[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 0—2°,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 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1970 American Association for Cancer Research. Bucovaz, Morrison, James, Dais, and Wood 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 50°.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 171.5-172° (with decomposition) was 0.29 g (62% of the 0—4°.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 174—175°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 5°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 75°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 5°,it yielded 0.5 g light-tan, solid material.