Methionine metabolism in mammals: S-adenosylhomocysteine hydrolase in rat intestinal mucosa

ARCHIVES

OF BIOCHEMISTRY

Methionine

BIOPHYSICS

Metabolism Hydrolase JAMES

Veterans

AND

Administration

171,

282-286

in Mammals: S-Adenosylhomocysteine in Rat Intestinal Mucosa’

D. FINKELSTEIN2 Hospital

(197%

AND

BARBARA

and Department of Medicine, Medicine, Washington, D.C. Received

June

George 20422

HARRIS Washington

University

School

of

18, 1975

Extracts of rat small intestinal mucosa can catalyze the synthesis of adenosylhomocysteine from homocysteine and adenosine. In order to demonstrate this reaction, adenosine deaminase, which competes for the substrate, must be inhibited or removed by purification. We have also demonstrated catabolism of adenosylhomocysteine during incubation with intestinal extracts. The immediate reaction products are adenosine and homocysteine. Thus, small intestinal mucosa contains both reactivities characteristic of S-adenosylhomocysteine hydrolase. This enzyme has been found in all mammalian tissues that have been studied.

bition did not correlate with the trypsin content of the intestinal preparation. Since we assayed enzyme activity by measuring the rate of synthesis of AdoHcy,4 we suggested the possibility that inhibition resulted from the consumption of the substrate adenosine by adenosine deaminase present in the crude intestinal preparations (1). In the present study, we confirm this hypothesis. S-Adenosylhomocysteine hydrolase is present in the small intestinal mucosa of the rat.

In a previous paper we noted our failure to detect S-adenosylhomocysteine hydrolase (EC 3.3.1.113 in extracts of rat small intestinal mucosa (1). The enzyme was demonstrable in all of the other tissues that we studied. We emphasized the need to confirm our observation since this was the first indication of a block in methionine metabolism in any organ. Prior investigations had established that every tissue possesses the capacity to synthesize S-adenosylmethionine and to metabolize homocysteine by conversion to cystathionine and/or by remethylation to methionine (2, 3). Our uncertainty concerning the presence or absence of S-adenosylhomocysteine hydrolase in intestinal mucosa resulted from our finding that this tissue, as well as spleen, contained a heat-labile factor that inhibited the hepatic enzyme. Inhi-

EXPERIMENTAL

Reagents. All chemicals were of reagent grade and were purchased from commercial sources. We used the method of Shapiro and Ehninger for the syntheses of S-adenosylhomocysteine from various radioactive substrates (4). These included [8‘Y!]adenosine, [8-3H]adenosine, [3,8-3Hz]adenosine and [YS]homocysteine. The latter was derived from 13%]methionine by means of the Baernstein degradation (2, 5). Extracts and enzyme preparations. We routinely used male, Sprague-Dawley rats. The animals were stunned and exsanguinated by carotid transection. The small intestine was removed and immediately rinsed with cold 0.01 M potassium phosphate, pH

1 Supported in part by Grant No. AM-13048 from the National Institutes of Health. * Medical Investigator, Veterans Administration. 3 This enzyme has been named S-adenosykhomocysteine hydrolase (EC 3.3.1.1) despite the fact that the thermodynamics of the reaction favor synthesis of S-adenosylhomocysteine rather than hydrolysis. In this paper we use the term adenosylhomocysteine synthase when we are studying the reaction in the direction of synthesis. Similarly, we employ the designation adenosylhomocysteine hydrolase in studies of the hydrolytic reaction.

4 Abbreviations mocysteine; MAPR, anoside. 282

Copyright All rights

0 1975 by Academic Press, of reproduction in any form

Inc. reserved.

PROCEDURES

used: AdoHey, S-adenosykho6-methylaminopurine-9-ribofur-

S-ADENOSYLHOMOCYSTEINE 7.4. Subsequently, we everted the intestine and removed the mucosa by gentle scraping. The crude mucosal homogenate was prepared in five volumes of the same buffer. Our partially purified preparation of hepatic enzyme was the O-80% ammonium sulfate fraction of de la Haba and Cantoni (61. Enzyme assays. The assay for AdoHcy hydrolase is a modification of our method for the measurement of AdoHcy synthase (1). The reaction media contained [8-‘4C]AdoHcy as substrate, in place of the radioactive adenosine and the homocysteine. The reaction was terminated with perchloric acid, and a standard amount of [8-3H]AdoHcy was added. Subsequently, we isolated the AdoHcy by ion-exchange chromatography on AG 50(H+). The ratio of 3H to 14C allowed us to calculate the quantity of unreacted substrate. Thus, we measured the rate of reaction by the rate of disappearance of the [8-‘4C]AdoHcy. Paper chromatography. We employed Whatman No. 1 paper and the following solvents: (A) n-butanobacetic acid/water, 4:1:5; (B) t-butanollmethyl ethyl ketone/ammonium hydroxide/water, 4:3:1:2; (C) isopropanol/88% formic acid/water, 7:1:2; (D) phenol/water/ammonium hydroxide, 160 g:40:1; (El t-butanobmethyl ethyl ketone/formic acid/water, 40:30:15:15; and (F) 95% ethanol/l M ammonium acetate (pH 5.01, 7:3. RESULTS

Nature

of Small

Intestinal

Inhibitor

Table I illustrates a typical experiment in which the addition of a crude extract of intestinal mucosa limited the synthesis of AdoHcy by the partially purified rat liver enzyme. We did not study in detail the quantitative relationship between the concentration of intestinal extract and the degree of inhibition. However in this experiment, as well as several others, the relationship appeared linear. Total inhibition of the synthesis of AdoHcy occurred with the addition of larger volumes of the extract. In additional experiments, we determined the nature of the radioactive metabolites present after the incubation of [814Cladenosine plus L-homocysteine with partially purified AdoHcy synthase and/or small intestinal extract. The major radioactive peaks in media exposed to the enzyme alone chromatographed with adenosine (unreacted substrate) and AdoHcy (product). In contrast, the media incubated with intestinal extract contained no radioactive AdoHcy and only 8% of the 14C label migrated with unreacted adenosine. The ma-

283

HYDROLASE TABLE INHIBITION

I

OF ADENOSYLHOMOCYSTEINE BY EXTRACTS

Volum;

otextract m 0 0.05 0.10 0.20

OF SMALL

SYNTHASE INTESTINE”

Product formation @mol/lO min) 0.411 0.378 0.259 0.115

o The designated volume of crude small intestinal extract was coincubated with 0.5 mg of enzyme partially purified from rat liver. We employed the routine conditions for assay in the direction of synthesis of AdoHcy (1).

jor radioactive fraction had a chromatographic mobility consistent with inosine. Incubation with enzyme together with intestinal extract revealed all three peaks. AdoHcy

Synthesis

by Small Intestine

From the above experiments it seemed clear that the inhibition of AdoHcy synthase by intestinal extracts resulted from the consumption of the substrate adenosine by an enzyme present in the extract. Adenosine deaminase seemed most probable. For this reason, we studied the effect of the addition of a known inhibitor of adenosine deaminase, 6-methylaminopurine-9-ribofuranoside (N6-methyladenosine), on the synthesis of AdoHcy by extracts of small intestine. By including this reagent in our routine assay system, we could demonstrate the synthesis of a compound which was inseparable from authentic AdoHcy by ion-exchange chromatography (AG 5O(H+)) or during paper chromatography in solvents A and B. Formation of this product required the presence of homocysteine and was a linear function of the concentration of intestinal extract (Table II). Since MAPR has little effect on the activity of partially purified AdoHcy synthase, we estimate that the specific activity of this enzyme in rat intestinal mucosa approximates 23 nmol (mg of protein)-’ (10 min)-*. Finally, we were able to demonstrate AdoHcy synthesis in the absence of MAPR by the intestinal protein fraction which is precipitated between 40 and 60% saturation with ammonium sulfate. However, this fraction retains significant aden-

284

FINKELSTEIN TABLE

SYNTHESIS

Volum;

II BY RAT

Product formation (nmoU30 mini Standard

0.05 0.10 0.25

0 0 0

+ MAPR 50.6 103.9 236.4

a The protein content of the crude intestinal extract was 15 mg/ml. We employed either the standard assay method for AdoHcy synthase or a modifkation in which we added 1 mM 6-methylaminopurine9-ribofuranoside to the reaction medium (+ MAPR).

osine deaminase, and the synthesis of AdoHcy increased from 7.6 to 354 nmol/30 min with the addition of MAPR. Hydrolysis

HARRIS

for the K,. This agrees well with a value of 30 PM obtained by similar studies with the partially purified liver enzyme. de la Haba and Cantoni reported that both products, adenosine and homocysteine, inhibit the hydrolysis of AdoHcy (6). We confirmed this observation. S-Adenosylmethionine also inhibited this reaction. When we added 0.2 mM adenosylmethionine to an assay system containing 15 PM AdoHcy we found that hydrolysis was inhibited by 68%. The degree of inhibition was decreased when we increased the concentration of substrate. Methionine, betaine and cystathionine had no effect on the hydrolysis of AdoHcy. PM

OF ADENOSYLHOMOCYSTEINE SMALL INTESTINE=

otextract m

AND

of AdoHcy

Our method measures the rate of disappearance of radioactive AdoHcy. Therefore, it is less specific for AdoHcy hydrolase than is the measurement of the synthesis of AdoHcy from homocysteine and adenosine. At least three additional enzymes catalyze degradative reactions of AdoHcy. L-Amino acid oxidase (EC 1.4.3.2) is present in mammalian tissues (7). In contrast, AdoHcy nucleosidase has been reported only in bacteria, and mammalian adenosine deaminase, unlike the enzyme from Aspergillus oryzae, cannot utilize AdoHcy as a substrate (8-10). In the present studies we found no evidence for any of these alternative reactions. Specifically we could not detect the predicted products, S-adenosyl-y-thio-cw-ketobutyrate, adenine, or S-inosylhomocysteine. In our system, the rate of disappearance of AdoHcy (15 PM) was proportional to the volume of small intestinal extract added, from 0.01 to 0.1 ml. With higher concentrations of extracts, we observed total degradation of the substrate. The rate of disappearance of AdoHcy is proportional also to the concentration of substrate. The consumption of substrate is almost complete at concentrations below 5 PM. Employing the reaction velocities at the higher substrate concentrations (lo-90 PM), we obtained a tentative value of 33

Identification Hydrolysis

of the Products

of AdoHcy

In order to define the fate of the homocysteine moiety, we characterized the product derived from the hydrolysis of [35Sladenosylhomocysteine. The radioactive product chromatographed with homocysteine in solvents A, C, and D. Following oxidation with per-formic acid, the 35S-labeled product was inseparable from homocysteic acid in these same solvents as well as in solvent B. Additional information, which confirmed that the compound was homocysteine, was obtained in experiments in which we incubated unlabeled AdoHcy, small intestinal extract and 0.2 mM N[ 14C]ethylmaleimide. Chromatograms in solvent E indicated the formation of a radioactive product inseparable from the chemically prepared adduct of homocysteine with N-ethylmaleimide. Formation of this compound required the addition of both AdoHcy and enzyme to the reaction media. We identified the product(s) derived from the adenosine moiety in studies with S-[14C8]adenosyl-L-homocysteine. chromatograms in solvents A, B, and E revealed a single radioactive peak that migrated with uric acid. Since urate was likely to be a final metabolite rather than the immediate derivative of AdoHcy, we studied the effect of the addition of inhibitors of adenosine deaminase (4 mM MAPR) and of xanthine oxidase (4 mM allopuri-

S-ADENOSYLHOMOCYSTEINE

nol). Both inhibitors reduced markedly the rate of disapearance of AdoHcy. Indeed, inhibition with MAPR was sufficient to preclude the identification of any metabolites. This is consistent with the finding that removal of adenosine (and homocysteine) is necessary in order to displace the equilibrium from the direction of AdoHcy synthesis to the direction of hydrolysis (6). Inhibition with allopurinol was less complete. We obtained sufficient radioactive product to establish that the chromatographic mobility was identical to hypoxanthine in solvents A, B, E, and F. Finally, we studied the products obtained when we incubated [3,8-3H,ladenosine together with S-[14C,1AdoHcy and the small intestinal extract. In the absence of inhibitor the 14C-containing product was uric acid and there was no 3H-labeled product. This was anticipated since the C-3 and C-8 positions are oxidized to keto groups in the conversion of adenosine to urate. When we added allopurinol to the reaction media both the 14C- and the 3H-labeled substrates gave rise to a common product that had the chromatographic properties of hypoxanthine. DISCUSSION

In previous studies we defined the pattern of activities of the enzymes of methionine in rat tissues (2, 3). All tissues contain methionine adenosyltransferase (EC 2.5.1.6), and we assumed that each possesses at least one of the many S-adenosylmethionine methyltransferases (11). Furthermore, every tissue has a capacity to metabolize homocysteine by means of cystathionine synthesis and/or remethylation. For this reason, we felt that it was important to amplify our finding that only small intestine, of 11 rat tissues studied, lacked S-adenosylhomocysteine synthase (1). If confirmed, this would raise the possibility of a “block” in methionine metabolism in intestinal mucosa. We considered several explanations for our inability to demonstrate AdoHcy synthase in small intestine. The enzyme could be present but masked by an additional property of the crude extract. Alternatively small intestine might contain an

285

HYDROLASE

alternate pathway for the metabolism of AdoHcy. At least three other enzymes are known to degrade AdoHcy. However, we did not detect the products predicted for any of these reactions. When we reexamined the metabolism of AdoHcy in rat small intestine, we found that extracts of this tissue catalyze the synthesis of AdoHcy from adenosine and homocysteine, if the competing adenosine deaminase reaction is either inhibited differentially with MAPR or removed by partial purification. Under these conditions, product formation is proportional to the concentration of enzyme. In addition, we measured the rate of disappearance of AdoHcy incubated with intestinal extract. The rate is a function of the concentrations of both AdoHcy and the extract. Homocysteine inhibits the reaction. Inhibition of adenosine deaminase also inhibits AdoHcy metabolism by the crude intestinal extracts. We have identified the products of the reaction. The sulfur amino acid portion yields homocysteine. In the absence of any inhibitor, the purine moiety is degraded to uric acid, paralleling the observations made with crude rat liver (12). When we partially inhibited the degradation of AdoHcy by the addition of allopurinol, the product was hypoxanthine. Finally, both adenosine and AdoHcy gave rise to the common products when incubated with crude intestinal extract with or without the addition of inhibitors. The above findings indicate clearly that intestinal mucosa contains an AdoHcy hydrolase which possesses many of the properties of the liver enzyme (6). We found no suggestion of any alternative reaction for the catabolism of AdoHcy. ACKNOWLEDGMENTS We thank Walter Kyle and Ann-Marie Pick their assistance in many aspects of this study.

for

REFERENCES 1. FINKELSTEIN,

Arch. 2. MIJDD, AND

J.

D.,

Biochem. S. H., LASTER,

4382-4392.

AND

Biophys.

FINKELSTEIN, L.

(1965)

HARRIS, B. J. 159, 160-165. J. D.,

J.

Bid.

(1973)

IRREVERRE,

F.,

Chem.

240,

286

FINKELSTEIN

3. FINKELSTEIN,

J. D.,

B. J. (1971) Arch. 92. 4. SHAPIRO,

Anal.

7. MILLER, Chem.

W.

E.,

AND

Siophys.

HARRIS, 146, 84-

HARRIS

8. DUERRE,

K.,

AND EHNINGER, 15, 323-333.

Biochem.

Chem. C. H.,

H.

D.

G.,

(1934)

AND

603-608.

AND

DUERRE,

244, 4273-4276.

J. Biol.

CANTONI,

234,

D.

J.

G. L.

J. A. (1969)

(1959)

J. Biol.

R. D.,

DUERRE,

J. Biochem.

(1966)

Chem.

J. A. (1962)

106,

J.

J. Biol.

Chem.

237, 3737-

3741. 9. WALKER,

S.

5. BAERNSTEIN, 451-456. 6. DE LA HABA,

Biol.

KYLE,

Biochem.

AND

10. SCHLENK, Biophys.

AND

J. A.

(1975)

Can.

53, 312-319.

AND ZYDEK, C. R. (1968) Biochem. Res. Commun. 31, 427-432. 11. MUDD, S. H., AND CANTONI, G. L. (1964) Comp. Biochem. Physiol. 15, l-47. 12. CORTESE, R., PERFETTO, E., ARCARI, P., PROTA, G., AND SALVATORE, F. (1974)Znt. J. Biochem. 5, 535-545. F.,