264

Biochimica et Biophysica Acta, 496 (1977) 264--271 © Elsevier/North-Holland Biomedical Press

BBA 28154

THE T R A N S S U L F U R A T I O N PATHWAY IN TETRAHYMENA PYRIFORMIS

AN Q. DANG and DAVID E. COOK

Department of Biochemistry, University of Nebraska Medical Center, 42nd and Dewey Avenue, Omaha, Nebr. 69105 (U.S.A.) (Received July 26th, 1976)

Summary Four enzymes necessary for the metabolism of methionine by the transsulfuration pathway, methionine adenosyltransferase (EC 2.5.1.6), adenosylhomocysteinase (EC 3.3.1.1), cystathionine ~-synthase (EC 4.2.1.22) and cystathionine 7-1yase (EC 4.4.1.1) were identified in Tetrahymean pyriformis. The ability of these cells to transfer 3sS from [3sS] methionine to form [3sS]cysteine was also observed and taken as direct evidence for the functional existence of this pathway in Tetrahymena. An intermediate in the pathway and an active methyl donor, S-adenosylmethionine, was qualitatively identified in Tetrahymena and its concentration was found to be greater in late stationary phase cells than in early stationary phase cells.

Introduction The metabolism of Tetrahymena pyriformis is similar to higher animals in many respects [1--4] and, therefore, has often been used as a model for biochemical and nutritional studies. Kidder and Dewey [5] first established that methionine is an essential amino acid for the growth of Tetrahymena as it is for rats and man [6]. This requirement for methionine by Tetrahymena can be spared by L-cysteine [7], L-cystine, DL-homocysteine, D-methionine [8] or L-cystathionine [9]. These observations suggest that methionine is a precursor of cysteine in Tetrahymena by way of the transsulfuration pathway. In the present communication direct evidence is presented for the existence of the transsulfuration pathway and its associated enzymes in Tetrahymena. The methyl donor S-adenosylmethionine, an intermediate of the pathway, was qualitatively identified and its concentration in Tetrahymena was found to vary with the growth phase of the cells.

265 Methods

Growth and harvesting of T. pyriformis. T. pyriformis, strain E-1 [10], were grown by established methods [1]. Cell numbers were determined with a Spencer Bright-line hemacytometer after fixation as previously described [1]. For in vivo metabolism studies, cells were grown in 2% proteose peptone (type I medium) [11]. For the isolation of S-adenosylmethionine and enzyme assays, cells were grown in the glucose-containing medium described by Warnock and Van Eys [12] except that Tween 80 was omitted (type II medium). Cells in the appropriate growth phase (Fig. 1) were harvested by centrifugation at 10 000 × g with a Sorvall KSB continuous flow system. This and all subsequent harvesting procedures were conducted at 0--4°C. The harvested cells were washed twice in 0.9% NaC1 [11,13], suspended at a ratio of 1 g wet wt. cells per 2 ml solution and disrupted by sonication at 100 W for 3 min with a Heat Systems (Plainfield, N.J.) ultrasonic cell disruptor, Model W185D. Microscopic examination indicated that essentially all cells were broken by this technique. For isolation of S-adenosylmethionine, cells were sonicated in 1.5 M perchloric acid. For enzyme studies, cells were sonicated in the following buffers: 100 mM phosphate buffer (pH 8.4) for methionine adenosyltransferase (EC 2.5.1.6) and adenosylhomocysteinase (EC 3.3.1.1), 50 mM phosphate buffer (pH 7.4) for cystathionine fl-synthase (EC 4.2.1.22) and 3 mM phosphate buffer (pH 6.9) for cystathionine 7-1yase (EC 4.4.1.1). The sonicated cell preparations are referred to as the "crude homogenates". Centrifugation of these preparations at 10 000 X g for 15 min gave the "10 000 X g supernatants" and centrifugation of these supernatant fractions at 100 000 X g for 1 h gave the "100 000 X g supernatants". Protein was determined by the method of Lowry et al. [14].

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Fig. 1. G r o w t h c u r v e o f Tetrahymena in 8 l o f t y p e II m e d i u m c o n t a i n i n g 0.1 m l a n t i f o a m B e m u l s i o n ( S i g m a C h e m i c a l Co.) p e r 1. T h e a r r o w s i n d i c a t e w h e n t h e c e l l s w e r e h a r v e s t e d . T h e p o i n t s s h o w n are t h e average: the vertical bars r e p r e s e n t +- S.D. (n = 3 - - 6 ) .

266

Isolation and identification of S-adenosylmethionine. S-Adenosylmethionine was isolated and quantitatively determined by the ion-exchange chromatography method of Shapiro and Ehninger [15]. S-Adenosylmethionine obtained by this procedure in 6 M HC1 was further purified by the phosphotungstic acid precipitation method of Schlenk et al. [16]. Paper chromatography and paper electrophoresis were done on Whatman No. 1 paper. Paper electrophoresis was run at 15--20 mA for 6 h in 5 mM ammonium acetate buffer (pH 7.3). The purified c o m p o u n d isolated from Tetrahymena and S-adenosylmethionine were located on the paper as coincident ultraviolet absorbing and ninhydrin positive spots. Enzyme assays. All enzyme assays were conducted at 37 ° C. The method for assaying methionine adenosyltransferase was that of Mudd et al. [17], except that the reaction mixture was buffered at pH 8.4 and contained 0.54 pCi L[CH3-14C] methionine, plus 0.1 ml of enzyme extract. The S-adenosyl-L-[14C]methionine produced was determined by the method of Lombardini et al. [18] in the molecular weight experiments. The procedure of Holcomb and Shapiro [19] was used for the enzyme assays. Adenosylhomocysteinase was assayed by determining the rate of formation of the free sulfhydryl group [20] formed from S-adenosylhomocysteine. The reaction mixture contained 4.5 pmol Sadenosylhomocysteine, 0.3 mmol phosphate buffer (pH 7.4) [21], and 0.5 ml enzyme extract in a total volume of 2 ml. The reaction mixture was incubated for 1 h under a nitrogen atmosphere. Cystathionine ~-synthase was assayed by the method of Kashiwamata et al. [22], and cystathionine -y-lyase by the method of Gaull et al. [23]. Other methods. Scintillation counting was done on a Unilux II scintillation counter (Nuclear Chicago) and the channels ratio method was used to correct for quenching. To determine the molecular weight of methionine adenosyltransferase, the 100 000 × g fraction was chromatographed on a previously calibrated 2.5 × 36 cm Sephadex G-100 column. The column was eluted with 20 mM phosphate buffer (pH 7.2) and the molecular weight of the enzyme protein was estimated by the method of Andrew [24]. Materials. L-Methionine, S-adenosylmethionine, S-adenosylhomocysteine, Lcysteine (HCl-hydrate), DL-homocysteine thiolactone (HC1), L-cystathionine, ninhydrine and N-ethyl maleimide were obtained from Sigma Chemical Company, St. Louis, Mo.; L-[CH3-14C] methionine from Amersham/Searle Corporation, Arlington Heights, Ill., and New England Nuclear, Boston, Mass.; and L[3-~S] methionine from Amersham/Searle. All other compounds were of highest purity commercially available. Results

S-A denosylmethionine It has been demonstrated that Tetrahymena incorporate radioactivity from L-[~4C]methionine into the choline portion of phosphatidylcholine and that exogenous S-adenosyl-L-[14C]methionine can serve as a methyl donor for lecithin synthesis in isolated microsomal preparations from Tetrahymena [25]. These observations indicate the presence of S-adenosylmethionine-dependent transferase activity in Tetrahymena and suggest that S-adenosylmethionine is a

267 TABLE I C O N C E N T R A T I O N O F S - A D E N O S Y L M E T H I O N I N E AS A F U N C T I O N O F G R O W T H P H A S E IN T E T R A H Y M E N A COMPARED TO THE C O N C E N T R A T I O N IN R A T L I V E R T h e c o n c e n t r a t i o n in T e t r a h y m e n a w a s d e t e r m i n e d as d e s c r i b e d in M e t h o d s . T h e c o n c e n t r a t i o n in r a t liver ( m a l e S p r a g u e - D a w l e y r a t s , 2 5 0 - - 3 0 0 g) w a s d e t e r m i n e d b y h o m o g e n i z i n g ( P o t t e r - E l v e h j e m h o m o g e n i z e r ) 1 0 ~ 1 3 g o f r a t Hver in 2 0 - - 2 6 m l 1.5 M p e r c h l o r i c acid. S u b s e q u e n t p r o c e d u r e s w e r e the s a m e as d e s c r i b e d in M e t h o d s f o r Tetrahymena. T h e results are e x p r e s s e d m e a n ± S.D. ( n u m b e r o f e x p e r i m e n t s ) Tetrahymena

R a t liver

G r o w t h phase

Concentration nmol/cclls wet wt.)

Early stationary Late stationary

57 ± 11 (3) 94 +- 14 (3)

P ~ 0.05

Condition

Concentration ( n m o l / g liver, w e t w t . )

Fed

122 ± 25 (3) *

* This c o n c e n t r a t i o n is similar to the 1 0 0 - - 1 5 0 n m o l / g liver, w e t w e i g h t , p r e v i o u s l y r e p o r t e d f o r r a t liver [ 2 6 ] .

constituent of Tetrahymena. In the present study, a compound was isolated from Tetrahymena that comigrated as a single spot with S-adenosylmethionine in two different solvent systems. The R~ values were 0.15 for ascending chromatography in n-butanol/acetic acid/water (60 : 15 : 25, v/v) and 0.30 for descending chromatography in ethanol/water/acetic acid (65 : 34 : 1, v/v). Both compounds also migrated 4.4 cm toward the cathode in paper electrophoresis. These results confirm the presence of S-adenosylmethionine in Tetrahymena. The results in Table I show that the concentration of S-adenosylmethionine in Tetrahymena is a function of the growth phase of the organism. It is interesting to note that the concentration of this methyl donor is higher in the late stationary phase than in the early stationary phase and that this latter concentration is similar to that found in normal rat liver.

Enzyme activities The activity of enzymes of the transsulfuration pathway not previously shown to be present in Tetrahymena are given in Table II. The activities for T A B L E II A C T I V I T I E S OF T R A N S S U L F U R A T I O N P A T H W A Y ENZYMES FROM L A T E S T A T I O N A R Y PHASE TETRAHYMENA T h e m e t h o d s of isolation a n d assay are d e s c r i b e d in M e t h o d s . Fraction

Methionine adenosyltransferase (Spee. act. * X 104 )

Adenosylhomocysteinase (Spec. act. * X 10)

Crude homogenate 10 0 0 0 X g 100 000 Xg

0 . 9 5 -+ 0.17 1 . 5 4 -+ 0 . 4 5 4.01 ± 1.05

1.38 -+ 0 . 4 3 2 . 2 4 ± 0.11 2.20 ± 0.15

C y s t a t h i o n i n e ** ~-synthase (Spec. act. * X 10 -2 )

Cystathionine ~-lyase (Spec. act. *)

2.67

4.24 10.33 17.10

* Specific a c t i v i t y , e x p r e s s e d as n m o l o f p r o d u c t f o r m e d / m i n p e r m g p r o t e i n . ** C y s t a t h i o n i n e fl-synthase a c t i v i t y c o u l d n o t be d e t e r m i n e d in e i t h e r c r u d e h o m o g e n a t e s o r 10 0 0 0 X g f r a c t i o n s b e c a u s e s u b s t a n c e s p r e s e n t in t h e s e f r a c t i o n s i n t e r f e r e d w i t h the d e t e r m i n a t i o n o f c y s t a t h i o n i n e . Also, 0 . 5 r a m CuSO 4 a d d e d to t h e s e c y s t a t h i o n i n e fi-synthase assay m i x t u r e s did n o t i n h i b i t c y s t a t h i o n i n e T-lyase a c t i v i t y as d e s c r i b e d b y K a s h i w a m a t a [ 2 2 ] f o r t h e e n z y m e i s o l a t e d f r o m rats.

268

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Fig. 2. E n z y m e a c t i v i t y as a f u n c t i o n o f t e m p e r a t u r e a n d p H . T h e c u r v e s in p a n e l A s h o w t h e a c t i v i t i e s o f t h e i n d i c a t e d e n z y m e s as a f u n c t i o n o f p H . C y s t a t h i o n i n e ~{-lyase a c t i v i t y (e - - e ) w a s m e a s u r e d as d e s c r i b e d in M e t h o d s e x c e p t t h a t t h e i n d i c a t e d p H w a s o b t a i n e d b y a d j u s t i n g t h e p H o f t h e 1 0 0 m M T r i s b u f f e r u s e d . M e t h i o n i n e a d e n o s y l t r a n s f e r a s e a c t i v i t y ( o - . - . - o ) w a s m e a s u r e d as d e s c r i b e d in M e t h o d s e x c e p t t h a t the i n d i c a t e d p i t w a s o b t a i n e d b y a d j u s t i n g t h e p H o f t h e 2 0 0 m M T r i s b u f f e r u s e d . T h e c u r v e s in p a n e l B s h o w t h e a c t i v i t i e s o f t h e s a m e e n z y m e s as a f u n c t i o n of t e m p e r a t u r e . T h e a c t i v i t i e s w e r e m e a s u r e d as d e s c r i b e d in M e t h o d s e x c e p t t h e r e a t i o n w a s c o n d u c t e d at t h e t e m p e r a t u r e i n d i c a t e d . T h e p o i n t s s h o w n are t h e a v e r a g e ; t h e v e r t i c a l b a r s r e p r e s e n t ~- S.D. (n = 3).

these enzymes were found to be highest in the 100 000 × g supernatant. These results indicate that the isolated enzymes are non-particulate in nature.

Some characteristics of the Tetrahymena enzymes Glutathionine was found not to be necessary for the activity of the Tetrahymena-derived methionine adenosyltransferase as has been reported for the human liver enzyme [17]. Based on gel filtration techniques, the molecular weight of this enzyme isolated from Tetrahymena was found to be approx. 74 000. This is about double the 40 000--43 000 molecular weight, determined by gel filtration, for the enzyme isolated from yeast [27], but only about half the 157 000 molecular weight, estimated by sedimentation techniques, for the yeast enzyme [28]. Both methionine adenosyltransferase and cystathionine 7lyase isolated from Tetrahymena were found to have a pH optimum of about 8.4 (Fig. 2). This is similar to the pH optimum found for cystathionine 7-1yase isolated from rat liver and human brain [17] and for methionine adenosyltransferase isolated from human liver [17]. The temperature optimum of Tetrahymena cystathionine 7-1yase was found to be 47°C whereas that for methionine adenosyltransferase was observed to be at least 56°C (Fig. 2).

Transfer of 3sS from [3sS] methionine to cysteine in vivo To obtain direct evidence for the transsulfuration pathway in Tetrahymena, 20.4 #Ci of L-[ 3 sS] methionine in 0.2 mmol of carrier methionine were added

269

to cells grown to the late stationary phase in type I medium. 2 h later the cells were harvested (83 • 106 cells) and analyzed for [3sS] cysteine content as outlined in Scheme 1. Under these conditions, 49 dpm and 12 dpm of [3sS] cysteine Scheme 1. Scheme for the isolation and handling of the perchloric acid-soluble and perchloric acidinsoluble (protein) fractions obtained from Tetrahymena grown in type I medium in the presence of [ 3 SS ] methionine.

HARVESTED CELLS s o n i c a t e d in 0.9% NaC1

I

suspended 1.5 M p e r c h l o r i c acid a n d e x t r a c t e d at r o o m t e m p e r a t u r e for lh 15 0 0 0 × g for 15 rain

SUPERN~ATANT neutralized w i t h KOH, 1 h at 0°C

PRECIPITATE suspended in H 2 0 and dialyzed against 2.7 • 103 volumes of H 2 0 for 5 days

15 0 0 0 × g 15 m i n

15 0 0 0 × g 15 rain

SUPERNATANT lyophilized and dissoved in citrate b u f f e r for a m i n o acid acid analysis [ 2 9 , 3 0 ]

PRECIPITATE

PERCHLORIC ACID-SOLUBLE FRACTION

S U P E R N ~ P I T A T E hydrolyzed for 20 h in 6 M HC1, dried a n d dissolved in citrate b u f f e r for a n i m o acid analysis [29,30] PERCHLORIC ACID-INSOLUBLE (PROTEIN) FRACTION

per 104 cells were incorporated into the perchloric acid-soluble fraction and the protein fraction, respectively. These results indicate that Tetrahymena can transfer 3sS from methionine to cysteine. The amount of radioactivity found in the cellular cysteine plus cystine pool undoubtedly reflects the slow rate of methionine uptake by Tetrahymena [31]. It is also important to remember that the observed values indicate the concentration of 35S that existed in the cysteine plus cystine pool at the time of harvesting and do not necessarily give any indication of the flux of 3sS from methionine through the transsulfuration pathway.

270

Discussion In Tetrahymena the last two enzymes of the transsulfuration pathway, cystathionine synthase and cystathionine 7-1yase were found to have higher activities than the two preceding enzymes studied (Table II). This might reasonably be expected since in yeast the equilibrium of the reaction catalyzed by adenosylhomocysteinase strongly favors the synthesis of S-adenosylhomocysteine [21,32], a potent inhibitor of several transmethylation reactions [33--36]. Such an inhibition would result in the buildup of S-adenosylmethionine which in turn could cause inhibition of methionine adenosyltransferase [27]. At present, however, it is difficult to interpret how the higher concentration of Sadenosylmethionine in late stationary phase versus early stationary phase cells (Table I) is related to the control of metabolic processes in Tetrahymena, such as the synthesis of glycogen from methionine [3] and transmethylation [25], since S-adenosylmethionine is, in other organisms, used for the synthesis of compounds like epinephrine and spermidine [37], both of which are found in Tetrahymena [38,39]. Acknowledgement We thank Dr. Theodore Mahowald for the use of the amino acid analyzer and Dr. Robert Ramaley for assistance in the molecular weight determinations. References 1 2 3 4 5 6 7 S 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Cook, D.E., Rangaraj, N.I., Best, N. and Wilken, D.R. (1968) Arch. Biochem. Biophys. 127, 72--77 Raugi, J.G., Liang, T. and Blum, L.I. (1975) J. Biol. Chem. 2 5 9 , 4 4 5 - - 4 6 0 Elson, C., Shrago, E., Sondheimer, E. and Yatvin, M. (1973) Biochim. Biophys. Acta 297, 125--134 Connett, R.J. and Blum, J.J. (1971) Biochemistry 10, 3299--3309 Kidder, G.W. and Dewey, V.C. (1945) Arch. Biochem. 6 , 4 2 5 - - 4 3 2 Rose, W.C. (1949) Fed. Proc. Am. Soc. Exp. Biol. 8 , 5 4 6 - - 5 5 2 Hogg, J.F. and Elliot, A.M. (1951) J. Biol. Chem. 1 9 2 , 1 3 1 - - 1 3 9 Genghof, S.D. (1949) Arch. Biochem. 23, 85--89 Genghof, S.D. (1951) Arch. Biochem. Biophys. 3 4 , 1 1 2 - - 1 2 0 Borden, D., Whitt, G.S.and Nanney, D.L. (1973) J. Protozool. 20, 693--700 Shrago, E., Brech, W. and T e m p l e t o n (1967) J. Biol. Chem. 242, 4060--4066 Warnock, L.G. and Van Eys, S. (1962) J. Cell. Comp. Physiol. 60, 53--60 Elson, C. and Richard, K. (1972) J. Nutr. 102, 1209--1215 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 1 9 3 , 2 6 5 - - 2 7 5 Shapiro, S.K. and Ehninger, J.D. (1966) Anal. Biochem. 15, 323--333 Schlenk, F. and DePalma, R.E. (1957) J. Biol. Chem. 229, 1051--1057 Mudd, H.S., Finkelstein, J.D., Irreverre, F. and Laster, L. (1965) J. Biol. Chem. 240, 4 3 8 2 - - 4 3 9 2 Lombardini, B.J., Coulter, A.W. and Talalay, P. (1970) Mol. Pharmacol. 6 , 4 8 1 - - 4 9 9 Holcomb, E.R. and Shapiro, S.K. (1975) J. Bacte~iol. 1 2 1 , 2 6 7 - - 2 7 1 Miller, C.H. and Deurre, J.A. (1968) J. Biol. Chem. 243, 92--97 Finkelstein, J.D. and Harris, B. (1973) Arch. Biochem. Biophys. 1 5 9 , 1 6 0 - - 1 6 5 Kashiwamata, S. and Greenberg, D.M. (1970) Biochim. Biophys. Acta 2 1 2 , 4 8 8 - - 5 0 0 Gaull, G.E., Rassin, D.K. and Sturman, J.A. (1969) Neuropadiatrie 1 , 1 9 9 - - 2 2 6 Andrew, P. (1965) Biochem. J. 9 6 , 5 9 5 - - 6 0 6 Smith, J.D. and Law, J.H. (1970) Biochim. Biophys. Acta 202, 141--152 Lombardini, B.J. and Talalay, P. (1971) Adv. Enz. Regul. 9 , 3 4 9 - - 3 8 4 Chou, T.C. and Talalay, P. (1972) Biochemistry 11, 1065--1073 Mudd, S.H. (1963) J. Biol. Chem. 238, 21-56--2163 Moore, S., Spackman, D.H. and Stein, W.H. (1958) Anal. Chem. 30, 1185--1206 Mahowald, T.A., Noltmann, E.A. and Kuby, S.A. (1962) J. Biol. Chem. 237, 1138--1145

271 31 32 33 34 35 36 37

Hoffmann, E.K. and Rasmussen, L. (1972) Biochim. Biophys. Acta 266, 206--216 de la Haba~ G. and Cantoni, G.L. (1959) J. Biol. Chem. 234~ 603--608 Zappia, V., Zydek-Cwick, C.R. and Schlenk, F. (1969) J. Biol. Chem. 244~ 4499--4509 Akamatsu, Y. and Law, J.H. (1970) J. Biol. Chem. 245, 709--713 Deguchi, T. and Barchas, J. (1971) J. Biol. Chem. 246, 3175--3181 Coward, J.K., D h r s o - S c o t t , M. and Sweet, W.D. (1972) Biochem. Pharm. 21, 1200--1203 Meister, A. (1965) in Biochemistry of the Amino Acids, 2nd Edn. Vol. II, p. 757--818, Academic Press, New Y o r k 38 Janakidevi, K., Dewey~ V.C. and Kidder, G.W. (1966) J. Biol. Chem. 241, 2576--2578 39 Arlock, P., Heby, O. and Holm, B. (1969) J. Protozool. 16, Suppl. 33