Reaction of Polycyclic Hydrocarbon-Cysteine Conjugates ...Preparation of Pyrophosphate-32P. The K432P2O7 was prepared from carrier-free orthophosphate-32P (New England Nuclear Corporation,

[CANCER RESEARCH 30, 155-161, January 1970]

Reaction of Polycyclic Hydrocarbon-Cysteine Conjugates withthe Aminoacyl-RNA Synthetase System1

E. T. Bucovaz, J. C. Morrison, H. L. James, C. F. Dais, and J. L. Wood

Department of Biochemistry, University of Tennessee Medical Units, Memphis, Tennessee 38103

SUMMARY

A number of arylcysteine derivatives, which are metabolitesof polycyclic aromatic hydrocarbons, have been synthesized.These amino acid analogs were tested for their ability to actas substrates for aminoacyl-RNA synthetases in an in vitrosystem using enzyme fractions of bakers' yeast. These studieshave shown that 5-(p-chlorophenyl)-L-cysteine, 5-(9 ,10dihydro-9-hydroxy-l 0-phenanthryl)-L-cysteine,5-(5 , 6-dihydro-6-hydroxy-5-benz(a)anthryl)-L -cysteine,5- (5 , 6- dihydro-6-hydroxy-5-dibenz(a , h)anthryl- L-cysteine,and S-(7-benz(a)anthryl)-methyl-L-cysteine are activated andtransferred to tRNA. The arylcysteines were observed tocompete with a variety of natural amino acids. This investigation revealed that chlorophenylcysteine competed witharginine; dihydrohydroxyphenanthrylcysteine competed withglutamic acid, phenylalanine, and histidine for activatingenzymes; dihydrohydroxybenzanthrylcysteine likewise competed with arginine and phenylalanine; and dihydrohydroxy-dibenzanthrylcysteine competed with methionine and leucine.Thus, the structure of the hydrocarbon moiety conjugatedwith the sulfhydryl group of cysteine determines inparticular the synthetases involved in amino acid activation.Moreover none of the cysteine conjugates were activated bythe cysteinyl-RNA synthetase. Hydrocarbons are bound toprotein by this pathway at sites different from thoseresulting from direct interaction.

INTRODUCTION

Protein binding of administered carcinogenic hydrocarbonshas been well established (16). The implication of suchmetabolic events in the process of carcinogenesis has beeninferred (30). This report describes a pathway by whichcarcinogens as conjugates of amino acids can be bound toproteins via the mechanism utilized for amino acid incorporation.

The in vitro incorporation of aromatic hydrocarbon-cysteine conjugates, 5-(l, 2, 3, 4-tetrahydro-2-hydroxy-l -

naphthyl)-L-cysteine and PCP-cysteine2 into microsomal

protein of rat liver was reported in earlier communications from this laboratory (7-9). Subsequently,we reported that tetrahydrohydroxynaphthylcysteine appearsto be activated by the valyl and isoleucyl RNA synthetases ofrat liver or yeast, and the conjugate is transferred to therespective species of tRNA of yeast (6). Furthermore, theconjugate was incorporated into ribosomal protein by arat-liver preparation. According to prevailing theories of aminoacid metabolism, tetrahydrohydroxynaphthylcysteine wouldbe transferred to some of the positions which are normallyoccupied by valine and isoleucine in the forming peptidechain.

These findings provide a pathway by which aromatichydrocarbons can be bound to specific sites during synthesisof protein molecules. The sites are distinct from thoseinvolved in direct binding of hydrocarbons or their metabolites to intact tissue proteins.

Typical cysteine conjugates of chlorobenzene, phenan-threne, and the carcinogens methylbenzanthracene and di-benzanthracene have been prepared and studied for theirbehavior in the protein-biosynthesizing system. This paperreports the activation and transfer of the hydrocarbon-cys-teine conjugates to tRNA. Preliminary reports have beenpresented (24-26).

MATERIALS AND METHODS

Preparation of Enzyme Fractions A, B, and C. Bakers'

yeast, 3 Ib, was frozen for 6 to 8 hr in approximately 3liters of an ether-C02 mixture to break the cells. Beforethawing, the excess ether was decanted from the yeast, andthe residual ether was removed by vacuum at room temperature. The resulting homogenate was cooled to 0â€”2Â°,15 g

KC1 were added, and the mixture was stirred for 12 to 14hr. Following the KC1 addition step, the homogenate wascentrifuged at 7,700 X g for 20 min to remove all debris;

This investigation was supported in part by USPHS Research GrantCA-01228 from the National Cancer Institute and by USPHSResearch Grant AM-09131 from the National Institute of Arthritisand Metabolic Diseases.

Received November 22, 1968; accepted June 3, 1969.

2The abbreviations used are: tRNA, transfer ribonucleic acid; PPj,inorganic pyrophospnate; PCP-cysteine, 5-(p-chlorophenyl)-L-cysteine;DHP-cysteine, 5-(9 ,10-dihydro-9-hydroxy-10-phenanthryl)-L-cysteine;DHD-cysteine, S-(5 , 6-dihydro-6-hydroxy-5-dibenz(a , h)anthryl)-L--cysteine; DHB-cysteine, S-(5 ,6-dihydro-6-hydroxy-5-benz(a)an-thryl)-L-cysteine; BM-cysteine, S-(7-benz(a)anthrylmethyl)-L-cysteine.

JANUARY 1970 155

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Bucovaz, Morrison, James, Dais, and Wood

the supernatant liquid was filtered through cheesecloth andthen centrifuged at 105,000 X g for 1 hr. The volume of thesupernatant liquid recovered varied between 350 and 375 ml,depending upon the water content of the yeast. A series ofammonium sulfate precipitation steps followed. Throughoutthese fractionation steps the temperature was maintained at0â€”4Â°.Initially, 15.36 g solid ammonium sulfate were added

to 300 ml of the crude enzyme fraction. After 3 days theprecipitate formed was removed by centrifugation and discarded. The supernatant fraction was adjusted to 60% saturation upon the addition of 33.3 g ammonium sulfate/100 ml,followed by thorough mixing. After 2 hr, the mixture wascentrifuged at 7,700 X g for 20 min. The supernatant layerwas discarded, and the precipitate was dissolved in 180 ml ofwater, resulting in a total volume of 230 to 360 mldepending on the amount of protein precipitated. Thispreparation was termed the "60% extract." Three fractions

were obtained by a second ammonium sulfate fractionation:Fraction B, 0 to 42%; Fraction C, 42 to 50%; and Fraction A,supernatant fraction equivalent to 50 to 60% saturation. The3 fractions were adjusted to 0.01 M Tris, pH 7.25, and thendialyzed against Tris of the same concentration and pH toremove ammonium sulfate and other small-molecular-weightcomponents. Fractions A, B, and C were used in thisinvestigation as sources of enzymes.

Transfer RNA of Bakers' Yeast. Transfer RNA was

obtained from the General Biochemicals Company (ChagrinFalls, Ohio) and was used as the acceptor of the arylcys-teines and naturally occurring amino acids.

Preparation of Pyrophosphate-32P. The K432P2O7 wasprepared from carrier-free orthophosphate-32P (New England

Nuclear Corporation, Boston, Mass.) by pyrolysis (19).

ATP-PPi of Exchange Reaction. The assays were carried outas described in Table 1. The ATP-32PPj exchange reaction

was terminated by the addition of 1 ml 10% trichloraceticacid. The mixture was briefly centrifuged to remove theprecipitated protein, and the ATP was absorbed on DarcoG-60 (Matheson, Coleman and Bell, East Rutherford, N. J.)according to the method of DeMoss and Novelli (10).

Preparation of ,s-substituted Cysteines. PCP-cysteineL-cysteine was prepared by the method of du Vigneaud et al.(12).

Epoxides were prepared according to the procedure ofNewman and Blum (28). The epoxides were conjugated withcysteine by a new procedure which yields crystalline products.For labeled products cysteine-35 S was used.

9 , 10-Dihydro-9 , 10-epoxyphenanthrene was prepared fromdiphenyl-2 , 2'-dialdehyde (28) by reaction with

tris(dimethylamino)phosphine. For conjugation with cysteine,0.3 g 9 , 10-dihydro-9 , 10-epoxyphenanthrene, 0.4 gL-cysteine hydrochloride monohydrate, and 0.4 g sodiumbicarbonate were mixed with 15 ml water and 15 ml freshlydistilled dioxane. The mixture was stirred at 65-70Â° for 2 hr.

After cooling, 10 ml water were added to the mixture, and thepH was adjusted to 6 to 7 by addition of 10% HC1. Themixture was flash-evaporated under vacuum at 30Â°to a 5-ml

volume and cooled in an ice bath for 30 min. White crystals,m.p. 158â€”161Â°(with decomposition), weighing 0.44 g

separated. These were extracted with two 15-ml portions ofdry benzene at 50Â°.The residue, 0.4 g, was extracted with 25

ml hot methanol. The extract was concentrated to 5 ml andtreated with water. Lustrous crystals separated. The yield ofS-(9,10-dihydro-9-hydroxy-10-phenanthryl)-L-cysteine meltingat 171.5-172Â° (with decomposition) was 0.29 g (62% of the

theoretical amount). Recrystallization did not change themelting point. A paper chromatogram developed withbutanol:acetic acid:water (1:2:1) had a single ninhydrin-positive spot at Rp 0.7.

5 , 6-Dihydro-5 , 6-epoxybenz(a)anthracene was preparedaccording to the procedure of Newman and Blum (28). Theepoxide, 0.2 g, and 0.23 g of L-cysteine hydrochloridemonohydrate were reacted essentially as described for thephenanthrene conjugate above. A 29% yield of S-(5 ,6-dihydro-6-hydroxy-5-benz(a)anthryl)-L-cysteine melting at174â€”175Â°was obtained. The elemental analysis values for the

compound agreed with the theoretical values. Chromatographyin butanol:acetic acid:water (1:2:1) gave a single spot at RF0.75.

2-Phenylphenanthrene-2 , 3-dialdehyde was prepared fromdibenzanthracene by oxidation of the hydrocarbon withosmium tetroxide followed by treatment of the resulting diolwith periodate (15). A more efficient procedure resulted fromozonolysis of dibenz(a,h)anthracene by the method ofMonconi et al. (27). The yield of dialdehyde was 55% of thetheoretical amount. 5 , 6-Dihydro-5 , 6-epoxydibenz(a,A)an-thracene was prepared from the dialdehyde. To 0.2 g of thedialdehyde in 2 to 3 ml dry benzene was added 0.2 g oftris(dimethylamino)phosphine. The system was kept undernitrogen. The mixture was shaken for 5 ml at 55% and thenwas cooled to 5Â°to produce crystals. The supernatant liquor

was decanted. The residue was washed 3 times with coldcyclohexane and then recrystallized from cyclohexane: benzene (70:30) to yield 0.17 g of light green needles. These hadan indefinite melting point as observed by Boyland and Sims(4). The yield of 'product was 88% of the theoretical.

5-(5 , 6-Dihydro-6-hydroxy-5-dibenz(aJi)anthryl-L-cysteinewas prepared from a mixture of L-cysteine hydrochloridemonohydrate, 0.6 g, sodium bicarbonate, 0.6 g, and theepoxide, 0.3 g, in 20 ml water and 20 ml freshly distilleddioxane. The mixture was stirred for 1.5 hr at 75Â°and then

adjusted to pH 7.0 at room temperature by slow addition of5% HC1. When the solution was evaporated to a 50-ml volumeand cooled at 5Â°,it yielded 0.5 g light-tan, solid material. The

dried, crude product was extracted with 20 ml dry benzene at40Â°for 2 hr. The residue was treated with 20 ml dry methanol

and filtered to remove cystine. The methanolic solution wasplaced on a 7-x 5/8-inch Florisil column and was eluted withmethanol:dioxane (80:20) in 10-ml fractions. Fractions 4through 15 were combined and evaporated to 5 ml. Crystallization was effected by slow addition of water to a hotsolution. Crystals weighing 0.06 g (15% yield) and melting at173.4-174Â° were obtained. The compound gave a single spot

(RF 0.86) on a paper chromatogram developed with butanol:acetic acid: water (2:1:1). Boy land and Sims (4) reported anRF of 0.6 for their preparation of the compound.

5<7-Benz(a)anthrylmethyl)-L-cysteine was prepared by themethod of Wood and Fieser (33).

156 CANCER RESEARCH VOL. 30



Polycyclic Hydrocarbon-Cysteine Conjugates

The structures of the 5-substituted cysteines prepared asdescribed in this section are shown in Chart 1.

-S-CH2-CH-COOH

NHZS-(p-CHLOROPHENYL)-L-CYSTEINE

(PCP-CYSTEINE)

S-(9, IO- DIHYDRO-9-HYDROXY-IO-PHENANTHRYO-L-CYSTEINE(DHP-CYSTEINE)

S-(5,6-DIHYORO-6-HYDROXY-5-BENZ(a)ANTHRYL)-L-CYSTEINE(DHB-CYSTEINE)

S-(5,6-DIHYDRO-6-HYDROXY-5-DIBENZ(a,h)ANTHRYL)-L-CYSTEINE(OHD-CYSTEINE)

CH2S-CYS

S-(7-BENZ(a)ANTHRYL)-METHYL-L-CYSTEINE(BM-CYSTEINE)

Chart 1. Chemical structures of hydrocarbon-cysteine conjugates.

Formation of Aminoacyl-tRNA. The reaction mixture isdescribed in Table 1. As indicated, certain tubes used to testcompetition between the arylcysteine and natural amino acidcontained 4 Â¿Â¿molesunlabeled arylcysteine or natural aminoacid in addition to the other components. The reactionmixtures were incubated for 10 min at 37Â°.The reaction

was stopped by submersing the tubes in a NaCl-ice waterbath, and 0.1-ml aliquots of the mixtures were pipetted ontopaper discs, dried, and analyzed in a manner similar to themethods described by Holley et al. (18) and Nishimura andNovelli (29).

Miscellaneous Methods. Protein concentrations were determined by the method of Lowry et al. (21). Orthophosphatewas determined by the method of Fiske and SubbaRow(14). Radioactivity was measured in a Packard Tri-Carbliquid scintillation counter with the use of the scintillationliquid described by Bray (5).

RESULTS

Earlier reports of work done with amino acid analogs havecontained generally a tacit assumption that activation and

Table 1

A TP-32PPÂ¡exchange produced by enzyme fractions from yeastThe complete system contained, in 1.0 ml, 0.5 mmole KF; 5.0

mmoles MgClj; 200 mmoles Tris-HCl, pH 7.5; 5 mmoles disodiumATP; 5 mmoles K432P2O7; 0.2 mg enzyme Fraction A, B, or C; and4 AmÃ³lesof the L-form of natural amino acid or arylcysteine. Thetime of incubation was 30 min at 37 . Tubes with all componentsexcept amino acid were prepared as controls. The results shown are acomposite of 38 ATP- PPÂ¿exchange experiments carried out oneach of the enzyme fractions.

Specific activity of enzyme fraction(Â¿imolesPPj/mg protein)

GroupIIISubstrateDHB-cy

steineDHD-cysteinePCP-cysteineDHP-cysteineHistidineGlutamic

acidLeucineTyrosineValineThreoninePhenylalanineBM-cy

steineMethionineIsoleucineLysineGlycineTyrosineAsparagineA0.390.080.040.060.050.124.101.771.380.280.550.600.032.130.290.270.180.21B0.180.040.460.400.090.252.020.530.480.060.200.050.060.080.050.050.090.14C0.020.050.260.060.040.180.820.460.060.030.280000000

III Cysteine 6.54 0.12

IVProlineAspartii-acidSerineGlutamineArginineAlanine0000000.030.040.030.03000.030.020000

transfer was catalyzed by the same enzyme system whichoperated with the corresponding natural amino acid. Sinceour studies involved use of cysteine analogs, our fractiona-tion of the 60% extract system was originally done toconcentrate the cysteine synthetase activity. It soon developed that there was no activation and transfer of any of ouraryl hydrocarbon-cysteine conjugates by the cysteine synthetase system. Nevertheless the analogs were activated,transferred to tRNA, and incorporated into protein by the60% extract. An ammonium sulfate fractionation of thecrude extract of bakers' yeast performed to reduce endog

enous exchange activity produced 3 active fractions. Table 1shows the ATP-32PPj exchange activity for natural amino

acids and analogs in Fractions A, B, and C. The analogs,which are all cysteine derivatives, differ only in the hydrocarbon substituted on the sulfur. A comparison of theactivities of the arylcysteines and natural amino acids inthese 3 enzyme fractions revealed distinct patterns of enzyme activity. These patterns are arranged into 4 groups.Group I has enzyme activity in all 3 fractions both foranalogs and natural amino acids listed. Group II containsthose with no measurable activity in Fraction C. Group III

JANUARY 1970 157




â€¢TI

shows the pattern of cysteine activity notably concentratedin Fraction A. Group IV contained little or no ATP-32PPi

exchange activity in any fraction. This probably reflectslability of the amino acid synthetases concerned.

With certain synthetases there is little correlation betweenthe level of pyrophosphate exchange and the extent oftransfer of that amino acid to tRNA. An example is thearginyl-tRNA synthetase which does not catalyze an observable exchange between ATP and pyrophosphate buttransfers the amino acid extensively to tRNA (see Table 4).Mitra and Mehler (22) have shown that the arginyl-tRNAsynthetase of the Escherichia coli system requires arginine-specific tRNA addition before measurable pyrophosphateexchange activity can be observed. Addition of tRNA to theyeast system did not stimulate activity however.

It is apparent from these patterns of enzyme distribution inFractions A, B, and C that aminoacyl-tRNA synthetasesother than the cysteine are involved in the activation andtransfer of the several arylcysteine analogs studied. Thecysteinyl-tRNA synthetase could not have been solely responsible for activation of any of the arylcysteines tested,since activity for this enzyme occurred almost entirely inFraction A while activity toward the analogs was found inall 3 fractions.3

In order to relate further the enzymes which function forthe amino acid analogs with those for the natural aminoacids, a system utilizing the appropriate enzyme fraction, Aor B, was assayed for transfer of labeled conjugates totRNA. Addition of a 10-fold concentration of unlabeledanalog or natural amino acid was made in a duplicatesystem. Table 2 shows the competition of amino acids withPCP-cysteine and DHP-cysteine. Of the natural amino acids,only arginine produced a marked decrease in the transfer ofPCP-cysteine- S to tRNA; only histidine, phenylalanine, orglutamic acid competed with the conjugate in experimentswith DHP-cysteine- s S. As expected, when a labeled arylcys-

teine was diluted with an unlabeled one, the resultingdecrease in specific activity was reflected in the decrease inthe specific activity of the conjugate bound to tRNA.

Table 3 shows that competition was observed when eitherunlabeled tyrosine or arginine was added to incubationmixtures containing DHD-cysteine-3 5S. Likewise unlabeledarginine or phenylalanine competed with DHB-cysteine-35 S,and methionine and leucine competed with BM-cysteine-35S.

The insolubility of the conjugate and high control values inthe absence of ATP complicate the interpretation of thestudies with BM-cysteine. It was particularly significant thatno appreciable competition was observed when unlabeledcysteine was added to the incubation mixtures containingany of the arylcysteines-35S.

Additional support that these arylcysteines are not activated bythe cysteinyl-RNA synthetase was provided from unpublished studiesby James and Bucovaz. In these studies the arylcysteines were shownnot to be activated or transferred to tRNA in reaction mediumscontaining the purified cysteinyl-RNA synthetase of bakers' yeast asthe only source of enzyme.

Table 2

Effect of duution by unlabeled amino acids on transfer ofanalogs to tRNA by Fraction B enzymes

The systems contained, in 1.0 ml, 2 mg yeast tRNA; 100 /Â¿molesTris-HCl, pH 7.5; 25 mmoles MgCl2; 2 mmoles disodium ATP; 0.5mmole EDTA; 0.4 /miÃ³le PCP-cysteine-3 5S (containing 3.49 X 10scpm) or 0.4 Mmole DHP-cysteine- SS (containing 4.05 X 10 cpm);

0.2 mg Fraction B and 4 AmÃ³lesof the amino acid diluento wereadded where indicated. The incubation time was 10 min at 37 . Thereaction was stopped by cooling the tubes in an ice-salt mixture.Other assay conditions are described under "Materials and Methods."

Componentsomitted Amino acid diluent

Specificradioactivity(mamÃ³le/pinole)

Fraction B and PCP-cysteine-3 5S system

None 0.22ATP 0.09None PCP-cysteine 0.11None Arginine 0.09None Cysteine 0.18None Leucine 0.18

Fraction B and DHP-cysteine-35S system

None 0.18ATP 0.03None DHP-cysteine 0.05None Histidine 0.07None Phenylalanine 0.06None Glutamic acid 0.08None Cysteine 0.17None Leucine 0.18

In Table 4 inhibition of transfer of the natural amino acidsby incubation in the presence of a 10-fold concentration ofthe arylcysteines is shown. The results were entirely consistent with those of Tables 2 and 3. Arginine uptake by tRNAwas competitively inhibited by PCP-cysteine, DHB-cysteine,and DHD-cysteine. Phenylalanine was inhibited byDHP-cysteine and DHB-cysteine, methionine and leucine wasinhibited by BM-cysteine, glutamic acid and histidine wasinhibited by DHP-cysteine, and tyrosine was inhibited byDHD-cysteine.

DISCUSSION

The results obtained with each of the 5 arylcysteines support the concept that polycyclic hydrocarbons, once conjugated through the sulfhydryl moiety of cysteine, can beactivated and transferred to tRNA by synthetase enzymes.The conjugates inhibit the transfer of specific amino acids,probably through competition for the corresponding synthetases.

Although these hydrocarbon moieties conjugated withcysteine constitute analogs with structures uniquely differentfrom that of cysteine or any of the other natural aminoacids, it is not surprising that the conjugates function in theprotein biosynthesis system. Other amino acid analogs suchas p-fluorophenylalanine (11), trifluoroleucine (13), and aza-tryptophan (32) are utilized in the synthesis of new protein.The enzyme must recognize the side chain as the only distinguishing feature between the amino acids, but recognitionof side-chain characteristics is not absolute (20). When the





Table 3

Effect of dilution by unlabeled amino acids on transfer ofanalogs to tRNA by Fraction A enzyme

Xcpm);Other than the radioactive arylcysteines, the system was the same asthat of Table 2 except for the substitution of 0.2 mg Fraction A forFraction B.

SpecificComponents radioactivity

omitted Amino acid diluent(m/umole//jmole)NoneATPNoneNoneNoneNoneNoneNoneATPNoneNoneNoneNoneNoneNoneATPNoneNoneNoneNoneNoneFraction

A and DHD-cy steine-3 sSsystemDHD-cysteineTyrosineArginineCysteineLeucineFraction

A and DHB-cysteine-3sSsystemDHB-cy

steineArgininePhenylalanineCysteineLeucineFraction

A and BM-cysteine-3s SsystemBM

-cysteineMethionineLeucineValineCysteine0.250.090.100.150.100.230.250.420.140.140.180.140.400.420.610.470.450.480.490.600.61

amino acid is provided in high concentration isoleucineamino-acyl-tRNA synthetase activates valine although it doesnot transfer valine to tRNA (1). Valine inhibits transfer ofisoleucine to isoleucine-specific tRNA. Under some conditionsthreonine is activated by the valyl-tRNA synthetase (1, 17).Therefore, a portion of the R-group of an activatable analogmust have characteristics similar to the R-group of a naturalamino acid. The effect of the size of the side-chain grouphas not been assessed. At present there is no evidence thatthe bulk of the substituted group inhibits activation andtransfer (31). Variations in the hydrocarbon ring system arereflected in differences in the selectivity by the synthetases.The phenanthrene conjugate, DHP-cysteine, competed withglutamic acid, histidine, and phenylalanine. The benzanthra-cene conjugate, DHB-cysteine, which has the structure of abenzphenanthrene, competed only with phenylalanine andarginine. The addition of still another benzene ring to form adibenzanthracene conjugate produced a competition withtyrosine and arginine.

Although final judgment must await testing with highlypurified enzymes for individual amino acids, we can assumefrom the competition experiments shown in Tables 2 and 3that more than one amino acid synthetase may activate andtransfer a given arylcysteine to tRNA. As far as is nowknown, once the arylcysteine is esterified with tRNA, theanalog presumably would be transferred to a position in theprotein designated for the natural amino acid for which thetRNA is normally specific. Transfer of such a conjugate fromits tRNA ester to ribosomal protein has been demonstratedwith 5-{l ,2,3,4-tetrahydro-2-hydroxy-l-naphthyl)-L-cys-teine (6â€”9).Similar misreading has recently been observedby Fenster and Anker (13). Trifluoroleucine was recognizedas leucine, isoleucine, and phenylalanine both in the tRNA-charging reaction and in polypeptide synthesis. Presumably,trifluoroleucine was incorporated at sites on the protein ordinarily occupied by these natural amino acids.

Table 4

Effect of dilution of arylcysteines on transfer of labeled amino acids to tRNA

Each incubation mixture contained 2 mg tRNA; 100 mmoles Tris-HCl, pH 7.25; 25 mmoles MgCl2; 2mmoles disodium ATP; 0.5 mmole EDTA; 0.4 jumÃ³leof the specified amino acid-14C and 0.5 mg protein

of the 60% extract. In addition 4.0 AmÃ³lescysteine conjugate or the unlabeled natural amino acid wereadded as indicated with water to make a total volume of 1 ml. The mixtures were incubated for 10min at 37Â°after addition of the enzyme. The extent of transfer was determined as described under"Materials and Methods."

DiluentaddedI4C-Labeledamino

acidGlutamicHistidineTyrosineArgininePhenylalanineMethionineLeucineComplete"system1.6822.5310.8848.535.681.534.32Less"ATP0.840.630.771.472.520.480.84"Cold

"a

aminoacid0.533.050.593.164.760.690.74CysteineconjugatesPCP"1.4723.6810.5329.585.741.583.79DHP"0.7418.6310.8848.424.221.484.53DHB"1.4723.5810.6325.052.941.375.16DHDo1.7922.328.0726.845.681.584.21BM"1.5822.6311.5848.325.900.792.63

"Specific activity: mamÃ³leamino acid/Mmole tRNA/10 min.

JANUARY 1970 159




The primary event in the metabolism of a polycyclichydrocarbon is an oxidation reaction. The oxidation productreacts with tissue components. Boyland and Sims (3, 4) haveprovided much indirect evidence that the initial metabolismproduct for hydrocarbons without an aliphatic side chain isan epoxide, or at least a compound which behaves chemically like an epoxide. Such epoxides react spontaneouslywith available sulfhydryl groups. Tissue sulfhydryl groupswhich are available in the cell include cysteine, glutathione,and protein. The reaction with glutathione is catalyzed by aliver enzyme (2); the reaction is degraded to an hydroxy-dihydroarylcysteine, which is acetylated and excreted as apremercapturic acid. This represents a pathway of detoxica-tion and excretion.

Protein-5-hydroxydihydrohydrocarbon conjugates are likewise degraded stepwise to hydroxydihydroarylcysteines (23).Thus, arylcysteine derivatives can arise as intermediates inseveral pathways of metabolism and are available for incorporation into protein. Since the cysteine synthetase isinactive with the conjugates, activation, transfer, and incorporation must occur through misreading by synthetase corresponding to other amino acids. Such incorporation couldintroduce dibenzanthracene for example into a number ofpositions on the protein fabric, i.e., positions ordinarilyoccupied by arginine or tyrosine. On the other hand, if thedibenzanthracene epoxide reacts with cysteine already loadedon tRNA, the position of the hydrocarbon analog on theprotein molecule would be that ordinarily occupied by cysteine. The position of binding of the hydrocarbons to protein is thus related to the stage in the sequence of reactionsat which the conjugation of the hydrocarbon with a sulfhydryl group occurs.

Incorporation of a carcinogen into a protein by this pathway must be a rare event however because the level ofactivation and transfer of the conjugates have been observedto be less than that for natural amino acids. Also, the principal reactions of carcinogenic hydrocarbons in tissues mustbe the detoxication processes of mercapturic acid and phenolformation. Thus, the hydrocarbon conjugate molecules available for incorporation into protein must be few.

The development of a positive relationship of these pathways of metabolism to carcinogenesis would be highly spec-ulatory at present. Nevertheless, the existence of suchpathways is additional stimulus for further investigations intothe behavior of polycyclic aromatic hydrocarbons in metabolic systems.

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2. Booth, J., Boyland, E., and Sims, P. An Enzyme from Rat LiverCatalyzing Conjugations with Glutathione. Biochem. J., 79:516-524, 1961.

3. Boyland, E., and Sims, P. The Metabolism of 9 , 10-Epoxy-9 , 10-dihydrophenanthrene in Rats. Biochem. J., 95. 788-795, 1965.

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1970;30:155-161. Cancer Res E. T. Bucovaz, J. C. Morrison, H. L. James, et al. the Aminoacyl-RNA Synthetase SystemReaction of Polycyclic Hydrocarbon-Cysteine Conjugates with

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