Methylthioadenosine nucleoside phosphorylase activity in Drosophila melanogaster

Inr. J &orhsm.. Vol IO. pp. 901 to 904 Perpamon Press Ltd 1979. Printed in Great Britain

METHYLTHIOADENOSINE NUCLEOSIDE PHOSPHO~YLASE ACTIVITY IN DROSOPHILA MELANOGASTER* LEE SHUGART,MANUELTANCER~and JENNIFERMOORE$ Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830 U.S.A. (Received 23 March 1979)

Abstract-I.

An enzyme activity that brings about the phosphorolysis of methylthioadenosine has been isolated from ~r~~phj~~ rne~~n~g~ster and purified approx. 25-fold. 2. The products of the reaction have been identified as adenine and methylthioribo~-~-phosphate. 3. The enzyme catalyzes the reverse reaction with these compounds.

INTRODUflION

The several mechanisms by which MeSAd+ synthesis occurs in both procaryotes and eucaryotes are welf established (see Cacciapuoti et al. (1978) for details); however, the question of the biological function of this compound in cells has yet to be resolved. This is due in part to the un~rtainty about the identity of the product(s) of its eatabohc breakdown. MeSAdo is an inhibitor towards many of the transmethyiation reactions that utilize AdoMet (Zappia et al., 1969). The main pathway whereby MeSAdo is degraded by microorganisms involves a hydrolytic nucleosidase (Shapiro & Mather, 1978) which acts at the glycosidic bond and releases Ade plus MeS-Rib. The enzyme has been isolated and purified from the bacterium Escherichiu coli and demonstrated to have activity toward other thioether-containing compounds, including AdoHcy (Duerre, 1962; Ferro et al., 1976). A MeSAdo-nucleosidase requiring inorganic phosphate has been shown in eucaryotic organisms (Pegg & Williams-Ashman, 1969; Toohey, 1977) with a strict specificity toward its substrate. Pegg & Williams-Ashman (1969) reported an absolute phosphateion dependence for a 30-fold purified enzyme activity isolated from rat ventral prostate and concluded that the mechanism of the enzyme was likely phosphoroly*Research sponsored by the Ofice of Health and Environmental Research, U.S. Department of Energy, under Contract W-7405~enawith the Union Carbide Corporation. t Oak Ridge Associated Universities undergraduate research participant, Summer 1978. Present address: Princeton University, Princeton, NJ, U.S.A. $ Great Lakes Colleges Ass~iation/Associated Colleges of the Midwest student, Fall 1978. Present address: Ohio Wesleyan University, Delaware, OH, U.S.A. 4 Abbreviations: MeSAdo, S-methylthioadenosine; AdoMet, adenosyimethionine; MeS-Rib, S-methylthjorjbose; MeS-Rib-l-P. 5’-methylthioribose l-phosphate; Ade, adenine: AdoHey. adenosylhomocysteine; Ado. adenosine; Ino, inosine; Rib-l-P, ribose-l-phosphate; Hepes, 4-(2-hydroxyethyl)-l-pi~razineethanesulfonic acid; HPLC. highpressure liquid chromatography: BBOT. 2,5-his-[2-(%tertbutylbenzoxazolyl)]-thiophene; DEAE-cellulose, O-(diethylaminoethyI)cellulose; e, 1, N6-etheno: DTT. dithiothreitol: TLC. thin-layer chromatography. B.C. IO,1 I_-(-

tic. Their inability to find MeS-Rib-l-P was attributed to the instability of the compound and the presence of phosphatases in the enzyme preparation. Caccipuoti et af. (1978) found that phosphate enhanced a highly purified enzyme activity from human placental tissue and concluded that phosphate-ion stimulation plays some sort of cellular regulatory role. Their data implies that either the enzyme is phosphoroiytic in its action or the phosphate binds to serine residue(s) in the enzyme, promoting favorable conformational changes at the active sites. Toohey (1977, 1978) demonstrated an enzyme activity in malignant murine hematopoietic cells which catalyzes the phosphorolytic cleavage of MeSAdo to yield MeS-Rib-l-P and Ade. The cetlular catabolism of Ade is understood: Ade enters the purine pool (Vogels & Van Der Drift, 1976). The data of Shroeder et al. (1973) demonstrates that in microorganisms MeS-Rib is a terminal product that is excreted into the growth medium. In eucaryotes, the metabolic fate of MeS-Rib and its phosphorylated analogue is not known. Toohey (1977, 1978) finds that MeS-Rib-l-P can be further broken down enzymatically to an alkylthio group which is in some way involved in cell division. In this paper, the occurrence of a MeSAdo nucleoside phosphorylase in drosophila ~la~gaster is reported for the first time. A purification scheme for the enzyme is described. Some of its properties are reported, as is an analysis of the products of the enzymic reaction. MATERIALS

AND METHODS

Materials

All chemicals were reagent grade. [14C, CH,]AdoMet (sp. act. 56Ci/mol) was obtained from Amersham/Searle; AdoMet and AdoHcy from Sigma Chemical Co.; Hepes. cc-o-Rib-l-P, and Ade from CaIbiochem; DDT and L-Hey from Vega-Fox Biochemicals; Ado from P-L Biochemicab; chloroacetaldehyde (300/, in water) from Columbia Organic Chemical Co. MeSAdo and MeS-Rib were a gift of Dr J. Duerre, University of North Dakota School of Medicine. Alkaline phosphatase (Code BAPC) was ourchased from Worthington Biochemical Corp. Amine; A6 and Cellex-D were obtained from Bio-Rad Laboratories;

901

902

LEE SHLJGART, MANUELTANCERand JENNIFERMIXRE Table 1. Purification

Purification step* 250,000 g supernatant Heat treatment (3 min at 6o’C) DEAE-cellulose chromatography Ultrafiltration

(pool)

procedure

Specific activity (nmols Ade/ 30 min/mg protein)

Enzyme unit (nmols Ade/30 min)

Total volume (ml)

Protein (mgiml)

33

14.6

9

4324

I.0

I00

25

6.8

25

4250

2.8

98

69 4

0.7 4.5

44 215

2034 3870

5.0 24.0

47 89

Purification (fold)

Yield (““)

* See Methods for details. and Silica Gel 1B chromatographic sheets were from J. T. Baker Chemicals. An ultrafiltration auparatus (Model 202) was purchased from Amicon Corp. . . HPLC

HPLC was performed in a 0.6 x 24cm glass column (Laboratory Data Control, Model LC-6M-13) as previously described (Shugart, 1978). Under these conditions. retention times (min) were: Ino. 13; Ado, 15; AdoHcy, 19; Ade, 30; MeSAdo, 40; the e-derivative of MeSAdo, 73; and AdoMet. 112. TLC

Samples in 10~1 aliquots were spotted onto a silica gel cbromatographic sheet, placed in an Eastman Chromagram developing apparatus, and developed in a single dimension with a solvent of n-butanol:ethapol:water (52:32: 16 v/v) at room temperature. Total run time averaged 7 hr for the solvent front to travel a distance of 16 cm. The chromatograms were air-dried and then observed under U.V.light, sprayed with a stain for pentose detection (Saini, 1966), and (where applicable) scraped in fractions into vials containing 5 ml toluene:BBOT scintillation fluid and counted in a scintillation spectrometer (Nuclear Chicago, Mark I) for radioactivity. Enzyme purijication and assay D. melanogaster. Drosophila were 0 to l-day-old Oregon-R wild type provided by Dr K. B. Jacobson of the Biology Division. Oak Ridge National Laboratory. The heads. wings and legs had previously been removed by sifting, and the remaining thorax-abdomens (nubs) were kept at -85°C until used. MeSAdo nucleoside phosphorylase assay. A standard reaction mixture in a final volume of 50~1 consisted of 5 ~1 of substrate (25 nmols of MeSAdo or [i“C, CH,]MeSAdo) 15 ~1 of 0.1 M Hepes buffer (pH 7.5), 5 ~1 of potassium phosphate (pH 7.8) and 5 ~1 of enzyme. The reaction, which was initiated by the addition of enzyme, was incubated at 30°C for 30 min and terminated with 10~1 of 4 M formic acid. Specific activity of the enzyme is defined as nmols of product formed in 30 min per mg of protein under the assay conditions specified. Product formation was measured as follows: 1. An aliquot (50~1) was injected onto the Aminex A6 HPLC column. and the amount of Ade enzymatically produced was determined spectrophotometrically. 2. The entire reaction mixture was applied quantitatively with 1 ml of water to a Dowex AG 50W column in the H + form (1 ml settled bed volume in a disposable Pasteur pipette). The material that passed through the column was collected and represents MeS-Rib-l-P (Toohey, 1978). Enzyme purification. Table 1 summarizes the purification scheme. Unless specified, all steps were performed at 6°C. Oregon-R Drosophila nubs (20g) were homogenized in 50 ml of buffer [lo mM Tris-HCI (pH 7.4) 10 mM magne-

sium acetate, 50mM KCI, 0.25 M sucrose, 107; (v/v) glycerol and 1 mM DTT) for 5 min. The homogenate was centrifuged at 10,OOOgfor 30 min; then the supernatant solution was filtered through Micracloth and recentrifuged at 250,000 g for 2 hr. The supernatant solution was heated in water bath with continuous mixing for 3 min at 6o’C and then rapidly cooled. The resulting precipitate was removed by centrifugation and the supernatant solution was applied directly to a DEAE-cellulose column (1.2 x 29cm) that had been previously equilibrated in the homogenizing buffer. Subsequently, the sample was eluted with a 2OOml linear KCI gradient from 0.05 to 0.35 M in the homogenizing buffer. Column fractions containing enzyme activity were pooled and concentrated by ultrafiltration. The enzyme preparation was resuspended in 0.1 M Hepes buffer (pH 7.5) containing 50% glycerol (v/v) and stored at -85°C. Miscellaneous

Protein concentrations of enzyme preparations were esttmated by the method of Lowry et al. (1951). Column fractions from DEAE-cellulose which contained enzyme activity were concentrated, suspended in 0.1 M Hepes buffer (pH 7.5) and reconcentrated by use of an Amicon Model 202 ultrafiltration apparatus at 6’C with a type UM-10 membrane. ft4C, CHqlMeSAdo was prepared by the method of Schlenk & Bhninger (1964). -In-a small glass test tube, 5 nmols of [t“C, CH,]AdoMet were mixed with 2.5 pmols of unlabeled AdoMet and 25 ~1 of 1 mN HCl, then sealed with Parafilm. After gradual warming, the solution was olaced in a boiling-water bath for 30 min. The resulting mixture was injected onto the Aminex A6 HPLC column. and the material eluting as MeSAdo was collected, lyophilyzed to dryness, and resuspended in water. The specific activity of this material (cpm/nmol) was determined. The e-derivative of MeSAdo was prepared by the method of Secrest et al. (1972). RESULTS Properties

of the enzyme

Assay conditions. Optimum assay conditions for MeSAdo nucleoside phosphorylase activity in D. melatwgaster were found to be essentially as reported by Pegg and Williams-Ashman (1969). An absolute dependence on phosphate ion was found at all stages of purification. Purtjication procedure. It was observed that highspeed centrifugation for long periods of time (overnight) and subsequent heat treatment resulted in total loss of enzyme activity. Activity was still present after ccntrifugation, suggesting a destabilization of the enzyme in the centrifugation step which results in

MeSAdo 40-

f/V

catabolism

0

IL/

_A-/-

z Km 16

‘OmM P

0.05

0.10

l/[FliOSPHATE]

0.15 mM-’

A-Z 0.005

K,

/

K,

I CO6 “M,’ /

,

\,

tion, Ade production was not observed when Ado, AdoHcy, AdoMet or the e-derivative of MeSAdo replaced MeSAdo as a substrate in the assay reaction mixture. Other enzyme activities. The presence of an AdoHcy hydrolase activity could be detected in the Drosophila preparation before DEAE-cellulose column chromatography but not after ultrafiltration. This activity was assayed for in the direction of AdoHcy synthesis (Richards et al., 1978). In addition, the final preparation contained an adenosine deaminase activity which had no activity toward MeSAdo, AdoHcy or AdoMet. Identification of products. The phosphorolytic cleavage of MeSAdo is assumed to proceed according to the equation MeSAdo

mM

, 0

20 l/[MeSAdo]

40

60

mM-’

Fig. 1. Etfect of phosphate and MeSAdo concentrations on MeSAdo nucleoside phosphorylase: The assay reaction mixture in a final volume of 50~1 contained 1.5 pmoles of Hepes buffer (pH 7.5). To determine the effect of phosphate ion concentration on enzyme activity, we held MeSAdo constant at 5.6 nmols in the reaction mixtures (Fig. la); the phosphate ion was held constant at 2.5 pmols (Fig. I b) for determination of the effect of MeSAdo concentration. The reactions were initiated with approx. 2 pg of enzyme protein (sp. act. 215 nmols Ade/30 min/mg protein) and allowed to incubate at 30°C for 10 min. The amount of Ade formed during this period of time (nmols/lO min) was determined spectrophotemetrically on an Aminex A6 HPLC column.

903

in Drosophila

+ P,

Under the assay conditions specified (see Methods) with [i4C, CH,]MeSAdo as a substrate, stoichiometric amounts of Ade and a radioactive product were detected. In an effort to isolate and identify the radioactive product, we chromatographed various enzymic reactions and known compounds on silica gel sheets. The R, values obtained by TLC are given in Table 2. Only one radioactive product from the enzymatic cleavage of [14C, CHJMeSAdo is detectable by this method, and it has an R, value of 0.05. If the radioactive product is isolated from the TLC sheet, treated with alkaline phosphatase, and rechromatographed, it migrates as authentic MeS-Rib. Reverse enzyme reaction The radioactive product from the forward reaction was recovered from TLC sheets (Rf, 0.05) and used along with Ade as substrate to demonstrate the reverse enzymatic reaction MeS-Rib-l-P

total loss of activity during the heat step. However, if high-speed centrifugation was limited to 2 hr, the subsequent stability of the enzyme to heat for short periods of time was similar to results reported by Cacciapuoti et al. (1978) and Toohey (1978). The enzyme activity was found to elute from the DEAEcellulose column at 0.21 M KC1 and the increase in specific activity upon ultrafiltration may be due in part to the loss of low molecular weight inhibitor(s). It was stable during storage at - 85°C in 50% glycerol (v/v) and the amount of product formed by the enzyme at this stage of purification was dependent upon enzyme concentration and time. Eflect of substrate concentration on enzyme. A double-reciprocal plot of initial velocities vs phosphate ion concentrations gave an approx. apparent K, of 6 mM (Fig. la). The other substrate, MeSAdo, gave a nonlinear graph from which two apparent K,‘s were derived: one with an approx. value of 0.005 mM and the other of 0.06 mM (Fig. I b). Inhibitors. Inhibition studies were performed as a test of the specificity of the enzymes. The concentration of each compound tested was equal to that of MeSAdo in the assay. The addition of Ado, AdoMet, AdoHcy, the e-derivative of MeSAdo, ATP or putrescine revealed no measurable inhibition. In addi-

enzyme+ Ade + MeS-Rib-l-P.

+ Ade

enzymeb MeSAdo

+ P,

Table 2. R, values from silica gel TLC of radioactive products of MeSAdo nucleoside phosphorylase reaction and related compounds Material Reaction mixture Reaction mixture Reaction mixture Rechromatography Rib-l-P Rib MeSAdo MeS-Rib

chromatographed

R,

I* 2 3 of reaction

0.70t 0.05t; 0.70t 0.05t 0.82t 0.50 0.60 0.70 0.82

mixture

3$

* Standard enzymic reaction mixtures were prepared (see Methods) with [‘4C]CH,-MeSAdo (198 cpm/nmol) as the labeled substrate and allowed to react with the MeSAdo nucleoside phosphorylase for zero min (reaction mixture 1). until approx. half of the substrate was consumed (reaction mixture 2). or until all the substrate was consumed (reaction mixture 3). t R, values determined by radioactivity. $ The material with an R, value of 0.05 (reaction mixture 3) was recovered from the TLC sheet, treated with alkaline phosphatase (data not shown) and rechromatographed on silica gel TLC.

904

LEE SHUGART,MANUELTANCERand JENNIFERMOORE

The reaction mixture in a final volume of 50 ~1 contained 1.5pmols of Hepes buffer (pH 7.5), 25 nmols of Ade, and 40”/, of the radioactive product recovered from TLC. The assay was initiated with 5 ~1 of enzyme (sp. act. 215 nmols Ade/30 min/mg proteins), allowed to proceed for 1 hr at 3o”C, and terminated with 10~1 of 4 M formic acid. A SOA aliquot was injected onto an Aminex A6 HPLC column. The U.V. absorption of the eluate at 254 nm was recorded and fractions were collected and counted for radioactivity. Based on the retention time and spectrophotometric characteristics of authentic MeSAdo, the data from the A254nm profile showed that 0.73 nmols of this material were present. The data for radioactivity of the same material gave 0.71 nmols of [14C, CHJMeSAdo. There was no MeSAdo present in the control (i.e. 4 M formic acid added at zero reaction time) or in reaction mixtures containing MeS-Rib in place of the radioactive product.

sion (Toohey, 1977, 1978) will be studied in flies at various stages of development. Acknowledgement-The authors wish to acknowledge the technical expertise of Barbara Chastain, who assisted in these experiments.

REFERENCES CACCIAPUOTIG., OLIVA A. & ZAPPIA V. (1978) Studies on phosphate-activated S-methylthioadenosine nucleosidase from human placenta. Int. .I. Biochem. 9, 31-41. DUERRE J. (1962) A hydrolytic nucleosidase acting on S-adenosyl homocysteine and on S-methylthioadenosine. J. biol. Chem. 237, 3737-3741. FERROA. J., BARRETTA. & SHAPIROS. K. (1976) Kinetic properties and the effect of substrate analogues on S-methyl thioadenosine nucleosidase from E. co/i. Biochim. biophys. Acta 438, 487-494.

LIAU M. C., LIN G. W. & HURLBERTR. B. (1977) Patrol purification and characterization of tumor and liver S-adenosyl methionine synthetase. Cancer Rex 37,

DISCUSSION

In D. melanogaster an enzyme activity can be isolated that cleaves MeSAdo and produces an equivalent amount of Ade. It has an absolute requirement for phosphate ion. When [14C, CHJMeSAdo is the substrate, a radioactive product can be isolated by silica gel TLC flable 2) which, upon treatment with alkaline phosphatase, is readily converted to Me!% Rib. The enzyme catalyzes the synthesis of MeSAdo from Ade and the radioactive product but not from Ade and MeS-Rib. These data suggest that the enzyme catalyzes the reversible reaction ’ MeS-Rib-l-P MeSAdo + Pi - enzyme

+ Ade;

427435.

LOMBARDINI J. B., CHOWJ. C. & TALALAYP. (1973) Regulatory properties of adenosine triphosphate-L-methionine S-adenosyl transferase of rat liver. Biochem. J. 135, 43-57.

LOWRY0. H., ROSENBROUGH N. J., FARRA. L. & RANDALL R. J. (1951) Protein measurements with Folin phenol reagent. J. biol. Chem. 193, 265-275. F’EGG A. E. & WILLIAMS-ASHMAN H. G. (1969) Phosphatestimulated breakdown of S-methylthioadenosine by rat ventral prostate. Biochem. J. 115, 241-247. RICHARDSH. H., CHIANGP. K. & CANTONIG. L. (1978) Adenosyl homocysteine hydrolase. Crystallization of the purified enzyme and its properties. J. biol. Chem. 253, 4475-4480. SAINI

this activity is similar in many respects to the enzyme isolated from rat ventral prostate (Pegg & WilliamsAshman, 1969) and also demonstrated in malignant murine hematopoietic cells (Toohey, 1977, 1978). It differs from that reported by Cacciapuoti er a[. (1978) in human placental tissue by its absolute requirement for phosphate ion. The deviation from linearity of the kinetic data in the double-reciprocal plot with MeSAdo as the variable substrate (Fig. lb) is suggestive of two enzyme activities or alternatively of two interconvertible forms of the same enzyme. It should be noted that similar observations have been reported for the regulatory enzyme AdoMet synthetase from a variety of eucaryotic tissues (Lombardini et al., 1973; Liau et al., 1977). Further, the inability of the enzyme to utilize Ado, AdoHcy, AdoMet and the e-derivative of MeSAdo as substrates is indicative of its strict specificity. This is especially intriguing since the same compounds do not act as inhibitors even though they possess similar structural characteristics. Additional purification of the enzyme and more detailed analyses of kinetic data obtained in the presence of products and inhibitors should clarify these observations. Finally, the use of a genetically well-defined and biochemically understood organism such as D. melanogaster should allow us to assess the metabolic fate of MeS-Rib-l-P. In particular, the role of this compound in alkylthio group formation and cell divi-

A. S. (1966) Some technical improvements in the paper chromatography of sugars. A method of sample desalting and a sensitive staining reagent. J. Chromat. 24, 484486.

SCHLENKF. & EHNINGERD. J. (1964) Observations on the metabolism of 5’-methylthioadenosine. Archs Biothem. Biophys. 106, 95-100. SCHROEDERH.

R., BARNESC. T., BOHINSKIR. C. & MALLETTEM. S. (1973) Biological production.of 5-methylthioribose. Can. J. Microbial. 19, 1347-1354. SECXESTJ. A., BARRIOJ. R., LEONARDN. J. & WEBERG. (1972) Fluorescent modification of adenosine-containing coenzymes. Biological activities and spectroscopic properties. Biochemistry 11, 3499-3506. SHAPIROS. K. & MAT&R A. N. (1978) The enzymatic decomposition of S-adenoyl methionine. J. biol. Chem. 233, 631433. SHUGARTL. R. (1978) Kinetic studies of E. co/i tRNA(urac&5-)-methvltransferase. Biochemistry 17, 1068-1072. Toot& J. (19?7) Methylthio group cleavage from methylthioadenosine. Description of an enzyme and its relationship to the methylthio group requirement of certain cells in culture. Biochem. biophys. Res. Commun. 78, 1273-1280.

TOOHEYJ. (1978) Methylthio adenosine nucleoside phosphorylase deficiency in methylthio-dependent cancer cells. Biochem. biophys. Res. Commun. 83, 27-35. V~GELSG. D. & VAN DER DRIFT C. (1976) Degradation of purines and pyrimidines by microorganisms. Bact. Rev. 40, 403468. ZAPPIAV.. ZYDEK-CWICKC. R. & SCHLENKF. (1969) The specificity of S-adenosylmethionine derivatives in methyl transfer reactions. J. biol. Chem. 244, 449%4509.