Reaction of Polycyclic Hydrocarbon-Cysteine Conjugates with the Aminoacyl-RNA Synthetase System1

[CANCER RESEARCH 30, 155-161, January 1970] Reaction of Polycyclic Hydrocarbon-Cysteine Conjugates with the 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 naphthyl)-L-cysteine and PCP-cysteine2 into microsomal protein of rat liver was reported in earlier communica A number of arylcysteine derivatives, which are metabolites tions from this laboratory (7-9). Subsequently, of polycyclic aromatic hydrocarbons, have been synthesized. we reported that tetrahydrohydroxynaphthylcysteine appears These amino acid analogs were tested for their ability to act to be activated by the valyl and isoleucyl RNA synthetases of as substrates for aminoacyl-RNA synthetases in an in vitro rat liver or yeast, and the conjugate is transferred to the system using enzyme fractions of bakers' yeast. These studies respective species of tRNA of yeast (6). Furthermore, the have shown that 5-(p-chlorophenyl)-L-cysteine, 5-(9 ,10 conjugate was incorporated into ribosomal protein by a dihydro-9-hydroxy-l 0-phenanthryl)-L-cysteine, rat-liver preparation. According to prevailing theories of amino 5-(5 , 6-dihydro-6-hydroxy-5-benz(a)anthryl)-L -cysteine, acid metabolism, tetrahydrohydroxynaphthylcysteine would 5- (5 , 6- dihydro-6-hydroxy-5-dibenz(a , h)anthryl- L-cysteine, be transferred to some of the positions which are normally and S-(7-benz(a)anthryl)-methyl-L-cysteine are activated and occupied by valine and isoleucine in the forming peptide transferred to tRNA. The arylcysteines were observed to chain. compete with a variety of natural amino acids. This investi These findings provide a pathway by which aromatic gation revealed that chlorophenylcysteine competed with hydrocarbons can be bound to specific sites during synthesis arginine; dihydrohydroxyphenanthrylcysteine competed with of protein molecules. The sites are distinct from those glutamic acid, phenylalanine, and histidine for activating involved in direct binding of hydrocarbons or their metab enzymes; dihydrohydroxybenzanthrylcysteine likewise com olites to intact tissue proteins. peted with arginine and phenylalanine; and dihydrohydroxy- Typical cysteine conjugates of chlorobenzene, phenan- dibenzanthrylcysteine competed with methionine and leucine. threne, and the carcinogens methylbenzanthracene and di- Thus, the structure of the hydrocarbon moiety conjugated benzanthracene have been prepared and studied for their with the sulfhydryl group of cysteine determines in behavior in the protein-biosynthesizing system. This paper particular the synthetases involved in amino acid activation. reports the activation and transfer of the hydrocarbon-cys- Moreover none of the cysteine conjugates were activated by teine conjugates to tRNA. Preliminary reports have been the cysteinyl-RNA synthetase. Hydrocarbons are bound to presented (24-26). protein by this pathway at sites different from those resulting from direct interaction. MATERIALS AND METHODS INTRODUCTION Preparation of Enzyme Fractions A, B, and C. Bakers' Protein binding of administered carcinogenic hydrocarbons yeast, 3 Ib, was frozen for 6 to 8 hr in approximately 3 has been well established (16). The implication of such liters of an ether-C02 mixture to break the cells. Before metabolic events in the process of carcinogenesis has been thawing, the excess ether was decanted from the yeast, and inferred (30). This report describes a pathway by which the residual ether was removed by vacuum at room tempera carcinogens as conjugates of amino acids can be bound to ture. The resulting homogenate was cooled to 0â€”2Â°,15g proteins via the mechanism utilized for amino acid incor KC1 were added, and the mixture was stirred for 12 to 14 poration. hr. Following the KC1 addition step, the homogenate was The in vitro incorporation of aromatic hydrocarbon- centrifuged at 7,700 X g for 20 min to remove all debris; cysteine conjugates, 5-(l, 2, 3, 4-tetrahydro-2-hydroxy-l -

2The abbreviations used are: tRNA, transfer ribonucleic acid; PPj, This investigation was supported in part by USPHS Research Grant inorganic pyrophospnate; PCP-cysteine, 5-(p-chlorophenyl)-L-cysteine; CA-01228 from the National Cancer Institute and by USPHS DHP-cysteine, 5-(9 ,10-dihydro-9-hydroxy-10-phenanthryl)-L-cysteine; Research Grant AM-09131 from the National Institute of Arthritis DHD-cysteine, S-(5 , 6-dihydro-6-hydroxy-5-dibenz(a , h)anthryl)-L- and Metabolic Diseases. -cysteine; DHB-cysteine, S-(5 ,6-dihydro-6-hydroxy-5-benz(a)an- Received November 22, 1968; accepted June 3, 1969. thryl)-L-cysteine; BM-cysteine, S-(7-benz(a)anthrylmethyl)-L-cysteine.

JANUARY 1970 155

the supernatant liquid was filtered through cheesecloth and separated. These were extracted with two 15-ml portions of then centrifuged at 105,000 X g for 1 hr. The volume of the dry benzene at 50Â°.The residue, 0.4 g, was extracted with 25 supernatant liquid recovered varied between 350 and 375 ml, ml hot methanol. The extract was concentrated to 5 ml and depending upon the water content of the yeast. A series of treated with water. Lustrous crystals separated. The yield of ammonium sulfate precipitation steps followed. Throughout S-(9,10-dihydro-9-hydroxy-10-phenanthryl)-L-cysteine melting these fractionation steps the temperature was maintained at at 171.5-172Â° (with decomposition) was 0.29 g (62% of the 0â€”4Â°.Initially, 15.36 g solid ammonium sulfate were added theoretical amount). Recrystallization did not change the to 300 ml of the crude enzyme fraction. After 3 days the melting point. A paper chromatogram developed with precipitate formed was removed by centrifugation and dis butanol:acetic acid:water (1:2:1) had a single ninhydrin- carded. The supernatant fraction was adjusted to 60% satura positive spot at Rp 0.7. tion upon the addition of 33.3 g ammonium sulfate/100 ml, 5 , 6-Dihydro-5 , 6-epoxybenz(a)anthracene was prepared followed by thorough mixing. After 2 hr, the mixture was according to the procedure of Newman and Blum (28). The centrifuged at 7,700 X g for 20 min. The supernatant layer epoxide, 0.2 g, and 0.23 g of L-cysteine hydrochloride was discarded, and the precipitate was dissolved in 180 ml of monohydrate were reacted essentially as described for the water, resulting in a total volume of 230 to 360 ml phenanthrene conjugate above. A 29% yield of S-(5 ,6- depending on the amount of protein precipitated. This dihydro-6-hydroxy-5-benz(a)anthryl)-L-cysteine melting at preparation was termed the "60% extract." Three fractions 174â€”175Â°wasobtained. The elemental analysis values for the were obtained by a second ammonium sulfate fractionation: compound agreed with the theoretical values. Chromatography Fraction B, 0 to 42%; Fraction C, 42 to 50%; and Fraction A, in butanol:acetic acid:water (1:2:1) gave a single spot at RF supernatant fraction equivalent to 50 to 60% saturation. The 0.75. 3 fractions were adjusted to 0.01 M Tris, pH 7.25, and then 2-Phenylphenanthrene-2 , 3-dialdehyde was prepared from dialyzed against Tris of the same concentration and pH to dibenzanthracene by oxidation of the hydrocarbon with remove ammonium sulfate and other small-molecular-weight osmium tetroxide followed by treatment of the resulting diol components. Fractions A, B, and C were used in this with periodate (15). A more efficient procedure resulted from investigation as sources of enzymes. Transfer RNA of Bakers' Yeast. Transfer RNA was ozonolysis of dibenz(a,h)anthracene by the method of Monconi et al. (27). The yield of dialdehyde was 55% of the obtained from the General Biochemicals Company (Chagrin theoretical amount. 5 , 6-Dihydro-5 , 6-epoxydibenz(a,A)an- Falls, Ohio) and was used as the acceptor of the arylcys- thracene was prepared from the dialdehyde. To 0.2 g of the teines and naturally occurring amino acids. dialdehyde in 2 to 3 ml dry benzene was added 0.2 g of Preparation of Pyrophosphate-32P. The K432P2O7 was tris(dimethylamino)phosphine. The system was kept under prepared from carrier-free orthophosphate-32P (New England nitrogen. The mixture was shaken for 5 ml at 55% and then was cooled to 5Â°to produce crystals. The supernatant liquor Nuclear Corporation, Boston, Mass.) by pyrolysis (19). was decanted. The residue was washed 3 times with cold ATP-PPi of Exchange Reaction. The assays were carried out as described in Table 1. The ATP-32PPj exchange reaction cyclohexane and then recrystallized from cyclohexane: ben zene (70:30) to yield 0.17 g of light green needles. These had was terminated by the addition of 1 ml 10% trichloracetic an indefinite melting point as observed by Boyland and Sims acid. The mixture was briefly centrifuged to remove the (4). The yield of 'product was 88% of the theoretical. precipitated protein, and the ATP was absorbed on Darco 5-(5 , 6-Dihydro-6-hydroxy-5-dibenz(aJi)anthryl-L-cysteine G-60 (Matheson, Coleman and Bell, East Rutherford, N. J.) was prepared from a mixture of L-cysteine hydrochloride according to the method of DeMoss and Novelli (10). monohydrate, 0.6 g, sodium bicarbonate, 0.6 g, and the Preparation of ,s-substituted Cysteines. PCP-cysteine epoxide, 0.3 g, in 20 ml water and 20 ml freshly distilled L-cysteine was prepared by the method of du Vigneaud et al. dioxane. The mixture was stirred for 1.5 hr at 75Â°and then (12). adjusted to pH 7.0 at room temperature by slow addition of Epoxides were prepared according to the procedure of 5% HC1. When the solution was evaporated to a 50-ml volume and cooled at 5Â°,it yielded 0.5 g light-tan, solid material. The Newman and Blum (28). The epoxides were conjugated with cysteine by a new procedure which yields crystalline products. dried, crude product was extracted with 20 ml dry benzene at For labeled products cysteine-35 S was used. 40Â°for 2 hr. The residue was treated with 20 ml dry methanol 9 , 10-Dihydro-9 , 10-epoxyphenanthrene was prepared from and filtered to remove cystine. The methanolic solution was diphenyl-2 , 2'-dialdehyde (28) by reaction with placed on a 7-x 5/8-inch Florisil column and was eluted with tris(dimethylamino)phosphine. For conjugation with cysteine, methanol:dioxane (80:20) in 10-ml fractions. Fractions 4 0.3 g 9 , 10-dihydro-9 , 10-epoxyphenanthrene, 0.4 g through 15 were combined and evaporated to 5 ml. Crystalli L-cysteine hydrochloride monohydrate, and 0.4 g sodium zation was effected by slow addition of water to a hot bicarbonate were mixed with 15 ml water and 15 ml freshly solution. Crystals weighing 0.06 g (15% yield) and melting at distilled dioxane. The mixture was stirred at 65-70Â° for 2 hr. 173.4-174Â° were obtained. The compound gave a single spot After cooling, 10 ml water were added to the mixture, and the (RF 0.86) on a paper chromatogram developed with butanol: pH was adjusted to 6 to 7 by addition of 10% HC1. The acetic acid: water (2:1:1). Boy land and Sims (4) reported an mixture was flash-evaporated under vacuum at 30Â°to a 5-ml RF of 0.6 for their preparation of the compound. volume and cooled in an ice bath for 30 min. White crystals, 5<7-Benz(a)anthrylmethyl)-L-cysteine was prepared by the m.p. 158â€”161Â°(with decomposition), weighing 0.44 g method of Wood and Fieser (33).

156 CANCER RESEARCH VOL. 30

The structures of the 5-substituted cysteines prepared as Table 1 described in this section are shown in Chart 1. A TP-32PPÂ¡exchange produced by enzyme fractions from yeast The complete system contained, in 1.0 ml, 0.5 mmole KF; 5.0 mmoles MgClj; 200 mmoles Tris-HCl, pH 7.5; 5 mmoles disodium -S-CH2-CH-COOH ATP; 5 mmoles K432P2O7; 0.2 mg enzyme Fraction A, B, or C; and NHZ 4 AmÃ³lesof the L-form of natural amino acid or arylcysteine. The S-(p-CHLOROPHENYL)-L-CYSTEINE (PCP-CYSTEINE) time of incubation was 30 min at 37 . Tubes with all components except amino acid were prepared as controls. The results shown are a composite of 38 ATP- PPÂ¿exchange experiments carried out on each of the enzyme fractions. Specific activity of enzyme fraction (Â¿imolesPPj/mg protein) GroupIIISubstrateDHB-cy S-(9, IO- DIHYDRO-9-HYDROXY-IO-PHENANTHRYO-L-CYSTEINE (DHP-CYSTEINE) steineDHD-cy steinePCP-cy steineDHP-cy steineHistidineGlutamic

acidLeucineTyrosineValineThreoninePhenylalanineBM-cy

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

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

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

III Cysteine 6.54 0.12

IVProlineAspartii- CH2 acidSerineGlutamineArginineAlanine0000000.030.040.030.03000.030.020000 S-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 is transfer was catalyzed by the same enzyme system which described in Table 1. As indicated, certain tubes used to test operated with the corresponding natural amino acid. Since competition between the arylcysteine and natural amino acid our studies involved use of cysteine analogs, our fractiona- contained 4 Â¿Â¿molesunlabeledarylcysteine or natural amino tion of the 60% extract system was originally done to acid in addition to the other components. The reaction mixtures were incubated for 10 min at 37Â°.The reaction concentrate the cysteine synthetase activity. It soon devel oped that there was no activation and transfer of any of our was stopped by submersing the tubes in a NaCl-ice water aryl hydrocarbon-cysteine conjugates by the cysteine syn bath, and 0.1-ml aliquots of the mixtures were pipetted onto thetase system. Nevertheless the analogs were activated, paper discs, dried, and analyzed in a manner similar to the transferred to tRNA, and incorporated into protein by the methods described by Holley et al. (18) and Nishimura and 60% extract. An ammonium sulfate fractionation of the Novelli (29). crude extract of bakers' yeast performed to reduce endog Miscellaneous Methods. Protein concentrations were deter enous exchange activity produced 3 active fractions. Table 1 mined by the method of Lowry et al. (21). Orthophosphate shows the ATP-32PPj exchange activity for natural amino was determined by the method of Fiske and SubbaRow acids and analogs in Fractions A, B, and C. The analogs, (14). Radioactivity was measured in a Packard Tri-Carb which are all cysteine derivatives, differ only in the hydro liquid scintillation counter with the use of the scintillation carbon substituted on the sulfur. A comparison of the liquid described by Bray (5). activities of the arylcysteines and natural amino acids in these 3 enzyme fractions revealed distinct patterns of en RESULTS zyme activity. These patterns are arranged into 4 groups. Group I has enzyme activity in all 3 fractions both for Earlier reports of work done with amino acid analogs have analogs and natural amino acids listed. Group II contains contained generally a tacit assumption that activation and those with no measurable activity in Fraction C. Group III

JANUARY 1970 157

Bucovaz, Morrison, James, Dais, and Wood

shows the pattern of cysteine activity notably concentrated Table 2 in Fraction A. Group IV contained little or no ATP-32PPi Effect of duution by unlabeled amino acids on transfer of exchange activity in any fraction. This probably reflects analogs to tRNA by Fraction B enzymes lability of the amino acid synthetases concerned. The systems contained, in 1.0 ml, 2 mg yeast tRNA; 100 /Â¿moles With certain synthetases there is little correlation between Tris-HCl, pH 7.5; 25 mmoles MgCl2; 2 mmoles disodium ATP; 0.5 mmole EDTA; 0.4 /miÃ³le PCP-cysteine-3 5S (containing 3.49 X 10s the level of pyrophosphate exchange and the extent of cpm) or 0.4 Mmole DHP-cysteine- SS (containing 4.05 X 10 cpm); transfer of that amino acid to tRNA. An example is the 0.2 mg Fraction B and 4 AmÃ³lesof the amino acid diluento were arginyl-tRNA synthetase which does not catalyze an obser added where indicated. The incubation time was 10 min at 37 . The vable exchange between ATP and pyrophosphate but reaction was stopped by cooling the tubes in an ice-salt mixture. Other assay conditions are described under "Materials and Methods." transfers the amino acid extensively to tRNA (see Table 4). Mitra and Mehler (22) have shown that the arginyl-tRNA synthetase of the Escherichia coli system requires arginine- Specific Components radioactivity specific tRNA addition before measurable pyrophosphate omitted Amino acid diluent (mamÃ³le/pinole) exchange activity can be observed. Addition of tRNA to the Fraction B and PCP-cysteine-3 5S system yeast system did not stimulate activity however. It is apparent from these patterns of enzyme distribution in None 0.22 ATP 0.09 Fractions A, B, and C that aminoacyl-tRNA synthetases None PCP-cysteine 0.11 other than the cysteine are involved in the activation and None Arginine 0.09 transfer of the several arylcysteine analogs studied. The None Cysteine 0.18 cysteinyl-tRNA synthetase could not have been solely re None Leucine 0.18 Fraction B and DHP-cysteine-35S system sponsible for activation of any of the arylcysteines tested, None 0.18 since activity for this enzyme occurred almost entirely in ATP 0.03 Fraction A while activity toward the analogs was found in None DHP-cysteine 0.05 all 3 fractions.3 None Histidine 0.07 In order to relate further the enzymes which function for None Phenylalanine 0.06 the amino acid analogs with those for the natural amino None Glutamic acid 0.08 None Cysteine 0.17 acids, a system utilizing the appropriate enzyme fraction, A None Leucine 0.18 or B, was assayed for transfer of labeled conjugates to tRNA. Addition of a 10-fold concentration of unlabeled analog or natural amino acid was made in a duplicate In Table 4 inhibition of transfer of the natural amino acids system. Table 2 shows the competition of amino acids with PCP-cysteine and DHP-cysteine. Of the natural amino acids, by incubation in the presence of a 10-fold concentration of the arylcysteines is shown. The results were entirely consis only arginine produced a marked decrease in the transfer of PCP-cysteine- S to tRNA; only histidine, phenylalanine, or tent with those of Tables 2 and 3. Arginine uptake by tRNA was competitively inhibited by PCP-cysteine, DHB-cysteine, glutamic acid competed with the conjugate in experiments with DHP-cysteine- s S. As expected, when a labeled arylcys- and DHD-cysteine. Phenylalanine was inhibited by DHP-cysteine and DHB-cysteine, methionine and leucine was teine was diluted with an unlabeled one, the resulting inhibited by BM-cysteine, glutamic acid and histidine was decrease in specific activity was reflected in the decrease in inhibited by DHP-cysteine, and tyrosine was inhibited by the specific activity of the conjugate bound to tRNA. DHD-cysteine. Table 3 shows that competition was observed when either unlabeled tyrosine or arginine was added to incubation mixtures containing DHD-cysteine-3 5S. Likewise unlabeled DISCUSSION arginine or phenylalanine competed with DHB-cysteine-35 S, and methionine and leucine competed with BM-cysteine-35S. The results obtained with each of the 5 arylcysteines sup port the concept that polycyclic hydrocarbons, once conju The insolubility of the conjugate and high control values in gated through the sulfhydryl moiety of cysteine, can be the absence of ATP complicate the interpretation of the activated and transferred to tRNA by synthetase enzymes. studies with BM-cysteine. It was particularly significant that The conjugates inhibit the transfer of specific amino acids, no appreciable competition was observed when unlabeled probably through competition for the corresponding syn cysteine was added to the incubation mixtures containing any of the arylcysteines-35S. thetases. Although these hydrocarbon moieties conjugated with cysteine constitute analogs with structures uniquely different from that of cysteine or any of the other natural amino acids, it is not surprising that the conjugates function in the protein biosynthesis system. Other amino acid analogs such Additional support that these arylcysteines are not activated by as p-fluorophenylalanine (11), trifluoroleucine (13), and aza- the cysteinyl-RNA synthetase was provided from unpublished studies tryptophan (32) are utilized in the synthesis of new protein. by James and Bucovaz. In these studies the arylcysteines were shown not to be activated or transferred to tRNA in reaction mediums The enzyme must recognize the side chain as the only dis containing the purified cysteinyl-RNA synthetase of bakers' yeast as tinguishing feature between the amino acids, but recognition the only source of enzyme. of side-chain characteristics is not absolute (20). When the

158 CANCER RESEARCH VOL. 30

Table 3 amino acid is provided in high concentration isoleucine amino-acyl-tRNA synthetase activates valine although it does Effect of dilution by unlabeled amino acids on transfer of not transfer valine to tRNA (1). Valine inhibits transfer of analogs to tRNA by Fraction A enzyme isoleucine to isoleucine-specific tRNA. Under some conditions X threonine is activated by the valyl-tRNA synthetase (1, 17). cpm); Therefore, a portion of the R-group of an activatable analog Other than the radioactive arylcysteines, the system was the same as must have characteristics similar to the R-group of a natural that of Table 2 except for the substitution of 0.2 mg Fraction A for amino acid. The effect of the size of the side-chain group Fraction B. has not been assessed. At present there is no evidence that Specific the bulk of the substituted group inhibits activation and Components radioactivity transfer (31). Variations in the hydrocarbon ring system are (m/umole//jmole)NoneATPNoneNoneNoneNoneNoneNoneATPNoneNoneNoneNoneNoneNoneATPNoneNoneNoneNoneNoneFractionomitted Amino acid diluent reflected in differences in the selectivity by the synthetases. systemDHD-cysteineTyrosineArginineCysteineLeucineFractionA and DHD-cy steine-3 sS The phenanthrene conjugate, DHP-cysteine, competed with glutamic acid, histidine, and phenylalanine. The benzanthracene conjugate, DHB-cysteine, which has the structure of a benzphenanthrene, competed only with phenylalanine and arginine. The addition of still another benzene ring to form a dibenzanthracene conjugate produced a competition with tyrosine and arginine. A and DHB-cysteine-3sSsystemDHB-cy Although final judgment must await testing with highly purified enzymes for individual amino acids, we can assume from the competition experiments shown in Tables 2 and 3 steineArgininePhenylalanineCysteineLeucineFraction that more than one amino acid synthetase may activate and transfer a given arylcysteine to tRNA. As far as is now known, once the arylcysteine is esterified with tRNA, the A and BM-cysteine-3ssystemBM S analog presumably would be transferred to a position in the protein designated for the natural amino acid for which the tRNA is normally specific. Transfer of such a conjugate from -cysteineMethionineLeucineValineCysteine0.250.090.100.150.100.230.250.420.140.140.180.140.400.420.610.470.450.480.490.600.61its tRNA ester to ribosomal protein has been demonstrated with 5-{l ,2,3,4-tetrahydro-2-hydroxy-l-naphthyl)-L-cysteine (6â€”9).Similar misreading has recently been observed by Fenster and Anker (13). Trifluoroleucine was recognized as leucine, isoleucine, and phenylalanine both in the tRNA- charging reaction and in polypeptide synthesis. Presumably, trifluoroleucine was incorporated at sites on the protein ordi narily 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; 2 mmoles 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 were added as indicated with water to make a total volume of 1 ml. The mixtures were incubated for 10 min at 37Â°after addition of the enzyme. The extent of transfer was determined as described under "Materials and Methods."

addedI4C-Labeledamino Diluent "a conjugatesPCP"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 aminoacid0.533.050.593.164.760.690.74Cysteine acidGlutamicHistidineTyrosineArgininePhenylalanineMethionineLeucineComplete"system1.6822.5310.8848.535.681.534.32Less"ATP0.840.630.771.472.520.480.84"Cold

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

JANUARY 1970 159

The primary event in the metabolism of a polycyclic 5. Bray, G. A. A Simple Efficient Liquid Scintilla tor for Counting hydrocarbon is an oxidation reaction. The oxidation product Aqueous Solutions in a Liquid Scintillation Counter. Anal. Bio reacts with tissue components. Boyland and Sims (3, 4) have chem., 1: 279-285, 1960. provided much indirect evidence that the initial metabolism 6. Bucovaz, E. T., Morrison, J. C., and Wood, J. L. Variations in product for hydrocarbons without an aliphatic side chain is Attachment of a Cysteine Conjugate to Soluble Ribonucleic Acid. J. Biol. Chem., 241: 5114-5118, 1966. an epoxide, or at least a compound which behaves chem 7. Bucovaz, E. T., and Wood, J. L. Activation and Incorporation of ically like an epoxide. Such epoxides react spontaneously p-Chlorophenylcysteine into Yeast Soluble-RNA. Federation with available sulfhydryl groups. Tissue sulfhydryl groups Proc., 22: 644, 1963. which are available in the cell include cysteine, glutathione, 8. Bucovaz, E. T., and Wood, J. L. The Effect of Aromatic Hydro and protein. The reaction with glutathione is catalyzed by a carbons upon Cysteine Incorporation into Protein. Federation liver enzyme (2); the reaction is degraded to an hydroxy- Proc., 23: 267, 1964. dihydroarylcysteine, which is acetylated and excreted as a 9. Bucovaz, E. T., and Wood, J. L. Activation and Incorporation of premercapturic acid. This represents a pathway of detoxica- Arylcysteines into Ribosomal Protein. J. Biol. Chem., 239: tion and excretion. 1151-1155, 1964. Protein-5-hydroxydihydrohydrocarbon conjugates are like 10. DeMoss, J. A., and Novelli, G. D. An Amino Acid Dependent Exchange Between P-Labeled Inorganic Pyrophosphate and wise degraded stepwise to hydroxydihydroarylcysteines (23). ATP in Microbial Extracts. Biochim. Biophys. Acta, 22: 49-61, Thus, arylcysteine derivatives can arise as intermediates in 1956. several pathways of metabolism and are available for incor 11. Dunn, T. F., and Leach, F. R. Incorporation of p-Fluorophenyl- poration into protein. Since the cysteine synthetase is alanine into Protein by a Cell-free System. J. Biol. Chem 242- inactive with the conjugates, activation, transfer, and incor 2693-2699, 1967. poration must occur through misreading by synthetase corre 12. du Vigneaud, V., Wood, J. L., and Binkley, F. Acetylation in sponding to other amino acids. Such incorporation could vivo of p-Bromophenyl-D-Cysteine. J. Biol. Chem., 138: introduce dibenzanthracene for example into a number of 369-374, 1941. positions on the protein fabric, i.e., positions ordinarily 13. Fenster, E. D., and Anker, H. S. Incorporation into Polypeptide occupied by arginine or tyrosine. On the other hand, if the and Charging on Transfer Ribonucleic Acid of the Amino Acid dibenzanthracene epoxide reacts with cysteine already loaded Analog, 5,5,5 -Trifluoroleucine by Leucine Auxotrophs of Escherichia coli. Biochemistry, 8: 269-274, 1969. on tRNA, the position of the hydrocarbon analog on the 14. Fiske, C. H., and SubbaRow, Y. The Colorimetrie Determination protein molecule would be that ordinarily occupied by cys of Phosphorus. J. Biol. Chem., 66: 375-400, 1925. teine. The position of binding of the hydrocarbons to pro 15. Hadler, H. I., and Kryger, A. C. K Region Fission and Addition tein is thus related to the stage in the sequence of reactions Products of 7 , 12-Dimethylbenz[a] anthracene. J. Org. Chem., at which the conjugation of the hydrocarbon with a sulfhy 25: 1896-1901, 1960. dryl group occurs. 16. Heidelberger, C. Studies on the Molecular Mechanism of Carcino Incorporation of a carcinogen into a protein by this path genesis. J. Cellular Comp. Physiol., 64(Suppl. 1): 129-148, way must be a rare event however because the level of 1964. activation and transfer of the conjugates have been observed 17. Hiish, D. I., and Lipmann, F. The Divergence in Reactivity of to be less than that for natural amino acids. Also, the prin Aminoacyl Transfer Ribonucleic Acid Synthetases of Escherichia cipal reactions of carcinogenic hydrocarbons in tissues must coli with Hydroxylamine. J. Biol. Chem., 243: 5724-5730, be the detoxication processes of mercapturic acid and phenol 1968. formation. Thus, the hydrocarbon conjugate molecules avail 18. Holley, R. W., Apgar, J., Doctor, B. P., Farrow, J., Marini, M. A., and Merrill, S. H. A Simplified Procedure for the Preparation of able for incorporation into protein must be few. Tyrosine- and Valine-Acceptor Fractions of Yeast "Soluble Ribo The development of a positive relationship of these path nucleic Acid." J. Biol. Chem., 236: 200-202, 1961. ways of metabolism to carcinogenesis would be highly spec- 19. Jones, M. E., Lipmann, F., Hilz, H., and Lynen, F. On the ulatory at present. Nevertheless, the existence of such Enzymatic Mechanism of Coenzyme A Acetylation with pathways is additional stimulus for further investigations into Adenosine Triphosphate and Acetate. J. Am. Chem. Soc., 75: the behavior of polycyclic aromatic hydrocarbons in meta 3285-3286, 1953. bolic systems. 20. Loftfield, R. B. The Frequency of Errors in Protein Biosynthesis. Biochem.. J., 89: 82-92, 1963. 21. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. REFERENCES Protein Measurement with the Folin Phenol Reagent. J. Biol. Chem., 193: 265-275, 1951. 1. Bergmann, F. H., Berg, P., and Dieckmann, M. The Enzymic 22. Mitra, S. K., and Mehler, A. H. The Role of Transfer Ribonucleic Synthesis of Amino Acyl Derivatives of Ribonucleic Acid. J. Acid in the Pyrophosphate Exchange Reaction of Arginine-Trans- Biol. Chem., 236: 1735-1740, 1961. fer Ribonucleic Acid Synthetase. J. Biol. Chem., 241: 2. Booth, J., Boyland, E., and Sims, P. An Enzyme from Rat Liver 5161-5162, 1966. 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