m o l e fl-phenylisopropylhydrazine, o.i ml 0. 5 M p h o s p h a t e buffer (pH 7.0) ,25/zl 0. 5 M MgC12, in a final v o l u m e of 0. 5 ml. After 15 min the incubation m i x t u r e was assayed for the h y d r a z o n e of adrenochrome.

Compound added

Relative activity (%)

None Cateehol 3,4-Dihydroxynorephedrine Dopamine Epinine Dopa d-Epinephrine 3,4-Dihydroxybenzoic acid N-Methylepinephrine p-Phenylenediamine Tyramine Tyrosine Synephrine Normetanephrine Chlorpromazine Octopamine

IOO 5 II 14 18 22 4° 45 65 16o 15 o 147 14 I 13o 13o i 12

Effect of inhibitors and activators A number of catechols were tested for their ability to inhibit the enzymic oxidation of epinephrine. All of the catechols examined inhibited the oxidation of the catecholamine (Table V). This suggested that these compounds were competing with epinephrine and that the enzyme is relatively nonspecific and can oxidize a wide variety of catechols. When monophenolic compounds were added to the incubation mixture they were found to stimulate the formation of adrenochrome (Table V). In addition to monophenols, p-phenylenediamine, a substrate for ceruloplasmin and T A B L E VI EFFECT

OF INHIBITORS

Partially purified cat salivary gland (125 #g protein) was incubated with inhibitor, 8 m # m o l e s [SH]epinephrine, o.I ml o. 5 M MgC1v 0.2 ml o. 5 M p h o s p h a t e buffer (pH 7.o), o.5/~mole flp h e n y l i s o p r o p y l h y d r a z i n e in a final v o l u m e of 0.5 ml for 15 min.

Inhibitor

Concentration

Inhibition (%)

Diethyldithiocarbamic acid a,a'-Dipyridyl a,a'-Dipyridyl Ascorbie acid GSH P o t a s s i u m cyanide Cytochrome c

5" lO-5 M 3" lO-4 M i • io -4 M 2. lO -6 M i • lO -6 M 2. lO -8 M 3" lO-4 M

47 30 io 48 62 50 o

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j. AXELROD

chloropromazine also enhanced enzyme activity. Reducing agents such as ascorbic acid and GSH inhibited the enzyme at low concentrations and KCN inhibited at high concentrations (Table VI). Diethyldithiocarbamic acid inhibited at lmv concentrations and a,~,-dipyridyl inhibited at higher concentration. Cvtochrome oxidase (EC 1.9.3.1 ) had no effect.

Distribution of the epinephrine oxidizing enzyme Enzyme activity was examined in the parotid gland of a number of mammalian species*. The cat parotid had the greatest activity followed by rat, guinea-pig, man, and rabbit (Table VII). No detectable enzyme activity was found in the dog salivary gland. About 7o% of the catecholamine oxidase was confined to the soluble supernatant fraction of the salivary gland. TABLE VII SPECIES

DISTRIBUTION

OF EPINEPHRINF

OXIDIZING

ENZYME

D i a l y z e d so luble s u p e r n a t a n t f r a c t i o n o b t a i n e d from 25 m g p a r o t i d g l a m l was i n c u b a t e d w i t h 2 mktmoles [SH]epinephrine, o.i m l o. 5 M p h o s p h a t e buffer (pH 7.o), 25 ltl 0. 5 M MgC1 a, o. 3/ *mol e f l - p h e n y l i s o p r o p y l h y d r a z i n e in a final v o l u m e of o. 5 ml. After 3 ° rain t h e r e a c t i o n m i x t u r e w a s e x a m i n e d for t h e h y d r a z o n e of a d r e n o c h r o m e .

Species

Cat

Relative activity

(%)

i oo

Rat Guinea-pig Man Rabbit Dog

4i 33 18 7 o

Catecholamine oxidase activity was studied in a number of tissues in the cat (Table VIII). The parotid and submaxillary gland had the highest activity by far. Small amounts of enzyme were found in the skin and lung and trace amounts in the spleen, plasma, adrenal gland, uterus, pancreas, intestine, kidney and thyroid, and negligible activity was observed in the diaphragm, testes, ovary, brain, liver and heart. DISCUSSION

The catecholamine oxidase enzyme in the salivary gland of the cat appears to be different from other catechol oxidase (EC 1.Io.3.1) previously reported 2-~. The adrenochrome-forming enzyme is highly localized in the soluble supernatant fraction of salivary gland of the cat and this would distinguish it from the widely distributed cytochrome oxidase system a, ceruloplasmin which is localized in plasma 4 and catechol * Dr. J. H. HAGEN has b r o u g h t m y a t t e n t i o n to a n e n z y m e in t h e p a r o t i d g l a n d of t he m o u s e t h a t o x i d i z e s c a t e c h o l a m i n e s (Thesis, U n i v e r s i t y of Oxford, 1958).

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TABLE VIII TISSUE

DISTRIBUTION

OF EPINEPHRINE

OXIDIZING

ENZYME

Soluble supernatant fraction obtained from 2o mg of tissue was incubated with o, i ml o.5 M phosphate buffer (pH 7.o), 2 re#moles [SH]epinephrine, 25 #10. 5 M MgCI~, 0. 3/*mole/5-phenylisopropylhydrazine. After 3° min incubation the mixture was examined for the hydrazone of adrenochrome. Tissue

Relative activity

Parotid Submaxillary Skin Lung Spleen Plasma Adrenal Uterus Pancrcas Intestine Kidney Thyroid Diaphragm Testes Ovary Brain Liver Heart

1oo 7° 12 6 4 4 3 3 2 2 2 2 i I i I o o

(%)

oxidase which is present in t h e p a r t i c u l a t e fraction of skin 5. F u r t h e r m o r e , inhibition studies would i n d i c a t e t h a t t h e s u b s t r a t e specificity of t h e catecholamine oxidase in t h e cat s a l i v a r y g l a n d is different from t h a t of other catechol oxidases. Tyrosine, a s u b s t r a t e for catechol oxidase does not i n h i b i t the s a l i v a r y g l a n d e n z y m e nor does p - p h e n y l e n e d i a m i n e , a s u b s t r a t e for ceruloplasmin, Like t h e o t h e r catechol oxidases, t h e e n z y m e in t h e s a l i v a r y gland a p p e a r s to c o n t a i n Cu e+ since it is m a r k e d l y i n h i b i t e d b y a copper complexing a g e n t , d i e t h y l d i t h i o c a r b a m i c acid. The catecholamine oxidase in t h e s a l i v a r y g l a n d can be c o n v e n i e n t l y a s s a y e d b y t r a p p i n g t h e u n s t a b l e a d r e n o c h r o m e f o r m e d from [3H]epinephrine w i t h ~-phenyli s o p r o p y l h y d r a z i n e as shown in Fig. I. This procedure m i g h t also be used in t h e m e a s u r e m e n t o f o t h e r catechol oxidases. HORITA has shown t h a t p y r u v a t e can c o u n t e r a c t t h e inhibition of m o n o a m i n e oxidase resulting from f l - p h e n y l i s o p r o p y l h y d r a z i n e a d m i n i s t r a t i o n in t h e i n t a c t a n i m a l 6. P y r u v a t e , like adrenochrome, p r o b a b l y forms a h y d r a z o n e with the h y drazine, t h u s p r e v e n t i n g its i n h i b i t o r y actions on m o n o a m i n e oxidase, fl-Phenylisop r o p y l h y d r a z i n e as well as other h y d r a z i n e s h a v e been shown to alleviate m e n t a l depressions. I t is c o m m o n l y believed t h a t these h y d r a z i n e s b r i n g a b o u t their t h e r a p e u t i c actions b y i n h i b i t i n g m o n o a m i n e oxidase. I n view of t h e observations t h a t /5-phenylisopropylhydrazine as well as other h y d r a z i n e s can i n t e r a c t w i t h a l d e h y d e s a n d ketones at a physiological p H , it is possible t h a t t h e anti-depressive action of these c o m p o u n d s m i g h t be due to a chemical i n t e r a c t i o n with n o r m a l l y occurring a l d e h y d e s a n d ketones. Biochim. Biophys. Acta, 85 (1964) 247-254

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J. AXELROD

H

I

H,~ OH I~ I

"2N-N- -CT7 "1, H

~.~_

H ~ l ~

OH

CH3

OXIDIZING ENZYME

m

CH3

I

I

I

~H

{

II

CHa Fig. i. The enzymic formation and trapping of adrenochrome.

PASTAN el al. 7 have observed t h a t when the t h y r o i d gland was i n c u b a t e d with epinephrine a pink c o m p o u n d was formed which was presumed to be adrenochrome. These investigators have also n o t e d t h a t small a m o u n t s of adrenochrome in the presence of a m i t o c h o n d r i a l p r e p a r a t i o n r a p i d l y oxidized N A D P H 2. It is possible t h a t adrenochrome formed in the salivary gland might serve to oxidize pyridine nucleotides. Previous work in this l a b o r a t o r y has accounted for more t h a n 95 % of the metabolites of epinephrine a n d none of these metabolites were identified as adrenochrome ~. It is highly unlikely t h a t the formation of adrenochrome in the salivary gland of the Cat would significantly c o n t r i b u t e to the overall m e t a b o l i s m of this catecholamine. W h e n laB]epinephrine ( 1 . 2 . i o 7 c o u n t s / m i n ) was injected together with 5 m g / k g fl-phenylisopropylhydrazine into the carotid artery of the cat, large a m o u n t s of epinephrine a n d m e t a n e p h r i n e were found in the p a r o t i d gland 5 nfin later. ]'here was no measurable a m o u n t of adrenochrome present in this gland, suggesting t h a t there is negligible formation of adrenochrome i n vivo in the salivary gland.

REFERENCES 1 j. AXELROD, R. W. ALm~:RS.aND C. I). CLEMENTE, J. Neurochem., 5 (I959) 68. 2 G. H. HOGEBOOMAND M. M. ADAMS,J. Biol. Chem., ~45 (I942) 273. a D. E. GRE~:NAND D. RICHTER, Biochem. J., 31 (I93I) 596. C. G. HOLMBERGAND E. B. LAURELL, Acta Chem. Scan&, 5 (I95I) 476. 5 A. 13. LERNER, Advan. Enzymol., ~4 (I953) 73. 6 A. HORITA, Ann. N . Y . Acad. Sci., lO7 (1963) 95 I. I. PASTAN, B. HERRING, P. JOHNSON AND J. B. FIELDS, J. Biol. Chem., 237 (I963) 287 . 8 E. H. LABROSSE, J. AXELROD, I. J. I(OPlN AND S. S. KET¥, J. Clin. Invest., 4° (I961) 253. Biochim. Biophys. Acta, 85 (1964) 247-254