Tyrosinase-produced quinones and the disappearance of kynurenine in larval extracts of Drosophila melanogaster

ARCHIVES

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BIOCEIEMISTRY

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67, 74-89 (19%')

Tyrosinase-Produced Quinones and the Disappearance of Kynurenine in Larval Extracts of Drosophila melanogaster’ Edward Glassma& 3 From the Merganthaler

Laboratory for Biology, The Johns Hopkins Baltimore, Maryland

University,

Received June 25, 1956

INTRODUCTION Thimann and Beadle (25) reported that the v+ and cn+ substances of Drosophila melanogaster could not be extracted from the living organism This under aerobic conditions unless a heat treatment was Crst applied. suggested that an enzymatic oxidation of these two substances was occurring. Indeed, it was later shown that the juices obtained from larvae or pupae could inactivate solutions with v+ or cn+ activity, but again only in the presence of oxygen (24). It was the purpose of the present investigation to study this phenomenon in the light of the more recent knowledge that the vf and en+ substances are kynurenine and 3-hydroxykynurenine, respectively. It was expected that the postulated enzymatic oxidation of the former might prove to be the hydroxylation to 3-hydroxykynurenine, a step long known to occur in vivo in Drosophila and other organisms, but not yet demonstrated in cell-free extracts. This expectation was not realized. As will be shown below, the disappearance of kynurenine from cell-free extracts of Drosophila larvae is due mainly to a non-enzymatic condensation of this compound with quinones formed by the action of tyrosinase on o-phenols abundantly present in this stage. The study has been extended to show that 1 Supported in part by a contract No. At(30-l)-1472 from the Il. S. Atomic Energy Commission. * Adam T. Bruce Fellow. This material is taken in part from a dissertation presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy. * Present address: Kerchkoff Laboratories of Biology, California Institute of Technology, Pasadena, Calif. 74

TTROSINASE-PRODUCED QUINONES

75

other aromatic amines related to kynurenine also show a similar chemical reaction with these quinones. The products are intensely colored and are termed “aminoquinone pigments.” The aminoquinone pigments derived from certain amino acids and o-benzoquinone generated from catechol have the general “monoaminoquinone” structure, I (9).

N-R II

I MATERIALS

AND METHODS

Sou.rces of Chemicals The L-kynurenine sulfate was prepared by the enzymatic method of Hayaishi (8). nn-Kynurenine sulfate was kindly donated by Dr. David Bonner, 3-hydroxynn-kynurenine by Drs. Adolph Butenandt and Takeo Sakan, 3-hydroxyanthranilic acid by Drs. Bernard Davis and Takeo Sakan, and 2,3-dihydroxy-nn-benzoylalanine hydrobromide by Drs. Takeo Sakan and Hideo Kikkawa. The remainder of the chemicals were of commercial origin.

CoLlection of Drosophila Material The strain used in these investigations was an Oregon-R wild-type stock maintained in this laboratory. The flies xere raised at room temperature in half-pint milk bottles on a standard Drosophila medium containing cornmeal, dextrose, and agar, supplemented with 3% of brewer’s yeast (w/v). Third instar larvae were collected by moistening their food and washing out those which crawled up the sides of the bottle. By repeatedly allowing the larger larvae to settle and decanting the lighter, smaller ones, almost pure collections of third instar larvae were obtained. In order to remove any debris still present, the larvae were placed in a watch glass and allon-ed to crawl out into a moist chamber, after which they were rewashed and stored at -15°C. until used. Pupae were obtained by allowing the larvae to pupate in the moist chamber. After specified intervals (12-24 hr.), these were harvested with water, dried, aged if desired, and stored at -15°C. until used. Adults were collected at frequent intervals and stored as described above.

Preparation of Extracts Adults and third instar larvae were homogenized in a “Ten Brock” glass homogenizer with 0.1 111potassium phosphate buffer, pH 7.c7.3. Pupae could not be homogenized in the glass tissue-grinder because of their hard outer covering and were therefore ground with sand and buffer in a chilled mortar. In all cases

76

EDWARD

GLASSMAN

the volume of buffer varied from one to two times the live weight. The resulting homogenate was centrifuged at 23,000 X g for 20-25 min. When it became apparent that the phenomenon observed was due to tyrosinase, the following method was adopted. Third instar larvae were homogenized and centrifuged. The supernatant was treated with saturated ammonium sulfate solution to 75yo of saturation, and the precipitated proteins were redissolved, using enough phosphate buffer, pH 7.0, to make the total volume equal to the original volume of the homogenate. This solution (about 50 ml.) was dialyzed for 2 or 3 days against 4-6 changes of large volumes of distilled water. The resulting protein precipitate, which contained all of the tyrosinase activity, was centrifuged, resuspended in water, and frozen overnight. This treatment caused the tyrosinase to become aggregated on heavy black granules. By pouring off the lighter undissolved protein, and extracting with buffer, preparations of insoluble tyrosinase could be obtained with 20% of the protein present in the crude homogenate, yet with only a 20% decrease in activity. For some purposes these insoluble preparations were excellent, since cent,rifugation removed the protein and left the supernatant solution clear. However, accurate pipetting of these granules was sometimes difficult. Occasionally, preparations were found in which the tyrosinase went back into solution, and these were used in some instances. In addition, it was found that if, following dialysis, the tyrosinase was suspended and frozen in 0.1 M phosphate buffer, pH 7, instead of distilled water, some of the enzyme went back into solution.

Assay of Tyrosinase The method used was a modification of the calorimetric procedure of Markert (13). Into a Klett calorimeter tube were placed 5.0 ml. of 0.1 M potassium phosphate buffer at pH 7.0, 0.01-0.1 ml. of the enzyme to be assayed, and 1.0 ml. of 0.02 211dihydroxyphenylalanine (DOPA). The solution was aerated by bubbling air through it when readings were not being taken. The intensity of the red DOPAchrome formed was determined at 30-sec. intervals in the Klett calorimeter while using a blue (# 42) filter. The initial rate was determined from the readings of the first 2 min., as the reaction rate after that time tended gradually to diminish. The unit of activity is defined as that quantity of enzyme which can produce a change of 1 Klett unit/min.

Measurement of the Disappeara.nce of Kynurenine Compounds

and Related

Aliquots of the reaction mixtures (details are given in each experiment) taken before and after incubation were deproteinized with sodium hydroxide and zinc acetate (11). The resulting precipitate of zinc hydroxide was removed by centrifugation, leaving a clear protein-free neutral supernatant for assay. In addition, the zinc hydroxide absorbs the various aminoquinone pigments so that they do not interfere with the assay of the reactants. It was found, however, that this procedure did not inactivate the tyrosinase, and it was necessary to centrifuge within 30 sec. after mixing, or to add 0.1 &f thiourea (which completely inhibits tyrosinase) to the zinc acetate used for deproteinization. The latter procedure was feasible

TYROSINASE-PRODUCED

77

QUINONES

only with compounds which absorb at wavelengths longer than 320 rnp, above which thiourea does not interfere. The following compounds were assayed by their ultraviolet absorption at wavelengths chosen so that there was a minimum of interference by other substances: kynurenine: ~~60 = 4400’; 3-hydroxykynurenine: ~310= 42405; anthranilic acid: ~09 = 2900,6 ~330= 15U01; 3-hydroxyanthranilic acid: csrk = 31204; p-aminobenzoic acid (PABA) : ~300= 7520; p-hydroxybenzoic acid at 250 rnp, and kynurenic acid: ez3p = 9800.6 Tryptophan except that

was assayed the quantities

according to the method used were halved.

of Spies and Chambers

(23),

-4bsorption Spectra All absorption spectra were taken with a Beckman DU or a DKZ recording spectrophotometer. When the spectra of some of the pigments were taken, it was necessary to minimize melanization by using low concentrations of tyrosinase “granules” (lo-25 unitsj, 2-2.5 pmoles of DOPA or catechol, 2-5 bmoles of amine, and enough buffer, pH 7.0, to make 3 or 4 ml. After pigment formation had proceeded to the point where the color was distinct, the solution was centrifuged and the absorption spectrum taken.

Oxygen Consumption Warburg manometry was carried out as recommended by Umbreit et ul. (27). Each reaction flask contained 5 pmoles of catechol or 2.5 pmoles of DOPA, either one or ten molar equivalents of the amine, 180-250 units of tyrosinase, and enough 0.1 Jf phosphate buffer to make 3.1 ml. The center well contained a small piece of fluted filter paper and 0.2 ml. of 20% KOH. The temperature was maintained at 25°C.; later, with the advent of warmer we&her, 30°C. was used.

Ammon.ia Production Ammonia was determined using Conway units for microdiffusion (2). The solution to be assayed was pipetted into the outer well of the sealed unit with 1 ml. of saturated KZCOJ solution. The ammonia was absorbed in the center well by 1.5 ml. of 0.01 N HCl. After 90 min., 1.0 ml. of the HCl from the center w-e11was assayed for ammonia by adding it to 8.5 ml. of distilled water. Thorough mixing was followed by the addition of 0.5 ml. of Nessler’s reagent. After 15 min. the yellow color which developed was read in a Klett calorimeter with a ~$42 (blue) filter.

Acid Production The hydrogen ions released during the reaction between catechol or DOPA and the various amines were measured by the amount of 0.01 N NaOH (from a microburet) required to keep the initial pH (6.7-7.3) of the reaction misture constant. 4 Mehler and Knox 6 Dalgliesh (3).

(16).

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EDWARD GLASSMAN

The reaction mixture consisted of 20 ml. of distilled water, IO-20pmoles of catechol OP DOPA, 20-100 pmoles of the amine, and 0.5 ml. of an enzyme solution containing 500 units/ml. (added last). The mixture was aerated with air passed through

soda-lime to remove CO* . RESULTS

Initial experiments confirmed the report of Tatum and Beadle (24) that the concentration of the vf substance (kynurenine) decreaseswhen it is incubated with larval extracts. In a typical experiment (Fig. 1) there was a net change in optical density at 360 mp of 0.527 (-A 0.204 of the control subtracted from -A 0.731 of the experimental), corresponding to a decrease of 0.35 pmole kynurenine/ml. of reaction mixture. Other experiments demonstrated that the greatest amount of kynurenine was removed when the reaction mixture was shaken, no kynurenine disappearing in the absence of oxygen, or if the homogenate was boiled prior to use. These data seemedto confirm the idea that an enzymatic oxidation or hydroxylation of kynurenine was involved. However, absorption spectra (Fig. 1) of the various solutions revealed that while the concentration of kynurenine, which absorbs at 260 and 360 rnp, decreased, no peak corresponding to an absorbing product appeared. To test the possibility that the product might have been removed during deproteinization, the zinc hydroxide precipitate derived from one experiment was dissolved in a small quantity of 2 N I-ICI, and the resulting suspension was centrifuged. It was found that the supernatant, when derived from extracts containing kyurenine, contained a brown pigment. The formation of t#hispigment from kynurenine had been obscured by the simultaneous blackening of the reaction mixture by tyrosinase action; its properties, however, precluded its being the brown eye pigment. of Drosophila. Further evidence against the hydroxylation of kynurenine was obtained when it, was found that this compound did not affect the amount and rate of oxygen consumed by the homogenate. In addition, compounds structurally or metabolically related to kynurenine, such as 3-hydroxykynurenine, ant#hranilic acid, 3-hydroxyanthranilic acid, and p-aminobenzoic acid (PABA), were also found to decrease markedly in amount when incubated with crude larval extracts, and also produced pigments. Negative results mere obtained with kynurenic acid, tryptophan, 2,3-dihydroxybenzoylalanine, and p-hydroxybenzoic acid, indicating that the free aromatic amino group is essential. This last fact, as well as the blackening of larval extracts upon shaking,

TYROSINASE-PRODUCED

QUINONES

79

3.c

2.l d 6

I .(

I

I

240

270

300

330

WAVELENGTH FIG. 1. Absorption

360

390

(rq.U

spectra of larval extracts incubated with or without kynurenine.

The larval extract contained 3.5 g. of larvae per 2.0 ml. of Drosophila Ringer’s solution. The reaction vessels contained 3.0 ml. of the extract, 1.0 mg. kynurenine sulfate (control had none), and 0.1 M phosphate buffer, pH 7.3, to give a final volume of 5.0 ml. The solutions were shaken in air at room temperature for 3.5 hr. Deproteinization and kynurenine assay is described in the text. Curves a. Homogenate with kynurenine, prior to incubation b. Homogenate alone, prior to incubation c. Homogenate with kynurenine, after incubation d. Homogenate alone, after incubation

led to the conclusion that these phenomena were most likely due to a condensation between aromatic amines and quinoid compounds formed during the action of tyrosinase on o-phenols, in a reaction similar to that reported to occur between amino acids and o-benzoquinone derived from catechol (7, 9, 10). The brown product observed above was the amino-

80

EDWARD

GL.4SSMAN

quinone pigment formed from this condensation. In support of this is the observation that inhibition of tyrosinase with 0.01111 sodium diebhyldithiocarbamate will simultaneously inhibit removal of kyurenine. In order to confirm t’his unequivocally, it was necessary to separate the tyrosinase from its substrate(s) by treating the crude homogenate with saturated ammonium sulfate as described above, omitting dialysis and the succeeding steps. When the amine is incubated with the resulting protein solution, one does not observe the blackening associated with the action of tyrosinase, nor the disappearance of kynurenine or other amine-containing compounds, nor aminoquinone pigment formation. If one now adds a substrate of t,yrosinase back to this preparation, all of these phenomena may again be observed. Table I shows that additions of randomly chosen compounds, such as diphosphopyridine nucleotide (DPN), ascorbic acid, or copper chloride, have no effect,, while DOPA, catechol, and phenol are quite effective, as is boiled crude larval extract. Confirmatory experiments demonstrated that substitution of crude pupal and adult homogenates of Drosophila for the larval tyrosinase did not result in pigment formation until additions of DOPA or catechol were made. This is so because these stages lack the quantity of endogenous TABLE The Eflects

of Tyrosinase

Substrates

I

on the Disappearance

of Kynurenine

Enzyme preparation: 2 g. larvae/3 ml. 0.1 M phosphate buffer at pH 7.3. After centrifugation, the proteins of the homogenate were precipitated with ammonium sulfate and redissolved as described in the text. The reaction vessels contained 1.0 ml. of crude homogenate, 0.5 mg. of kynurenine sulfate, the additions, and sufficient buffer to make up to 3.0 ml. The control lacked kynurenine sulfate. The vessels were shaken for 2 hr. at room temperature. Constituents

Enzyme “ “ ‘l ‘I ‘I “ ‘I ‘C

alone + kynurenine + “ + “ + “ + ‘I + “ f “ + “

+ + + + + + +

0.5 mg. DPN 0.5 mg. ascorbic acid 0.4 mg. CuClt 0.5 mg. DOPA 0.5 mg. catechol 0.05 ml. water-saturated phenol 0.5 ml. boiled extract of crude homogenate

4pmoles kynurennw/ml. reaction mixture

0.00 0.00 0.00 0.00 0.00 -0.30 -0.32 -0.17 -0.24

TYROSINASE-PRODUCED QUINONES

81

substrate found in the third instar larvae. These results have been confirmed, using tyrosinase prepared from acetone powders of mushrooms or blowfly larvae in a method essentially similar to that described above. OBSERVATIONS ON THE AMINOQUINONE

PIGMENTS

Stoichiometry It is difficult t,o come to any conclusion concerning the stoichiometry of these reactions. Even in the presence of excess amine it was found that a large fract,ion of the quinone is diverted to melanin production. Only by slowing down quinone formation (by using very small amounts of tyrosinase) was it possible to provide conditions for almost complete trapping of the quinone by the amine, but the reaction is not completed because of inactivation of the enzyme. These factors affect the apparent stoichiometry of the reaction, and it is difficult to ascertain whether the pigment contains one or two molecules of amine per molecule of quinone. Dianilinoquinones are produced from aniline and o-benzoquinone [cf. (7)], and some data in the present investigations indicate that the corresponding diaminoquinone may be formed. When DOPhquinone is used, two molar equivalents of the amine prove only slightly more effective than one equivalent. However, if catechol is used, then two molar equivalents of t,he amine sometimes doubled the amount of amine utilized. The fact that this occurred only rarely, prevented further study. A more typical experiment in which kynurenine content was varied is depicted in Fig. 2. Only a moderate increase in its utilization is observed between one and two molar equivalents of this aromatic amine. h curve similar to this was obtained by Jackson and Kendall (9) and by Trautner and Roberts (26) from calorimetric measurements of the pigment formed when the molar equivalents of aliphatic amines were varied. Trautner and Roberts (26) considered that this type of curve pointed to the formation of the diaminoquinone when the amine was in excess. However, these and ot,her data (4) make it seem more likely t,hat the slight increase in pigment is due to the fact that some of the quinone which would otherwise have been diverted to melanin formation reacts with the excess amine. Types of Pigments Table II summarizes the colors and absorption maxima of various water-soluble pigments. In obtaining these data, conditions were controlled so as to minimize melanization, and at the same time to utilize

82

EDWARD

GLASSMAN

enough of the amine so as not to interfere with the absorption spectrum of the colored complex. Another difficulty encountered was that some of the pigments (especially those derived from anthranilic acid and PABA with o-benzoquinone) are unstable, and their colors and spectra change rapidly with time. Thus, it cannot be said with certainty that the spectra reflect the initial chromophore group, for they might instead represent secondary arrangements. Other types of pigments are possible, depending upon the conditions. Black, amorphous solids insoluble in water and carbon tetrachloride, sparingly soluble in benzene, and soluble in ether, ethanol, acetone, and pyridine are produced from glycine and chemically generated o-benzoquinone (7) or from anthranilic acid and o-benzoquinone generated from catechol with potassium ferricyanide (four molar equivalents of the latter with equimolar equivalents of a catechol and anthranilic acid in 0.1 M phosphate buffer, pH 7.9). If the amounts of anthranilic acid are inCATECHOL

0

0.3 -

0

DOPA A-

A

I

I 2

I 3

MOLAR RATIO (

I 4

KYN;/TyR. SUB. )

2. The effect of varying kynurenine concentration on its utilization in the formation of aminoquinone pigments. In addition to varying amounts of kynurenine, each reaction vessels contained 50 units of tyrosinase, 2.5 rmoles of DOPA or catechol, and sufficient buffer, pH 7, to make 4.0 ml. The reaction vessels were shaken for 2 hr. at room temperature; these conditions are such that the reaction had stopped by this time. The data are presented as the decrease in micromoles of kynurenine against the molar ratio of kynurenine to tyrosinase substrate. FIG.

TYROSINASE-PRODUCED

TABLE

II

Color and Absorption Maxima of the Aminoquinone Amine

With DOPA-quinane Absorption P&&y lll%dlma

-

Glycine

None

Tryptophan Proline Anthranilic

None None Brown

415-?

PABA

Brown

410-420

Kynurenine 3-Hydroxykynurenine 3-Hydroxyanthranilic acid 2,3-Dihydroxybenzoylalanine

Brown Red-brown Red-brown

455 450 350, 453

acid

83

QUINONES

-

Pigments

With o-benzoquinone Absorption Pb$;t UlXUIll~

Reddish vio475480 let Reddish violet 475480 Purple 520 Purple, 516525, flatchanges to tens out with brown time Purple, 490-530, flatchanges to tens out with brown time ? Green 430 Green Red-brown 440 Green

485, 635

creased relative to the amounts of o-quinone (generated from catechol with potassium ferricyanide), there appears in addition to the black solid a smaller amount of a reddish purple compound. This is insoluble in water, 2 N HCl, or ether, but is soluble in concentrated acid, 2 N KOH, acetone, or ethanol. The amount obtained was too small for further characterization. A similar pigment is also formed in small amounts when tyrosinase is used as an oxidant of the o-phenol, if conditions are right (see, for example, those used to measure acid production). This pigment may be demonstrated by deproteinizing with zinc acetate and sodium hydroxide, dissolving the zinc hydroxide in 2 N HCl, and centrifuging down the insoluble pigment. It is formed only from aromatic amines; if an aliphatic amine or a “mixed” amine, such as kyurenine, is used, this particular pigment is not formed. It will be noticed in Table II that DOPAquinone does not combine with amino acids. An observation of t,he same type was made by Krueger (12), who showed that while o-benzoquinone can react with acetoacetic acid, the quinone derived from tyrosine (i.e., DOPAquinone) is inactive toward this acid. The reason for this is obscure. While steric factors might seem to be important, this is not likely in view of the fact that the quinone derived from 3,4dihydroxyphenyllactic acid, which differs

84

EDWARD

GLASSMAN

from DOPAquinone only by having a hydroxyl group substituted for the amino group on the side chain, apparently can condense with amino acids to form the colored complexes (7). Since the side chain of the quinone of 3,4-dihydroxyphenyllactic acid does not close to form an indole nucleus, as DOPAquinone does, the formation of this structure through the amino group on the side chain may be the determining factor in the reactivity of DOPAquinone with various amines. This would explain why tyrosine, which reacts readily with o-benzoquinone (7), does not react with the DOPAquinone to which it eventually gives rise. Oxichtive Deamination

of Amino Acids

Aminoquinone pigments derived from either aliphatic or aromatic amines can oxidatively deaminate a-amino acids, such as glycine (9, 10) or kynurenine. Purely aromatic amines, like PABA and anthranilic acid, are not deaminated by any aminoquinone pigment. Previous reports have indicated that secondary amines were also not deaminated (9). These data tend t#o subst,antiate the suggestion (26) that o.xidative deamination is due to rearrangement and loss of an active a-hydrogen following the formation of a Schiff base between the a-amino group and a keto group of the pigment. Oxygen Consumption One difference between aliphatic and aromatic amines is their effect on the amount of oxygen consumed during t#he oxidation of catechol or DOPA (normally 2 and 3.4 at,oms oxygen/mole substrate, respectively). Previous reports indicate that aliphatic amines, if present in equimolar concentrahion, do not affect the amount of oxygen normally consumed when catechol is oxidized by tyrosinase (6). If the amine is in excess, however, its oxidative deaminat,ion t,akes place and additional oxygen is consumed (9, 10, 26). On the other hand, in t,he present investigation it was found that one to ten molar equivalents of kynurenine or anthranilic acid increase the amount of oxygen consumed during the t.yrosinase oxidation of catechol or DOPA by 0.3-0.9 moles oxygen/mole substrate. Higher concentrations produced a more pronounce increase since more quinone is utilized. Excess aniline also causes a similar increase (9). The cause of the increase is unknown, but it cannot be ascribed to oxidative deamination of the amines, since (a) it occurs even in t,he presence of equimolar amounts of amine and catechol or DOPA, and (b) the consumption of oxygen ceases when the catechol or DOPA is depleted. Thus it appears that there may be additional factors op-

TPROSINASE-PRODUCED

erating during the formation amines.

85

QUINONES

of aminoquinone

pigments from aromatic

Acid Production Another difference between the aliphatic and aromatic amines is in the production of acid during pigment formation. When aliphatic amines produce pigments with the o-quinone that is enzymatically produced from catechol or homocatechol, almost one mole of hydrogen ion is released for every mole of quinone (9). However, aromatic amines (PABA, anthranilic acid) do not (Table III) and, in fact, suppress the acidity which normally arises during catechol oxidation by tyrosinase. Compounds containing an aliphatic and an aromatic amino group (kynurenine) do increase the acidity, indicating that kynurenineprobablyreacts with o-benzoquinone through the amino acid moiety instead of the aromatic amino group. TABLE Acid Production

during

III

Aminoquinone

Pigment

Formation

To 20 ml. of dist. water were added 10 or 20 pmoles of catechol or DOPA, 20106 pmoles of the amine, and 56 units of tyrosinase. The pH was kept constant by adding 0.01 N NaOH from a microburet. Amine added

None Proline Kynurenine hnthronilic

acid

PABA

Amine present jmoles -

20 40 50 50 40 50 40 50

-

None

-

Kynurenine Anthranilic PABA

50

acid

100 50 106 50 100

Tyrosinase substrate present Catechol DOPA pmoles wdes

20 10 20 20 20 10 20 10 20 10

-

-

-

10 10 10 10 10 10 10 10

NaOH required .umoles

7.0 4.0 17.0 9.3 12.0 5.6 None None None Kane 8.2 9.2 8.1 6.5 5.7 6.1 7.5 4.2

86

EDWARD GLASSMAN

In the absence of amine, about one mole of hydrogen ion is released per mole of DOPA. Since the amine had no consistent effect, interpretation is dif%cult, but it should be noted that melanization was much more extensive with DOPA than when catechol was used. DISCUSSION

The evidence in the present paper indicates that the “enzymatic oxidation” of kyurenine suggested by Tatum and Beadle (24) is not a primary one. Instead, the requirement for oxygen is due to the action of tyrosinase on its own substrates, which are present in the crude extract and which are oxidized to quinones. These in t,urn react nonenzymatically with aromatic amino groups so as to produce a pigmented complex and thereby destroy the v+ or cnf activity. That this explanation is correct is indicated by the fact t,hat removal of the endogenous substrates of tyrosinase present in the crude larval homogenate completely destroys the capacit,y of the homogenate to “oxidize” added kynurenine. Only by the addition of a sub&rate of tyrosinase is that activity restored. The same kind of reaction has also been shown to take place between the naturally occurring quinones and a variety of aromatic amines related 60 kynurenine, including %hydrosykynurenine, anthranilic acid, 3-hydroxyanthranilic acid, and p-aminobenzoic acid. These compounds may be considered to be substituted derivatives of aniline, which has long been known to condense with quinones [cf. (7, 19)]. It has also been shown that almost any free amino group, such as exists in primary or secondary amino acids (7,9,10,26), in protamine or nucleoprotamine (14), or in secondary amines (l), may react mibh o-benzoquinone derived from catechol to produce pigmented products. The present investigation has shown t,hat amino acids do not react either with DOPA quinone or wit,h the quinones which occur in the crude larval extracts of Drosophila; yet amino acids are capable of reacting with the quinones of other tyrosinase substrat,es found in insect,s (7). It would therefore appear t,hat DOPA may be the natural substrate of tyrosinase in Drosophda, alt#hough further work is necessary to substantiate this point. The decrease in ultraviolet absorption of the crude homogenate to which no kynurenine was added is due to t,he disappearance of a compound which has an absorption maximum at approximately 370 rnp (Fig. 1). The exact nature of this subdance is still unknown, but it does not disappear in the absence of oxygen, nor in the presence of 0.01 M

TYROSINASE-PRODUCED

QUINONES

87

sodium diethyldithiocarbamate, which is a potent inhibitor of tyrosinase (unpublished data). The unknown substance does not appear to be kynurenine, since the rate of disappearance of kynurenine is independent of this compound (4). It may well represent endogenous 3-hydroxykynurenine, but this has not been established. It is of interest that an extensive examination of cell-free extracts of various stages in the life cycle of Drosophila has failed to demonstrate any enzymes which can directly metabolize kynurenine. Neither a kynureninase, which hydrolyzes kynurenine to anthranilic acid and alanine, nor the transaminase which converts kyurenine to kynurenic acid could be shown to exist in larvae, pupae, or adults. These enzymes have been extracted from such diverse sources as mammalian liver, bacteria, and Neurospora [cf. (15)]. The nature of the mechanism which prevents the addition of amino acids or other compounds to the quinones that are formed during melanogenesis in via!0is not known, but is of considerable interest. Quinones are thought to be involved in the hardening of the insect cuticle (6, 17, 18), and thus have other functions besides the formation of melanin. Chemically, quinones are extremely reactive and have a strong tendency to form addSon compounds not only with those compounds containing free amino groups, but with sulfhydryl compounds (22) and cr-keto acids (12) as well. Furthermore, quinones are powerful oxidizing agents and are known to oxidize various dyes, pyridine nucleotides, ascorbic acid, and other compounds [cf. (2O)j. Tyrosinase-produced quinones have also been reported to inactivate certain enzymes (21). For this reason, biochemical investigations involving the larvae of Drosophila or other insects which contain both tyrosinase and an abundance of its endogenous substrate, would do well to take these phenomena into account. The possible physiological significance, if any, of the aminoquinone pigments is not clear. That quinones which occur naturally in Drosophila can react with aromatic amines to produce highly pigmented products suggests that some natural pigme& might be formed in oivo in this manner. Because 3-hydroxykynurenine is the precursor of the brown eye pigment of this species,it is possible that this pigment is one of these. Evidence in regard to this point is lacking, however, mainly because of the difficulties involved in characterizing the eye pigment [see (5)]. In any case the wide variety of pigments possible from various amines and quinones under differing conditions would argue for the possibility that some natural pigme& might be formed from t,hesecompounds in living systems.

88

EDWARD GLASSMAN ACKNOWLEDGMENTS

I should like to express my gratitude to Dr. Bentley Glass for his help during the course of this investigation, and to Dr. H. K. Mitchell for reading the manuscript. I should also like to thank my wife, Ann, for assistance in some of the experiments. SUMMARY

The “enzymatic oxidation” of the zl+and cnf compounds (kynurenine and 3-hydroxykynurenine, respectively) by crude larval extracts of D. melanogaster has been shown to be due to a non-enzymatic condensation of these aromatic amines with quinones formed during the act,ion of tyrosinase on its own substrates. These quinones are believed t,o derive from dihydroxyphenylalanine. Some data on the aminoquinone pigments derived from aromatic and aliphatic amines are also reported. It is suggested that the brown eye pigment of Drosophila may be related to the aminoquinone pigments. The reason why quinones, which are very reactive compounds, do not react with amino acids or other substances during melanogenesis in vivo remains a challenging question. REFERENCES 1. BEEVERS, H., AND JAMES, W. O., Biochem. J. 43, 636 (1948). Analysis and Volumetric Error.” D. Van 2. CONWAY, E. J., “Micro-diffusion Nostrand Co., Inc., New Pork, London, 1947. 3. DALOLIESH, C. E., Biochem. J. 62, 3 (1952). 4. GLASSMAN, E., Ph.D. Thesis. The Johns Hopkins University, Balbimore, Maryland, 1955. 5. GOODWIN, T. W., AND SRISUKH, S., Biochem. J. 47, 649 (1950). 6. HACKMAN, R. H., Biochem. J. 64, 371 (1953). 7. HACKMAN, R. H., AND TODD, A. R., Biochem. J. 66, 631 (1953). 8. HAYAISHI, O., Biochem. Preparations 8, 108 (1953). 9. JACKSON, H., AND KENDALL, L. P., Biochem. J. 44, 477 (1949). 10. JAMES, W. O., ROBERTS, E. A., BEEVERS, H., AND DE KOCH, P. C., Biochem. J. 43, 626 (1948). 11. KNOX, W. E., AND MEHLER, A. H., J. Biol. Chem. 187, 419 (1950). 12. KRUEQER, C., Arch. Biochem. and Biophys. 66, 398 (1955). 13. MARKERT, C. L., Genetics 36, 60 (1950). 14. MASON, H. S., AND PETERSON, E. W., J. Biol. Chem. 212, 485 (1956). 15. MEHLER, A. H., in “A Symposium on Amino Acid Metabolism” (W. D. McElroy and B. Glass, eds.), p. 882. The Johns Hopkins Press, Baltimore, 1955. 16. MEHLER, A. H., AND KNOX, W. E., J. Biol. Chem. 187, 431 (1960). 17. PRYOR, M. G. M., RUSSELL, P. B., AND TODD, A. R., Biochem. J. 40,627 (1946). 18. PRYOR, M. G. M., RUSSELL, P. B., AND TODD, A. R., Nalltre 169, 399 (1947).

TYROSINASE-PRODUCED

QUINONES

19. PUGH, C. E. ItI., AND RAPER, H. S., Biochem. J. 21, 1370 (1927). 20. SINGER, T. P., AND KEARNY, E. B., in “The Proteins” (H. Neurath 21. 22. 23. 24. 25. 26.

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and K. Bailly, eds.), Vol. II, Pt. A, p. 121. Academic Press, New York, 1951. SIZER, I. W., and BRINDLEY, C. O., J. Biol. Chem. 186, 323 (1950). SNELL, J.&I., AND WEISSBERGER,A.,J. AP,z. Chem. Sot. 61, 450 (1939). SPIES, R., AND CHAMBERS, D. C., Ana.1. Chew. 20, 30 (1948). TATUM, E. L., AND BEADLE, G. W., J. Gen. PhysioZ. 22, 239 (1938). THIMANN, K., AND BEADLE, G. W., Proc. Natl. Bead. Sci. U. S. 23, 143 (1937). TRAUTNER, E. hl., AND ROBERTS, E. A. H., dustralian J. Sci. 3, 356 (1950). UIVIBREIT, W. W., BURRIS, R. H., AND STAUFFER, J. F., “Manometric Techniques and Tissue Metabolism.” Burgess Publishing Co., Minneapolis, 1949.