Chem-Biul. interactions, ll(l975)
599-604
599
0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
EVIDENCE FOR A SECOND ARYLHYDROXAMIC ACID ACYLTRANSFERASE SPECIES IN THE SMALL INTESTINE OF THE RAT
CHARLES W. OLIVE
AND
CHARLES M. KING
Division of Cancer Research, Department of Medicine, Michael Reese Hospital and Medical Center and the University of Chicago Pritzker School of Medicine, Chicago, Ill. 60616 (U.S.A.) (Received February llth, 1975) (Revision received June 4th, 1975) (Accepted June Ilth, 1975)
SUMMARY
The small intestine of the Sprague-Dawley rat has been shown to contain two species of arylhydroxamic acid acyltransferases. These enzymes were separable by gel filtration on Sephadex G-100. The smaller species had the mobility of rat liver acyltransferase and was precipitated with antiserum directed against the liver enzyme. The larger species was not precipitated with this antiserum. These species differ in their relative abilities to utilize N-hydroxy-N-2-acetylaminofluorene (N-hydroxy-AAF) and N-hydroxy-N-Cacetylaminobiphenyl (N-hydroxy-AABP) as substrates, and in their inhibition by non-immune serum.
INTRODUCTION
The induction of tumors by chemicals1 and the toxicity of some drugs2 may result from the alteration of tissue macromolecules by reactive metabolites of these agents. Cytosol of rat liver contains an enzyme that catalyzes the introduction of the arylamine moiety of N-hydroxy-AAF into nucleic acid3J. More recent experiments suggest that this activation is dependent on the generation of reactive Nacetoxy arylamin% that are formed by enzymatic N-r0 acyltransfer5s6. This enzymatic activity has b:en demonstrated in several tissues of the rat including the liver, kidney, stomach, small intestine, mammary gland, colon, lung and spleen5-‘, and two or more tissues of the hamster, rabbit, guinea pig, monkey518 and humans. While the acyltransferase activities in the liver and kidney of Sprague-DawIey Abbreviations: N-hydroxy-AABP, N-hydmxy-N-4acetylaminobiphenyl; roxy-N-2-acetylaminofluorene.
N-hydroxy-AAF,
N-hyd-
600 and Fischer rats are comparable, the activities in other tissues (e.g., the small intestine, colon and lung) differ sharply between these two rat strains*. One explanation which might account for this observation is that tissue-speci~c acyltransferase species are present. The availability of antiserum to a partially purified preparation of liver acyltransferase presented a means by which this possibility might be examined. The present communication presents data, derived in part by use of this antiserum, that demonstrate the presence of two species of acyltransferase in the small intestine of the Sprague-Dawley rat. MATERIALS AND METHODS
The foilowing were obtained from the sources indicated: f9-r4C]&hydroxyAAF (International Chemical and Nuclear Corp., Cleveland, Ohio); dithioth~ito~ and yeast tRNA (Calbiochem Corp., Los Angeles, Ca.); Sephadex G-100 (Pha macia Laboratories, Inc., Piscataway, N.J.); DE-52 diethylaminoethyl cellulose (H. Reeve Angel Co., Clifton, NJ.); Freund’s complete adjuvant (Grand island Biological Co., Grand Island, N.Y.); Freund’s incomplete adjuvant (Difco Laboratories, Detroit, Mi.). [SHIN-Hydroxy-AABP was prepared from [sH]4-nitrobiphenylro. Hydroxamic acids were purified as required by conversion to a cupric cheiate” or by ether extraction of a solution of the hydroxamate in 0.5 N NaOH. Radiochemical purity was established as described previouslys. Male Sprague-Dawley-derived rats were raised and maintained in our facilities as described previously6. Male Syrian golden hamsters weighing approx. 100 g were obtained from the Lakeview Hamster Colony (Newfield, NJ.). Male PF/RB New Zealand White-derived rabbits were obtained from Pel-freez Bio-animals, Inc. (Rogers, Ark.). Rats and hamsters were killed with ether prior to the removal of the tissues; rabbits were killed by injection of air. Tissues were rinsed in chilled 0.05 M pyrophosphate:NaCl buffer, pH 7.0, containing 1 mM dithiothreitol, and cytosols for chromatography were prepared from 40% homogenates in this buffer by centrifugation of the homogenates at 105 000 x g for 1 h at 4”. Cytosols for use in immunoprecipitation experiments were prepared from 20% homogenates of the tissue in 0.05 M Tris:HCl, pH 7.4. For use as antigen, acyltransferase was partially purified from the liver cytosol of male Sprague-Dawley-derived rats (ARS/Sprague-Dawley, Madison, Wis.; Hoitzman Co., Madison, Wis.). This puri~cation was accomplished by salt fractionations, batchwise adsorption to diethylaminoethyl cellulose in 2 mM pyrophosphate: NaCl buffer (pH 6.8) containing 1 mM dithiothreitol, elution with 0.2 M NaCl, concentration by ultrafiltration, and column chromatography on Sephadex G-100 (ref. 6). This procedure resulted in enzyme having specific activities between 70 and 120 nmoles bound per mg protein. Six to eight zones of protein were detectable in this preparation after disc-gel electrophoresis at oH 8.3 using Amido-Black for staining of the protein 12- The enzyme preparation was dialyzed overnight against 0.15 M NaCl and sterilized by filtration through a Millipore filter having a 0.22 p pore size.
601 This material (1.2 mg protein in 0.3 ml) was emulsified with an equal volume of Freund’s complete adjuvant and injected into each rabbit. Six weeks later a second injection (2.5 mg protein in 0.8 ml) was given using Freund’s incomplete adjuvant. After an additional 6 weeks, a booster injection of saline-dialyzed preparation (2 mg protein in 1 ml) was given intravenously and the rabbit was bled 7-10 days later. Blood was obtained either from the ear vein or by direct cardiac puncture. Serum samples were prepared by centrifugation of clots formed at room temperature. Acyltransferase was assayed by determining the incorporation of the labeled aromatic nucleus of N-hydroxy-AAF or N-hydroxy-AABP into tRNA as described previouslye. Corrections were made for non-enzymatic incorporation of isotope. lmmunochemical precipitation of acyltransferase was achieved by incubation of enzyme preparations with immune serum for 1 h at 37” followed by low-speed centrifugation. Aliquots of the supernatant were assayed for acyltransferase activity and compared to the activities of control supernatants that had been incubated with non-immune serum. It should be noted that non-immune serum in sufficiently high quantities can totally inhibit the activity that is observed with either of the two substrates. The highest concentration of non-immune serum used with rat cytosols in the present experiments caused no more than 50% inhibition of the enzyme. RESULTS
Optimal quantities of antiserum precipitated 960; of the enzyme present in 105 000 x g supernatants of liver (Table I). Lesser amounts of acyltransferase were precipitated from the cytosols of other rat tissues. Approximately 67% of the acTABLE I PRECIPITATION OF ACYLTRANSFERPSE BY ANTIBODY TO PARTIALLY PURIFIED RAT LIVER ENZYME
Tisme
Species
Serutn:cyIosol
?i of activity precipitateda
Liver Kidney Stomach Small intestine Colon Spleen Lung Liver Liver Small intestme Small intestine Liver Small intestine
Rat Rat Rat Rat Rat Rat Rat Hamster Hamster Hamster Hamster Rabbit Rabbit
0.25” 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.50 0.25 0.50 0.50 0.50
96 + 2 (7) 76 f
7(3)
86 i 5 (3) 67 f 6(5) 92 :k 3 (3) 81 1’; 5(3) 77 f 4(3) 48
(2)
72 0 5 0 0
(2) (2) (2) (2) (2)
:I The percentage of acyltransferase precipitated with antiserum is shown. [9J”C]N-Hydroxy-AAF (5.5 @i/jlmole) was used as substrate. Average values rt standard deviation are given; the number of cytosols assayed is given in parentheses. Assays were carried out in duplicate. b The optimal ratio for precipitation of acyltransferase from rat liver cytosol was 0.25.
tivity present in a soluble extract of rat small intestine could be precipitated at the ratio of antiserum to cytosol that precipitated 95 ‘A of the liver enzyme. Cytosols obtained from liver and small intestine of the rabbit and hamster were tested at several ratios of antiserum to soluble extract for cross-~actjon with antiserum, The activity was precipitated from hamster liver extracts to a considerable extent at high ratios, but was not removed from cytosols of small intestine (Table I). No pr~ipitation of acyltransfera~ from extracts of rabbit liver or small intestine was observed. Repeated experiments have shown that the acyltransferase of rat small intestine was distributed in two peaks on Sephadex G-100. A typical elution profile is shown in Fig. la. The activity present in the first peak to be eluted was greater than or equal to that in the slower-moving peak, as assayed using N-hydroxy_AAF. No acyltrans4
8
n
2
3 FRACTION
Fig. 1. Sephadex G-100 chromatography of cytosol derived from 40% homogenates of (a) small intestine (35 ml) or (b) liver (30 ml), from rats was performed (4”) using a column (5 x 85 cm) equilibrated with 0.02 M pyrophosphate:NaCI buffer, pH 7, containing 1 mM dithiothreitol. Fractions were assayed for acyltransferase activity using [9-W.T]N-hydroxy-AAF (4.25 .@Zi/pmole) or ~aHl,~-hydroxy-AABP (28.1 ~Ci~~rnoie). Enzyme activities nre exprbwed as nmoies bound per 0.4 ml of solution. , Azsonm; 0, IV-hydroxy-AAF; 0, N-hydroxy-AABP.
603
ferase activity could be detected in soluble extracts of the contents of the small intestine. Chromatography of rat liver cytosol showed a single peak of acyltransferase activity which was eluted at the same volume as the smaller species from the small intestine when either N-hydroxy-AAF (Fig. 1b) or N-hydroxy-AABP was used in the assay mixture. When the slower moving, lower molecular weight species was preincubated with antiserum, more than 85 % of the acyltransferase was precipitated. Similar data were obtained using either of the two substrates. Antiserum was no more effective than control serum in reducing the activity of the larger acyltransferase species under identical experimental conditions. The activities of the acyitransferases of the small intestine were both reduced by the addition of non-immune serum. These partial inhibitions were, however, more pronounced with the larger of the two species. While this phenomenon required consideration in assessing the immunoprecipitation of these enzymes, it further demonstrated basic differences in the two species. In repeated experiments both peaks could be shown to have activity with IVhydroxy_AAF and IV-hydroxy-AABP (Fig. 1). The activity of each of the peaks was greater with the fluorenyl derivative, but the smaller species catalyzed the reaction with the biphenyl derivative mere efficiently. When the soluble fractions obtained from 40% homogenates of rat liver and small intes$ine were mixed and applied to a Sephadex G-100 column, the activity profile obtained was the sum of those obtained by applying liver and intestinal cytosols separately. DISCUSSION
Evidence is presented for the existence of two species of arylhydroxamic acid acyltransferase in extracts of rat small intestine. This conclusion is based on differences in their chromatographic behavior, precipitation by antiserum, sensitivity to nonimmune control serum, and utilization of the acylhydroxamic acids of fluorene and biphenyl as substrates. These observations support the idea that the smaller acyltransferase species is similar to the liver enzyme and is different from the larger species. The observed differences in immunoprecipitation of acyltransferase in hamster liver and small intestine support this interpretation of the data and suggest that multiple forms of the enzyme may exist in animals other than the rat. It is unlikely that the form of acyltransferase which is characteristic of the cytosol of the small intestine is derived from the bacterial flora associated with this tissue since this activity was not detected in the contents of the small intestine. It should also be noted that the liver soluble fraction contains no detectable larger species. This observation argues against the possibility that the larger form of the enzyme could result from dimerization of the liver-type enzyme. Furthermore, experiments in which cytosols of liver and small intestine were mixed prior to chromatography gave no evidence for conversion of the liver enzyme to a larger species. Bartsch et al.7have reported that the mammary gland of pregnant or lactating
Sprague-Dawley rats contained an acyltransferase that could utilize N-hydroxyAABP but not N-hydroxy_AAF as substrate. Other studies have shown Ihat some, but not all, tissues of the Sprague-Dawley rat had higher acyltransferase levels than did comparable tissues from animals of the Fischer strain*. These observations and the present studies suggest that the biochemical activation of arylhydroxamic acids be acyltransferase is a complex process involving multiple enzymes that differ in their ability to activate closely related substrates. Accordingly, it will be necessary to consider these factors in attempts to determine the role of these acyltransferases in the induction of tumors by arylamines. ACKNOWLEDGEMENTS
The authors wish to thank Dr. Raymond A. Cardona for the synthesis of [SHIN-hydroxy-AABP and Mr. Zenon M. Lortz for technical assistance. These studies were supported by a grant from the Jules J. Reingold Trust, NIH Research Grants CA 13179 and 15640 from the National Cancer Institute, and the Medical Research Institute Council of Michael Reese Medical Center.
REFERENCES 1 J. A. Miller and E. C. Miller, Chemical carcinogenesis: mechanisms and approaches to its control, J. Nat/. Cancer Inst., 47 (1971) No. 3: v-xiv. 2 J. R. Gillette, A perspective on the role of chemically reactive metabolites of foreign compounds in toxicity, 1. Correlation of changes of covalent binding of reactivity metabolites with changes in the incidence and severity of toxicity, B&hem. Phcwnmcol., 23 (1974) 2785-2794. 3 C. M. King and B. Phillips, Enzyme-catalyzed reactions of the carcinogen N-hydroxy-2-fluorenylacetamide with nucleic acid, Science, 159 (1968) 1351-1353. 4 J. R. DeBaun, J. Y. Rowley, E. C. Miller and J. A. Miller, Sulfotransferase activation of Nhydroxy-2-acetylaminofluorene in rodent livers susceptible and resistant to this carcinogen, Proc. Sot. Exptl. Bioi. Med., 129 (1968) 268-273. 5 H. Bartsch, M. Dworkin, J. A. Miller and E. C. Miller, Electrophilic N-acetoxyaminoarenes derived from carcinogenic N-hydroxy-N-acetylaminoarenes by enzymatic deacetylation and transacetylation in liver, Biochim. Biophys. Arta, 286 (1972) 272-298. 6 C. M. King, Mechanism of reaction, tissue distribution, and inhibition of arylhydroxamic acid acyltransferase, Cancer Res., 34 (1974) 1503-1515. 7 H. Bartsch, C. Dworkin, E. C. Miller and J. A. Miller, Formation of electrophilic N-acetoxyarylamines in cytosols from rat mammary gland and other tissues by transacetylation from the carcinogen N-hydroxy-4-acetylaminobiphenyl, Biochim. Biophys. Acta, 304 (1973) 42-55. 8 C. M. King and C. W. Olive, Comparative effects of strain, species and sex on the acyltransferase and sulfotransferase catalyzed activations of N-hydroxy-N-2-fluorenylacetamide. Cancer Rcs., 35 (1975) 906-912. 9 C. M. King, C. W. Olive and R. A. Cardona, Activation of carcinogenic arylhydroxamic acids ‘by human tissues, J. Natl. Cancer Inst., 55 (1975) 285-287. 10 E. Kriek, On the mechanism of action of carcinogenic aromatic amines, 11. Binding of N-hydroxy-N-acetyl&aminobiphenyl to rat liver nucleic acids in viva, Chem.-Biol. Interact., 3 (1971) 19-28. 11 M. Enomoto, P. Lotlikar, J. A. Miller and E. C. Miller, Urinary metabolites of 2-acetylaminofluorene and related compounds in the Rhesus monkey, Cancer Res., 22 (1962) 1336-1342. 12 Polyanalyst. An Analytical Tet~lpcrature-rcgulted Electrophoresis Apparatus, Buchler Instruments, Fort Lee, N. J., Sept. 1971.
599-604
599
0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
EVIDENCE FOR A SECOND ARYLHYDROXAMIC ACID ACYLTRANSFERASE SPECIES IN THE SMALL INTESTINE OF THE RAT
CHARLES W. OLIVE
AND
CHARLES M. KING
Division of Cancer Research, Department of Medicine, Michael Reese Hospital and Medical Center and the University of Chicago Pritzker School of Medicine, Chicago, Ill. 60616 (U.S.A.) (Received February llth, 1975) (Revision received June 4th, 1975) (Accepted June Ilth, 1975)
SUMMARY
The small intestine of the Sprague-Dawley rat has been shown to contain two species of arylhydroxamic acid acyltransferases. These enzymes were separable by gel filtration on Sephadex G-100. The smaller species had the mobility of rat liver acyltransferase and was precipitated with antiserum directed against the liver enzyme. The larger species was not precipitated with this antiserum. These species differ in their relative abilities to utilize N-hydroxy-N-2-acetylaminofluorene (N-hydroxy-AAF) and N-hydroxy-N-Cacetylaminobiphenyl (N-hydroxy-AABP) as substrates, and in their inhibition by non-immune serum.
INTRODUCTION
The induction of tumors by chemicals1 and the toxicity of some drugs2 may result from the alteration of tissue macromolecules by reactive metabolites of these agents. Cytosol of rat liver contains an enzyme that catalyzes the introduction of the arylamine moiety of N-hydroxy-AAF into nucleic acid3J. More recent experiments suggest that this activation is dependent on the generation of reactive Nacetoxy arylamin% that are formed by enzymatic N-r0 acyltransfer5s6. This enzymatic activity has b:en demonstrated in several tissues of the rat including the liver, kidney, stomach, small intestine, mammary gland, colon, lung and spleen5-‘, and two or more tissues of the hamster, rabbit, guinea pig, monkey518 and humans. While the acyltransferase activities in the liver and kidney of Sprague-DawIey Abbreviations: N-hydroxy-AABP, N-hydmxy-N-4acetylaminobiphenyl; roxy-N-2-acetylaminofluorene.
N-hydroxy-AAF,
N-hyd-
600 and Fischer rats are comparable, the activities in other tissues (e.g., the small intestine, colon and lung) differ sharply between these two rat strains*. One explanation which might account for this observation is that tissue-speci~c acyltransferase species are present. The availability of antiserum to a partially purified preparation of liver acyltransferase presented a means by which this possibility might be examined. The present communication presents data, derived in part by use of this antiserum, that demonstrate the presence of two species of acyltransferase in the small intestine of the Sprague-Dawley rat. MATERIALS AND METHODS
The foilowing were obtained from the sources indicated: f9-r4C]&hydroxyAAF (International Chemical and Nuclear Corp., Cleveland, Ohio); dithioth~ito~ and yeast tRNA (Calbiochem Corp., Los Angeles, Ca.); Sephadex G-100 (Pha macia Laboratories, Inc., Piscataway, N.J.); DE-52 diethylaminoethyl cellulose (H. Reeve Angel Co., Clifton, NJ.); Freund’s complete adjuvant (Grand island Biological Co., Grand Island, N.Y.); Freund’s incomplete adjuvant (Difco Laboratories, Detroit, Mi.). [SHIN-Hydroxy-AABP was prepared from [sH]4-nitrobiphenylro. Hydroxamic acids were purified as required by conversion to a cupric cheiate” or by ether extraction of a solution of the hydroxamate in 0.5 N NaOH. Radiochemical purity was established as described previouslys. Male Sprague-Dawley-derived rats were raised and maintained in our facilities as described previously6. Male Syrian golden hamsters weighing approx. 100 g were obtained from the Lakeview Hamster Colony (Newfield, NJ.). Male PF/RB New Zealand White-derived rabbits were obtained from Pel-freez Bio-animals, Inc. (Rogers, Ark.). Rats and hamsters were killed with ether prior to the removal of the tissues; rabbits were killed by injection of air. Tissues were rinsed in chilled 0.05 M pyrophosphate:NaCl buffer, pH 7.0, containing 1 mM dithiothreitol, and cytosols for chromatography were prepared from 40% homogenates in this buffer by centrifugation of the homogenates at 105 000 x g for 1 h at 4”. Cytosols for use in immunoprecipitation experiments were prepared from 20% homogenates of the tissue in 0.05 M Tris:HCl, pH 7.4. For use as antigen, acyltransferase was partially purified from the liver cytosol of male Sprague-Dawley-derived rats (ARS/Sprague-Dawley, Madison, Wis.; Hoitzman Co., Madison, Wis.). This puri~cation was accomplished by salt fractionations, batchwise adsorption to diethylaminoethyl cellulose in 2 mM pyrophosphate: NaCl buffer (pH 6.8) containing 1 mM dithiothreitol, elution with 0.2 M NaCl, concentration by ultrafiltration, and column chromatography on Sephadex G-100 (ref. 6). This procedure resulted in enzyme having specific activities between 70 and 120 nmoles bound per mg protein. Six to eight zones of protein were detectable in this preparation after disc-gel electrophoresis at oH 8.3 using Amido-Black for staining of the protein 12- The enzyme preparation was dialyzed overnight against 0.15 M NaCl and sterilized by filtration through a Millipore filter having a 0.22 p pore size.
601 This material (1.2 mg protein in 0.3 ml) was emulsified with an equal volume of Freund’s complete adjuvant and injected into each rabbit. Six weeks later a second injection (2.5 mg protein in 0.8 ml) was given using Freund’s incomplete adjuvant. After an additional 6 weeks, a booster injection of saline-dialyzed preparation (2 mg protein in 1 ml) was given intravenously and the rabbit was bled 7-10 days later. Blood was obtained either from the ear vein or by direct cardiac puncture. Serum samples were prepared by centrifugation of clots formed at room temperature. Acyltransferase was assayed by determining the incorporation of the labeled aromatic nucleus of N-hydroxy-AAF or N-hydroxy-AABP into tRNA as described previouslye. Corrections were made for non-enzymatic incorporation of isotope. lmmunochemical precipitation of acyltransferase was achieved by incubation of enzyme preparations with immune serum for 1 h at 37” followed by low-speed centrifugation. Aliquots of the supernatant were assayed for acyltransferase activity and compared to the activities of control supernatants that had been incubated with non-immune serum. It should be noted that non-immune serum in sufficiently high quantities can totally inhibit the activity that is observed with either of the two substrates. The highest concentration of non-immune serum used with rat cytosols in the present experiments caused no more than 50% inhibition of the enzyme. RESULTS
Optimal quantities of antiserum precipitated 960; of the enzyme present in 105 000 x g supernatants of liver (Table I). Lesser amounts of acyltransferase were precipitated from the cytosols of other rat tissues. Approximately 67% of the acTABLE I PRECIPITATION OF ACYLTRANSFERPSE BY ANTIBODY TO PARTIALLY PURIFIED RAT LIVER ENZYME
Tisme
Species
Serutn:cyIosol
?i of activity precipitateda
Liver Kidney Stomach Small intestine Colon Spleen Lung Liver Liver Small intestme Small intestine Liver Small intestine
Rat Rat Rat Rat Rat Rat Rat Hamster Hamster Hamster Hamster Rabbit Rabbit
0.25” 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.50 0.25 0.50 0.50 0.50
96 + 2 (7) 76 f
7(3)
86 i 5 (3) 67 f 6(5) 92 :k 3 (3) 81 1’; 5(3) 77 f 4(3) 48
(2)
72 0 5 0 0
(2) (2) (2) (2) (2)
:I The percentage of acyltransferase precipitated with antiserum is shown. [9J”C]N-Hydroxy-AAF (5.5 @i/jlmole) was used as substrate. Average values rt standard deviation are given; the number of cytosols assayed is given in parentheses. Assays were carried out in duplicate. b The optimal ratio for precipitation of acyltransferase from rat liver cytosol was 0.25.
tivity present in a soluble extract of rat small intestine could be precipitated at the ratio of antiserum to cytosol that precipitated 95 ‘A of the liver enzyme. Cytosols obtained from liver and small intestine of the rabbit and hamster were tested at several ratios of antiserum to soluble extract for cross-~actjon with antiserum, The activity was precipitated from hamster liver extracts to a considerable extent at high ratios, but was not removed from cytosols of small intestine (Table I). No pr~ipitation of acyltransfera~ from extracts of rabbit liver or small intestine was observed. Repeated experiments have shown that the acyltransferase of rat small intestine was distributed in two peaks on Sephadex G-100. A typical elution profile is shown in Fig. la. The activity present in the first peak to be eluted was greater than or equal to that in the slower-moving peak, as assayed using N-hydroxy_AAF. No acyltrans4
8
n
2
3 FRACTION
Fig. 1. Sephadex G-100 chromatography of cytosol derived from 40% homogenates of (a) small intestine (35 ml) or (b) liver (30 ml), from rats was performed (4”) using a column (5 x 85 cm) equilibrated with 0.02 M pyrophosphate:NaCI buffer, pH 7, containing 1 mM dithiothreitol. Fractions were assayed for acyltransferase activity using [9-W.T]N-hydroxy-AAF (4.25 .@Zi/pmole) or ~aHl,~-hydroxy-AABP (28.1 ~Ci~~rnoie). Enzyme activities nre exprbwed as nmoies bound per 0.4 ml of solution. , Azsonm; 0, IV-hydroxy-AAF; 0, N-hydroxy-AABP.
603
ferase activity could be detected in soluble extracts of the contents of the small intestine. Chromatography of rat liver cytosol showed a single peak of acyltransferase activity which was eluted at the same volume as the smaller species from the small intestine when either N-hydroxy-AAF (Fig. 1b) or N-hydroxy-AABP was used in the assay mixture. When the slower moving, lower molecular weight species was preincubated with antiserum, more than 85 % of the acyltransferase was precipitated. Similar data were obtained using either of the two substrates. Antiserum was no more effective than control serum in reducing the activity of the larger acyltransferase species under identical experimental conditions. The activities of the acyitransferases of the small intestine were both reduced by the addition of non-immune serum. These partial inhibitions were, however, more pronounced with the larger of the two species. While this phenomenon required consideration in assessing the immunoprecipitation of these enzymes, it further demonstrated basic differences in the two species. In repeated experiments both peaks could be shown to have activity with IVhydroxy_AAF and IV-hydroxy-AABP (Fig. 1). The activity of each of the peaks was greater with the fluorenyl derivative, but the smaller species catalyzed the reaction with the biphenyl derivative mere efficiently. When the soluble fractions obtained from 40% homogenates of rat liver and small intes$ine were mixed and applied to a Sephadex G-100 column, the activity profile obtained was the sum of those obtained by applying liver and intestinal cytosols separately. DISCUSSION
Evidence is presented for the existence of two species of arylhydroxamic acid acyltransferase in extracts of rat small intestine. This conclusion is based on differences in their chromatographic behavior, precipitation by antiserum, sensitivity to nonimmune control serum, and utilization of the acylhydroxamic acids of fluorene and biphenyl as substrates. These observations support the idea that the smaller acyltransferase species is similar to the liver enzyme and is different from the larger species. The observed differences in immunoprecipitation of acyltransferase in hamster liver and small intestine support this interpretation of the data and suggest that multiple forms of the enzyme may exist in animals other than the rat. It is unlikely that the form of acyltransferase which is characteristic of the cytosol of the small intestine is derived from the bacterial flora associated with this tissue since this activity was not detected in the contents of the small intestine. It should also be noted that the liver soluble fraction contains no detectable larger species. This observation argues against the possibility that the larger form of the enzyme could result from dimerization of the liver-type enzyme. Furthermore, experiments in which cytosols of liver and small intestine were mixed prior to chromatography gave no evidence for conversion of the liver enzyme to a larger species. Bartsch et al.7have reported that the mammary gland of pregnant or lactating
Sprague-Dawley rats contained an acyltransferase that could utilize N-hydroxyAABP but not N-hydroxy_AAF as substrate. Other studies have shown Ihat some, but not all, tissues of the Sprague-Dawley rat had higher acyltransferase levels than did comparable tissues from animals of the Fischer strain*. These observations and the present studies suggest that the biochemical activation of arylhydroxamic acids be acyltransferase is a complex process involving multiple enzymes that differ in their ability to activate closely related substrates. Accordingly, it will be necessary to consider these factors in attempts to determine the role of these acyltransferases in the induction of tumors by arylamines. ACKNOWLEDGEMENTS
The authors wish to thank Dr. Raymond A. Cardona for the synthesis of [SHIN-hydroxy-AABP and Mr. Zenon M. Lortz for technical assistance. These studies were supported by a grant from the Jules J. Reingold Trust, NIH Research Grants CA 13179 and 15640 from the National Cancer Institute, and the Medical Research Institute Council of Michael Reese Medical Center.
REFERENCES 1 J. A. Miller and E. C. Miller, Chemical carcinogenesis: mechanisms and approaches to its control, J. Nat/. Cancer Inst., 47 (1971) No. 3: v-xiv. 2 J. R. Gillette, A perspective on the role of chemically reactive metabolites of foreign compounds in toxicity, 1. Correlation of changes of covalent binding of reactivity metabolites with changes in the incidence and severity of toxicity, B&hem. Phcwnmcol., 23 (1974) 2785-2794. 3 C. M. King and B. Phillips, Enzyme-catalyzed reactions of the carcinogen N-hydroxy-2-fluorenylacetamide with nucleic acid, Science, 159 (1968) 1351-1353. 4 J. R. DeBaun, J. Y. Rowley, E. C. Miller and J. A. Miller, Sulfotransferase activation of Nhydroxy-2-acetylaminofluorene in rodent livers susceptible and resistant to this carcinogen, Proc. Sot. Exptl. Bioi. Med., 129 (1968) 268-273. 5 H. Bartsch, M. Dworkin, J. A. Miller and E. C. Miller, Electrophilic N-acetoxyaminoarenes derived from carcinogenic N-hydroxy-N-acetylaminoarenes by enzymatic deacetylation and transacetylation in liver, Biochim. Biophys. Arta, 286 (1972) 272-298. 6 C. M. King, Mechanism of reaction, tissue distribution, and inhibition of arylhydroxamic acid acyltransferase, Cancer Res., 34 (1974) 1503-1515. 7 H. Bartsch, C. Dworkin, E. C. Miller and J. A. Miller, Formation of electrophilic N-acetoxyarylamines in cytosols from rat mammary gland and other tissues by transacetylation from the carcinogen N-hydroxy-4-acetylaminobiphenyl, Biochim. Biophys. Acta, 304 (1973) 42-55. 8 C. M. King and C. W. Olive, Comparative effects of strain, species and sex on the acyltransferase and sulfotransferase catalyzed activations of N-hydroxy-N-2-fluorenylacetamide. Cancer Rcs., 35 (1975) 906-912. 9 C. M. King, C. W. Olive and R. A. Cardona, Activation of carcinogenic arylhydroxamic acids ‘by human tissues, J. Natl. Cancer Inst., 55 (1975) 285-287. 10 E. Kriek, On the mechanism of action of carcinogenic aromatic amines, 11. Binding of N-hydroxy-N-acetyl&aminobiphenyl to rat liver nucleic acids in viva, Chem.-Biol. Interact., 3 (1971) 19-28. 11 M. Enomoto, P. Lotlikar, J. A. Miller and E. C. Miller, Urinary metabolites of 2-acetylaminofluorene and related compounds in the Rhesus monkey, Cancer Res., 22 (1962) 1336-1342. 12 Polyanalyst. An Analytical Tet~lpcrature-rcgulted Electrophoresis Apparatus, Buchler Instruments, Fort Lee, N. J., Sept. 1971.
