Chem.-Biol. Interactions, 13 (1976) 47-55 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
PROTEIN MODIFICATIONS BY ACTIVATED CARCINOGENS II. THE ACETYLATION OF RIBONUCLEASE BY N-ACETOXY-3-FLUORENYLACETAMIDE *
E.J. BARRY and H.R. GUTMANN
Laboratory for Cancer Research, Veterans Administration Hospital, and the Department of Biochewktry. University of Minnesota, Minneapolis, Minn. 554Z 7 (U.S.A.) (Received June 16th, 1975) (Revision received October 6th, 1975) (Accepted October lOth, 1975)
SUMMARY
The reaction of IV-[‘H] acetoxy-3-fluorenylacetamide (IV-[‘H] acetoxy3-FAA), a potent carcinogen for the rat, with RNAase yielded three modified proteins separable from RNAase by ion exchange chromatography on Bio-Gel CM-30 with a gradient of increasing sodium ion concentration. Only minor amounts of RNAase were recovered. The modified proteins were labeled with 3H to a varying degree, and their order of elution was inversely related to the extent of labeling. The modification of the proteins was the result of the transfer of the acetyl group from IV-[3H]acetoxy-3-FAA to RNAase. The evidence for this conclusion was (a) the release of 84-86% of the radioactivity as [3H] acetic acid from the two major proteins upon acid hydrolysis and (b) the isolation of E-N-[~H]acetyl-L-lysine from enzymatic hydrolysates of these proteins. A comparison of the present data with those previously reported for the acetylaton of RNAase by the isomeric carcinogen, IV-acetoxy-a-FAA, showed that N-acetoxy-3-FAA is the more potent acetylating agent. The present study in conjunction with the previous results, suggests that structural alteration of cellular nucleophiles by acylation may be a biochemical mechanism underlying the biological activity of N-acetoxy3-FAA and related activated carcinogens.
__-_ * This work was supported by United the National Cancer Institute. Abbreviation : FAA, fluorenylacetamide.
States Public Health Service Grant CA02571
from
47
N-Hydroxy-&FAA is a potent cart oral and local administration [ 1,2]. Act cal carcinogenesis, arylhydroxamic ac se but must be activated by esterification furnishes, by spontaneous decomposition, N-acyl-N-arylnitrenium ion which and nucleic acida. These interactions provide the c accepted molecular m hanism for the initiation compounds. In order to test this c we prepared Nqcetoxy=S-FAA, a carcinogen, N-acetoxy-2-FAA, and tested its cart nicity by a variety of routes. The tests indicated that N-acetoxy-$-FAA more potent car&ogen than N-hydroxy-S-FAA upon local administration to the rat [ 2). The compound also produced the malignant transformation of fibroblasts of rat embryos in culture [4]. All of these data were in accord with the idea that the carcinogenicity of arylhydroxamic acids is dependent upon activation of the arylhydroxamic acid by esterification. However, several attempts to demonstrate that N-acetoxy-S-FAA reacts with nucleophilic accept0 through an N-acyl~N-arylnitrenium ion were unsuccessful [ 2,5]. In the search for an alternative mechanism for the action of N-acetoxy&FAA, we decided to explore the capacity of N-acetoxy_S-FAA to acetylate RNAase. We had found previously that the isomeric N-acetoxy-a-FAA interacts with RNAase in a model system not only by arylamidation but also by acetylation and that the acetylation of the e-amino group of lysine in RNAase alters the chromatographic properties of RNAase [S] . The data derived from the studies of the acetylation of RNAase by N-acetoxy-a-FAA form the basis of the present report. EXPERIMENTAL PROCEDURE NjsHj Acetoxy-a-FAA was prepared by the acetylation of N-hydroxy_SFM (101 mg, 132-134’) with [3H]acetic anhydride (25.5 mg, 50 mCi/ mmole, New England Nuclear) and 0.025 ml unlabeled acetic anhydride in pyridine (1 ml) [ 5 ] . A portion of the product (18 mg) was mixed with Nacetoxya-FAA ($9 m2, m.p. 104’) [5) and recrystallized from ethanol : water to give the labeled ester (m.p. 105-106.5”, 5.22 *10Qdpm/pmole). The purity of the labeled compound was established by UV spectrophotometry and by means of several radiochromato s [6] which showed a single radioactive peak coincident with the flu rice-quenching band given by authentic N-acetoxy-a-FAA. Ribonuclease A (XII-A, Sigma) was purified by ion exchange chromate raphy on BiomGelCM-30 (Bio-Rad) as previously described [S] . The reaction was carried out by dissolving N-[“HI acetoxy-S-FAA (23.75 rmoles) in 0.5 ml ethanol and adding the ester slowly Gvera period of 25 min to a solution of RNAase A (4.75 Fmoles) in distilled water (20 ml). The solution was stirred magnetically for 24 h at 25’. After cooling the reaction mixture in ice, 48
h residue was d
The hydrolysates were then radioactive fractions were co11 191%m.g. 247-249’. in the amoun to the pooled fractions. The were chromatographed on 8
R Nase,
0.20
0.10
E
z2
s-
Expt. 2 0.20
u g
0.10
m
2 Expt. 3 0.20
0.10
0
50
100
150
200
250
300
Effluent,ml Fig. 1, Chromatography on Bio-Gel CM-30 of the proteins precipitated with acetone from the reaction of RNAase with N-[3H]acetoxy-3-FAA. The gel was equilibrated in 0.005 M Tris-HCl (pH 8.0) poured into the column (1 x 20 cm) and washed with 200 ml of the buffer. The proteins dissolved in water were applied to the gel and eluted at 4’ with the linear concentration gradient of NaCl in 0.005 M TripHCl [6] indicated by the broken line in the upper section of the figure. The elution was carried out at a rate of 19 ml/h. Effluent fractions of 5-6 ml were collected and the proteins were located by their absorbance at 280 nm. The elution profile of native RNAase is indicated by the dotted line. The figure shows the elution profile of the proteins from three separate experiments in which RNAase was reacted with the 3H-labeled carcinogen as described in the text. The am< mts of proteins in the effluent fractions delineated by the arrows were determined by the modified Folin method [‘I] and are shown in Table I.
pmoles) (Table II) and represented that fraction of the RNAase which had remained largely unmodified. Proteins, B, C and D were displaced from the ion exchange resin at lower Na+ ion concentrations than either protein A or native RNAase (Fig. 1). This would be expected if the net positive charge of SO
TABLE I THE RELATIVE AMOUNTS GF THE MODIFIED PROTEINS IN THE ELUATE FOL LOWING ION EXCHANGE CHROMATOGRAPHY OF RNAase REACIED WITH N-[3H]ACETOXY-3-FAA a Experiment No.
Percent of reacted RNAse recovered b in peak A
in peak B -
1 2 3
6.5
24.6 20.7 12.0
in peak C -I_-_ 28.3 46.2 29.9
in peak D -.-~_
16.6
Apparent total recovery of (S) 52.9 66.9 58.6 (82.3)
a The reaction conditions were the same in all three experiments and are described in the text. b The designation of the peaks refers to the peaks of Fig. 1. The values repr
nt the Folin-reactive compounds in the e&ate fractions delineated by the arrows in Fig. c The values represent the sum of the Folin-reactive material in the pooled &ate frsctions corresponding to the principal peaks B, C and D of Fig: 1. The value in psrenth represents the Folin-reactive compounds in the total eluate. The recovery of native RNAase was 95%.
these modified proteins were progressively decreased relative to that of native RNAase or of protein A. The progressive decrease in the affinity of the modified proteins to the resin (D < C < B) was attributable to the transfer of the acetyl group from N-[3H]acetoxy-3-FAA to RNAase and the modified proteins. This conclusion was based on the observatiorrthat protein B was formed by the addition of approx- 1 mole of acetyP$o 1 mole of RNAase. Proteins C and D, in turn, appeared to result from the addition of 1 mole of acetyl to proteins B and C, respectively (Table II). The extent of the acetyl transfer appeared to vary somewhat since protein D was formed only in 1 of 3 experiments. The most probable explanation for the differences in the yield of the modified proteins lies in the limited solubility of the 3H-labeled TABLE II SPECXFIC RADIOACTIVITIES OF THE PROTEINS ISOLATED FROM THE REACTION OF RNAase with ~-~3H]AG~OXY-3-FAA flatoms sHllO0 pmoles protein b -~-___l-_~_~fll-~-~ 8 A 86 f 5 B 172$-g c 274 D v-m -0 -e _-_ a A, B, C and D refer to the proteins corres~nding to the peaks in the e~uti~n profit Fig. 1. b The values are the means 2 average deviations for the specific radioactivities of the pro teins isolated from experiments 1, 2 and 3 of Fig. 1 and Table 1. Protein isolated a
-
TABLE III RELEASE OF [3HlACETYL AND ACID HYDROLYSIS
GROUPS
FROM PROTEINS
Protein
Hydrolytic medium
Proteinbound jH dpm . 10e5
3H in steam distillate dpm . 10m4
B B C C
acid b alkaline c acid b alkaline c
2.38 2.38 5.54 5.54
20.0 4.1 47.8 9.3
----+ + + +
0.5 0.1 0.3 0.2
B AND C BY ALKALINE
Percent of bound 3H recovered as [ ‘HIacetic acid a -83.9 17.3 86.2 16.8
+ + + +
2.1 0.3 0.4 0.3
Percent of [ 3H]acetyl groups bound in amide linkage
66.6 69.4
a The [ 3H] acetic acid in the hydrolysate was determined by inverse isotope dilution as described in the text. The values are the means + average deviations of duplicate determinations on the proteins isolated from experiments 1, 2 and 3 listed in Fig. 1 and Table I. b Acid hydrolysis was performed in 6 N HzS04 at 100” for 6 h. c Alkaline hydrolysis was performed in 2 N NaOH at 25” for 18 h.
ester in the aqueous incubation system which contained 2.5% ethanol. Any decrease in the solubility of the labeled ester during the incubation would have the effect of changing the concentration of the acetylating agent in solution and thus the extent of modification. Modification by acetylation of RNAase was confirmed by the recovery of [3H] acetic acid following acid and alkaline hydrolysis of proteins B and C (Table III). The data permitted the calculation of a minimum value for the acetyl groups joined to proteins B and C through an amide linkage [6] . The calcsllations ‘indicated that 66 and 69% of the labeled acetyl groups in proteins B and C were bound to these proteins in amide linkage (Table III). Because of the high content of lysine in RNAase [lo], it was anticipated that the e-amino group of this amino acid would be the functional group that would accept the acetyl group of N-[3H]acetoxy-3-FAA. This was shown to be the case by the isolation of E-N-[~H]acetyl-L-lysine from enzymatic digests of proteins B and C. The [‘HI amino acid accounted for 57 to 63% of the tritiated compounds of small molecular weight present in Sephadex G-25 filtrates of enzymatic hydrolysates of proteins B and C (Table IV). It is evident that lysine is the principal amino acid of RNAase that forms an amide linkage with the acetyl group of N-acetoxy-3-FAA and that the formation of e-acetyl-L-lysine is primarily responsible for the change in the chromatographic properties of the RNAase. We have previously reported that N-acetoxy-a-FAA, a carcinogenic isomer of N-acetoxy-3-FAA, modified RNAase by acetylation of the e-amino group of lysine [6]. A comparison of the modification of RNAase by the two carcinogens showed that acetylation of RNAase by N-acetoxy-3-FAA was even more extensive than by ZV-acetoxy-2-FAA. This conclusion wa:3 based 52
TABLE IV ISOLATION OF E-N-[ 3H]ACETYL-L-LYSINE OF THE MODIFIED PROTEINS OBTAINED WITH A’-[ 3H] ACETOXY-3-FAA a Protein hydrolyzed
B C
“H associated with free amino acids b
dpm
Specific radioactivity of purified E-N[3H]acetylL-lysine dpmlmg
1.40 * 106 1.15 * 106
1.01 . 104 8.47 . lo3
FROM ENZYMATIC HYDRDLYSATES FROM THE INCUBIZTION OF RNAase
3H associated with E-N-[ 3H ]acetyl-L-lysine ‘
c-N-[__” ~ 3H]acctyl-L-iysine --“-_----
dpm
%
8.77 . 10s 6.52 . 10’
62.6 56.7
[ 3H]amino
x 166
acids
_
--
a The proteins were hydrolyzed successively with trypsin and pronase as described in the text. b The values refer to the total radioactivity in the pooled fractions obtained after chromatography of the pronase hydrolysates of proteins B and C on Sephadex G-25. r 87 and 77 mg of e-N-acetyl-L-lysine were added as carriers to the pooled fractions that were obtained after Sephadex G-25 chromatography of the pronase hydrolysates of proteins B and C, respectively.
on the following evidence. First, protein A, which represents largely unmodified RNAase, was detected only once in minor amounts when RNAase was incubated with N-acetoxyS-FAA. In contrast, major amounts of protein A were isolated consistently when RNAase was reacted with N-acetoxyS-FAA under identical conditions [6]. Second, protein D, which, as judged by its chromatographic properties and by its specific radioactivity, was the most extensively modified protein in the present experiments, was not detected in the reaction of RNAase with N-acetoxy-2-FAA. Third, the specific radioactivities of proteins B and C obtained from the reaction of RNAase with Nacetoxy-2-FAA [6] were one-half to one-third of the specific radioactivities of the corresponding proteins isolated from the reaction of RNAase with the isomeric N-acetoxy-3-FAA. The data of this report are compatible with a reaction mechanism previously proposed for N-acetoxyS-FAA [6] and applied here to the 3-isomer. According to this mechanism (Fig. 2), N-acetoxy-3-FAA acetylates RNAase, and presumably other proteins, by electrophilic attack on the e-amino group of lysine. The scheme also indicates the possibility of cyclic acetyl transfer by enzymatic reacetylation of the hydroxamate resulting from the spontaneous decomposition of the intermediate adduct. It should be noted that, theoretically, the reaction scheme shown in Fig. 2 applies also to the transfer of other acyl groups, such as the sulfate or phosphate group, to nucleophilic acceptors. Attempts L-3demonstrate the formation of a sulfate ester of N-hydroxy-3-FAA have been unsuccessful thus far, presumably because the test for the formation of such an ester was based on the arylamidation rather than on the acylation of a nucleophilic acceptor [2,5] .
53
Arylamidation of cellular macromolecules has heretofore been the only molecular mechanism considered to be pertinent to the induction of malignant transformation by Wacetoxy-2-FAA and related activated carcinogens [3]. Hawever, it has not been possible tu demonstra~ the ~l~idation of nucleophilic acceptors by ~ffacetoxy-3-~AA f2,5]. The acetylation of cellular macromolec~~ shown here is an afternative rnech~~ to ra~on~i~e the c~c~n~ganic activity of ~-ace~x~3-FAA. The potenti~ for tr~sfe~g the acetyl group is a property which N-acetoxy-3-FAA and N-acetoxy-2-FAA share with other structurally related carcinogens, such as N-acetoxy-4-acetyie aminobiphenyl, ~-~~~etox~2=acetyl~in~phan~threne and Smacetoxyxana thine [l&12] * In the case of the biphenyl and phen~thre~e de~v~~ves, it has been reported that acetylation of ~~o~~e p~dominates over arylamidation [Zl] . In the case of 3~cetoxyx~thine* nu data are available that would permit a ~ua~t~tativ~ comparison of the ace~lation and of the aryiamidation of the same nucleophife under comparable condiGona. The data of the present and the previous study [6] justify the view that, when acetylation 54
of a nucleophilic acceptor is the only demonstrable or the predominant reaction displayed by these carcinogens, structural alterations of cellular nucleophiles by acetylation ought to be considered in attempts to explain the mode of action of these compounds. REFERENCES 1 H.R. Gutmann, D.S. Leaf, Y. Yost, R.E. Rydell and C.C. Chen, Cancer Res., 30 (1970) 1485. 2 Y. Yost, H.R. Gutmann and R.E. Rydell, Cancer Res., 35 (1975) 4471. 3 J.A. Miller, Cancer RBS., 30 (1970) 559. 4 L.I. Sekely, D. Malejka-Giganti, H.R. Gutmann and R.E. Rydell, J. Natl. Cancer Inst., 50 (1973) 1337. 5 F.J. Zieve and H.R. Gutmann, Cancer Res., 31 (1971) 471. 6 E.J. Barry and H.R. Gutmann, J. Biol. Chem., 248 (1973) 2730. 7 E. Layne. Methods Enzymol., 2 (1957) 448. 8 E.D. Chase and J.L. Rabinowitz, Principles of Radioisotope Methodology, Burgess, Minneapolis, 1966, p. 442. 9 A. Neuberger and F. Banger, Biochem. J., 37 (1943) 515. 10 H.A. Scheraga and J.A. Rupley, in F.F. Nord, (Ed.), Advances in Enzymology, Interscience, New York, 1962, p. 161. 11 J.A. Miller and E.C. Miller, in F. Hornburger (Ed.), Progress in Experimental Tumor Research, Vol. 11, Karger, New York, 1967, p. 273. 12 G. Stiihrer and G. Salemnick, Cancer Res., 35 (1975) 122.
55
PROTEIN MODIFICATIONS BY ACTIVATED CARCINOGENS II. THE ACETYLATION OF RIBONUCLEASE BY N-ACETOXY-3-FLUORENYLACETAMIDE *
E.J. BARRY and H.R. GUTMANN
Laboratory for Cancer Research, Veterans Administration Hospital, and the Department of Biochewktry. University of Minnesota, Minneapolis, Minn. 554Z 7 (U.S.A.) (Received June 16th, 1975) (Revision received October 6th, 1975) (Accepted October lOth, 1975)
SUMMARY
The reaction of IV-[‘H] acetoxy-3-fluorenylacetamide (IV-[‘H] acetoxy3-FAA), a potent carcinogen for the rat, with RNAase yielded three modified proteins separable from RNAase by ion exchange chromatography on Bio-Gel CM-30 with a gradient of increasing sodium ion concentration. Only minor amounts of RNAase were recovered. The modified proteins were labeled with 3H to a varying degree, and their order of elution was inversely related to the extent of labeling. The modification of the proteins was the result of the transfer of the acetyl group from IV-[3H]acetoxy-3-FAA to RNAase. The evidence for this conclusion was (a) the release of 84-86% of the radioactivity as [3H] acetic acid from the two major proteins upon acid hydrolysis and (b) the isolation of E-N-[~H]acetyl-L-lysine from enzymatic hydrolysates of these proteins. A comparison of the present data with those previously reported for the acetylaton of RNAase by the isomeric carcinogen, IV-acetoxy-a-FAA, showed that N-acetoxy-3-FAA is the more potent acetylating agent. The present study in conjunction with the previous results, suggests that structural alteration of cellular nucleophiles by acylation may be a biochemical mechanism underlying the biological activity of N-acetoxy3-FAA and related activated carcinogens.
__-_ * This work was supported by United the National Cancer Institute. Abbreviation : FAA, fluorenylacetamide.
States Public Health Service Grant CA02571
from
47
N-Hydroxy-&FAA is a potent cart oral and local administration [ 1,2]. Act cal carcinogenesis, arylhydroxamic ac se but must be activated by esterification furnishes, by spontaneous decomposition, N-acyl-N-arylnitrenium ion which and nucleic acida. These interactions provide the c accepted molecular m hanism for the initiation compounds. In order to test this c we prepared Nqcetoxy=S-FAA, a carcinogen, N-acetoxy-2-FAA, and tested its cart nicity by a variety of routes. The tests indicated that N-acetoxy-$-FAA more potent car&ogen than N-hydroxy-S-FAA upon local administration to the rat [ 2). The compound also produced the malignant transformation of fibroblasts of rat embryos in culture [4]. All of these data were in accord with the idea that the carcinogenicity of arylhydroxamic acids is dependent upon activation of the arylhydroxamic acid by esterification. However, several attempts to demonstrate that N-acetoxy-S-FAA reacts with nucleophilic accept0 through an N-acyl~N-arylnitrenium ion were unsuccessful [ 2,5]. In the search for an alternative mechanism for the action of N-acetoxy&FAA, we decided to explore the capacity of N-acetoxy_S-FAA to acetylate RNAase. We had found previously that the isomeric N-acetoxy-a-FAA interacts with RNAase in a model system not only by arylamidation but also by acetylation and that the acetylation of the e-amino group of lysine in RNAase alters the chromatographic properties of RNAase [S] . The data derived from the studies of the acetylation of RNAase by N-acetoxy-a-FAA form the basis of the present report. EXPERIMENTAL PROCEDURE NjsHj Acetoxy-a-FAA was prepared by the acetylation of N-hydroxy_SFM (101 mg, 132-134’) with [3H]acetic anhydride (25.5 mg, 50 mCi/ mmole, New England Nuclear) and 0.025 ml unlabeled acetic anhydride in pyridine (1 ml) [ 5 ] . A portion of the product (18 mg) was mixed with Nacetoxya-FAA ($9 m2, m.p. 104’) [5) and recrystallized from ethanol : water to give the labeled ester (m.p. 105-106.5”, 5.22 *10Qdpm/pmole). The purity of the labeled compound was established by UV spectrophotometry and by means of several radiochromato s [6] which showed a single radioactive peak coincident with the flu rice-quenching band given by authentic N-acetoxy-a-FAA. Ribonuclease A (XII-A, Sigma) was purified by ion exchange chromate raphy on BiomGelCM-30 (Bio-Rad) as previously described [S] . The reaction was carried out by dissolving N-[“HI acetoxy-S-FAA (23.75 rmoles) in 0.5 ml ethanol and adding the ester slowly Gvera period of 25 min to a solution of RNAase A (4.75 Fmoles) in distilled water (20 ml). The solution was stirred magnetically for 24 h at 25’. After cooling the reaction mixture in ice, 48
h residue was d
The hydrolysates were then radioactive fractions were co11 191%m.g. 247-249’. in the amoun to the pooled fractions. The were chromatographed on 8
R Nase,
0.20
0.10
E
z2
s-
Expt. 2 0.20
u g
0.10
m
2 Expt. 3 0.20
0.10
0
50
100
150
200
250
300
Effluent,ml Fig. 1, Chromatography on Bio-Gel CM-30 of the proteins precipitated with acetone from the reaction of RNAase with N-[3H]acetoxy-3-FAA. The gel was equilibrated in 0.005 M Tris-HCl (pH 8.0) poured into the column (1 x 20 cm) and washed with 200 ml of the buffer. The proteins dissolved in water were applied to the gel and eluted at 4’ with the linear concentration gradient of NaCl in 0.005 M TripHCl [6] indicated by the broken line in the upper section of the figure. The elution was carried out at a rate of 19 ml/h. Effluent fractions of 5-6 ml were collected and the proteins were located by their absorbance at 280 nm. The elution profile of native RNAase is indicated by the dotted line. The figure shows the elution profile of the proteins from three separate experiments in which RNAase was reacted with the 3H-labeled carcinogen as described in the text. The am< mts of proteins in the effluent fractions delineated by the arrows were determined by the modified Folin method [‘I] and are shown in Table I.
pmoles) (Table II) and represented that fraction of the RNAase which had remained largely unmodified. Proteins, B, C and D were displaced from the ion exchange resin at lower Na+ ion concentrations than either protein A or native RNAase (Fig. 1). This would be expected if the net positive charge of SO
TABLE I THE RELATIVE AMOUNTS GF THE MODIFIED PROTEINS IN THE ELUATE FOL LOWING ION EXCHANGE CHROMATOGRAPHY OF RNAase REACIED WITH N-[3H]ACETOXY-3-FAA a Experiment No.
Percent of reacted RNAse recovered b in peak A
in peak B -
1 2 3
6.5
24.6 20.7 12.0
in peak C -I_-_ 28.3 46.2 29.9
in peak D -.-~_
16.6
Apparent total recovery of (S) 52.9 66.9 58.6 (82.3)
a The reaction conditions were the same in all three experiments and are described in the text. b The designation of the peaks refers to the peaks of Fig. 1. The values repr
nt the Folin-reactive compounds in the e&ate fractions delineated by the arrows in Fig. c The values represent the sum of the Folin-reactive material in the pooled &ate frsctions corresponding to the principal peaks B, C and D of Fig: 1. The value in psrenth represents the Folin-reactive compounds in the total eluate. The recovery of native RNAase was 95%.
these modified proteins were progressively decreased relative to that of native RNAase or of protein A. The progressive decrease in the affinity of the modified proteins to the resin (D < C < B) was attributable to the transfer of the acetyl group from N-[3H]acetoxy-3-FAA to RNAase and the modified proteins. This conclusion was based on the observatiorrthat protein B was formed by the addition of approx- 1 mole of acetyP$o 1 mole of RNAase. Proteins C and D, in turn, appeared to result from the addition of 1 mole of acetyl to proteins B and C, respectively (Table II). The extent of the acetyl transfer appeared to vary somewhat since protein D was formed only in 1 of 3 experiments. The most probable explanation for the differences in the yield of the modified proteins lies in the limited solubility of the 3H-labeled TABLE II SPECXFIC RADIOACTIVITIES OF THE PROTEINS ISOLATED FROM THE REACTION OF RNAase with ~-~3H]AG~OXY-3-FAA flatoms sHllO0 pmoles protein b -~-___l-_~_~fll-~-~ 8 A 86 f 5 B 172$-g c 274 D v-m -0 -e _-_ a A, B, C and D refer to the proteins corres~nding to the peaks in the e~uti~n profit Fig. 1. b The values are the means 2 average deviations for the specific radioactivities of the pro teins isolated from experiments 1, 2 and 3 of Fig. 1 and Table 1. Protein isolated a
-
TABLE III RELEASE OF [3HlACETYL AND ACID HYDROLYSIS
GROUPS
FROM PROTEINS
Protein
Hydrolytic medium
Proteinbound jH dpm . 10e5
3H in steam distillate dpm . 10m4
B B C C
acid b alkaline c acid b alkaline c
2.38 2.38 5.54 5.54
20.0 4.1 47.8 9.3
----+ + + +
0.5 0.1 0.3 0.2
B AND C BY ALKALINE
Percent of bound 3H recovered as [ ‘HIacetic acid a -83.9 17.3 86.2 16.8
+ + + +
2.1 0.3 0.4 0.3
Percent of [ 3H]acetyl groups bound in amide linkage
66.6 69.4
a The [ 3H] acetic acid in the hydrolysate was determined by inverse isotope dilution as described in the text. The values are the means + average deviations of duplicate determinations on the proteins isolated from experiments 1, 2 and 3 listed in Fig. 1 and Table I. b Acid hydrolysis was performed in 6 N HzS04 at 100” for 6 h. c Alkaline hydrolysis was performed in 2 N NaOH at 25” for 18 h.
ester in the aqueous incubation system which contained 2.5% ethanol. Any decrease in the solubility of the labeled ester during the incubation would have the effect of changing the concentration of the acetylating agent in solution and thus the extent of modification. Modification by acetylation of RNAase was confirmed by the recovery of [3H] acetic acid following acid and alkaline hydrolysis of proteins B and C (Table III). The data permitted the calculation of a minimum value for the acetyl groups joined to proteins B and C through an amide linkage [6] . The calcsllations ‘indicated that 66 and 69% of the labeled acetyl groups in proteins B and C were bound to these proteins in amide linkage (Table III). Because of the high content of lysine in RNAase [lo], it was anticipated that the e-amino group of this amino acid would be the functional group that would accept the acetyl group of N-[3H]acetoxy-3-FAA. This was shown to be the case by the isolation of E-N-[~H]acetyl-L-lysine from enzymatic digests of proteins B and C. The [‘HI amino acid accounted for 57 to 63% of the tritiated compounds of small molecular weight present in Sephadex G-25 filtrates of enzymatic hydrolysates of proteins B and C (Table IV). It is evident that lysine is the principal amino acid of RNAase that forms an amide linkage with the acetyl group of N-acetoxy-3-FAA and that the formation of e-acetyl-L-lysine is primarily responsible for the change in the chromatographic properties of the RNAase. We have previously reported that N-acetoxy-a-FAA, a carcinogenic isomer of N-acetoxy-3-FAA, modified RNAase by acetylation of the e-amino group of lysine [6]. A comparison of the modification of RNAase by the two carcinogens showed that acetylation of RNAase by N-acetoxy-3-FAA was even more extensive than by ZV-acetoxy-2-FAA. This conclusion wa:3 based 52
TABLE IV ISOLATION OF E-N-[ 3H]ACETYL-L-LYSINE OF THE MODIFIED PROTEINS OBTAINED WITH A’-[ 3H] ACETOXY-3-FAA a Protein hydrolyzed
B C
“H associated with free amino acids b
dpm
Specific radioactivity of purified E-N[3H]acetylL-lysine dpmlmg
1.40 * 106 1.15 * 106
1.01 . 104 8.47 . lo3
FROM ENZYMATIC HYDRDLYSATES FROM THE INCUBIZTION OF RNAase
3H associated with E-N-[ 3H ]acetyl-L-lysine ‘
c-N-[__” ~ 3H]acctyl-L-iysine --“-_----
dpm
%
8.77 . 10s 6.52 . 10’
62.6 56.7
[ 3H]amino
x 166
acids
_
--
a The proteins were hydrolyzed successively with trypsin and pronase as described in the text. b The values refer to the total radioactivity in the pooled fractions obtained after chromatography of the pronase hydrolysates of proteins B and C on Sephadex G-25. r 87 and 77 mg of e-N-acetyl-L-lysine were added as carriers to the pooled fractions that were obtained after Sephadex G-25 chromatography of the pronase hydrolysates of proteins B and C, respectively.
on the following evidence. First, protein A, which represents largely unmodified RNAase, was detected only once in minor amounts when RNAase was incubated with N-acetoxyS-FAA. In contrast, major amounts of protein A were isolated consistently when RNAase was reacted with N-acetoxyS-FAA under identical conditions [6]. Second, protein D, which, as judged by its chromatographic properties and by its specific radioactivity, was the most extensively modified protein in the present experiments, was not detected in the reaction of RNAase with N-acetoxy-2-FAA. Third, the specific radioactivities of proteins B and C obtained from the reaction of RNAase with Nacetoxy-2-FAA [6] were one-half to one-third of the specific radioactivities of the corresponding proteins isolated from the reaction of RNAase with the isomeric N-acetoxy-3-FAA. The data of this report are compatible with a reaction mechanism previously proposed for N-acetoxyS-FAA [6] and applied here to the 3-isomer. According to this mechanism (Fig. 2), N-acetoxy-3-FAA acetylates RNAase, and presumably other proteins, by electrophilic attack on the e-amino group of lysine. The scheme also indicates the possibility of cyclic acetyl transfer by enzymatic reacetylation of the hydroxamate resulting from the spontaneous decomposition of the intermediate adduct. It should be noted that, theoretically, the reaction scheme shown in Fig. 2 applies also to the transfer of other acyl groups, such as the sulfate or phosphate group, to nucleophilic acceptors. Attempts L-3demonstrate the formation of a sulfate ester of N-hydroxy-3-FAA have been unsuccessful thus far, presumably because the test for the formation of such an ester was based on the arylamidation rather than on the acylation of a nucleophilic acceptor [2,5] .
53
Arylamidation of cellular macromolecules has heretofore been the only molecular mechanism considered to be pertinent to the induction of malignant transformation by Wacetoxy-2-FAA and related activated carcinogens [3]. Hawever, it has not been possible tu demonstra~ the ~l~idation of nucleophilic acceptors by ~ffacetoxy-3-~AA f2,5]. The acetylation of cellular macromolec~~ shown here is an afternative rnech~~ to ra~on~i~e the c~c~n~ganic activity of ~-ace~x~3-FAA. The potenti~ for tr~sfe~g the acetyl group is a property which N-acetoxy-3-FAA and N-acetoxy-2-FAA share with other structurally related carcinogens, such as N-acetoxy-4-acetyie aminobiphenyl, ~-~~~etox~2=acetyl~in~phan~threne and Smacetoxyxana thine [l&12] * In the case of the biphenyl and phen~thre~e de~v~~ves, it has been reported that acetylation of ~~o~~e p~dominates over arylamidation [Zl] . In the case of 3~cetoxyx~thine* nu data are available that would permit a ~ua~t~tativ~ comparison of the ace~lation and of the aryiamidation of the same nucleophife under comparable condiGona. The data of the present and the previous study [6] justify the view that, when acetylation 54
of a nucleophilic acceptor is the only demonstrable or the predominant reaction displayed by these carcinogens, structural alterations of cellular nucleophiles by acetylation ought to be considered in attempts to explain the mode of action of these compounds. REFERENCES 1 H.R. Gutmann, D.S. Leaf, Y. Yost, R.E. Rydell and C.C. Chen, Cancer Res., 30 (1970) 1485. 2 Y. Yost, H.R. Gutmann and R.E. Rydell, Cancer Res., 35 (1975) 4471. 3 J.A. Miller, Cancer RBS., 30 (1970) 559. 4 L.I. Sekely, D. Malejka-Giganti, H.R. Gutmann and R.E. Rydell, J. Natl. Cancer Inst., 50 (1973) 1337. 5 F.J. Zieve and H.R. Gutmann, Cancer Res., 31 (1971) 471. 6 E.J. Barry and H.R. Gutmann, J. Biol. Chem., 248 (1973) 2730. 7 E. Layne. Methods Enzymol., 2 (1957) 448. 8 E.D. Chase and J.L. Rabinowitz, Principles of Radioisotope Methodology, Burgess, Minneapolis, 1966, p. 442. 9 A. Neuberger and F. Banger, Biochem. J., 37 (1943) 515. 10 H.A. Scheraga and J.A. Rupley, in F.F. Nord, (Ed.), Advances in Enzymology, Interscience, New York, 1962, p. 161. 11 J.A. Miller and E.C. Miller, in F. Hornburger (Ed.), Progress in Experimental Tumor Research, Vol. 11, Karger, New York, 1967, p. 273. 12 G. Stiihrer and G. Salemnick, Cancer Res., 35 (1975) 122.
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