225

Biochimica et Bi~physica Acta, 1077 (1991) 225-232 © 1991 Elsevier Science Publishers B.V. 0167-4838/91/$03.50 ADONIS 0167493891001517 BBAPRO 33884

Regulation of S-Adenosylmethionine synthetase activity in cultured human lymphocytes J a m e s D e L a R o s a * , M a l a k K o t b * a n d N i c h o l a s M. K r e d i c h Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, NC (U.S.A.)

(Received22 October1990) Key words: S-Adenosyhnethionine synthetase: Eazyme activation: Product inhibition: Human lymphocyte; Phosphate: Pyrophosphate

S-Adenosylmethionine (AdoMet), inorganic pyroplmsplmte (PPi) and inorganic phosphate (Pi) are potent product inhibitors of AdoMet synthetase and have been postulated to piny a role in increasing AdoMet levels and turnover in peripheral blood mononudeer cells (PBM) after sfimdation with phytehemagglatinin (PHA). Measurements of these metabofites in PHA-stimulated PBM showed the expected 2- to 3-fold increases in AdoMet after 8 II, and smaller ineneases in PPi alid Pi- Since the kinetic model requires substantial deereases in PPi ~ Pl in respollse to PXA, product inlu'biticm cannot explain the obsmved changes in AdoMet metabolism in this system. A 2.~fold increase in AdoMet syathetase catalytic activity was found ~ crmle extracts of PBM within 8 h of P H A ~ and wobably accounts for increased celhdar levels and utilization of AdoMet. Immtmottem~ai analyses with a m o n o c l o ~ antibody specific for the a / a ' submmits of human lymphocyte AdoMet synthetase showed that these increases in catalytic activity were not assoeinted wlih increases in immumrenctive lWeteia. The ratio of catalytic activity to kammtmeaetivity in stinndated cells was 4-foM higher than in mmlnndated controls and almost idemicai to tlud found in exlrncts frmu the human B-lymphocyte line WI-L2. Unstimutated PBM appear to eontain substantial muoums of AdoMet syntbetase a/a" sulbemitwith reduced or absent ~tAlytic activity, which can be activated by PHA-slbnulatioa. introduetinn S-Adenosylmethionine (AdoMet) is synthesized from ATP and L-methionine by the enzyme AdoMet synthetase (ATP: L-metbionine S-adenosyltransferase, EC 2.5.1.6; Scheme I) [1], ATP + L-methionine ~ AdoMet + Phosphate + Pyrophosphate Scheme I

* Present addresses: Veterans Administration Medical Center, Memphis. "IN 38104. U.S.A. Abbreviations: AdoMet, S-adenosyI-L-methionine; APS, adenosine 5'-phosphosulfate: BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbant assay; HPLC, h i g h * p e r f ~ liquid chromatography; Hepes, N-2-hydroxyethylpiperazine-N'-2-erhanesulfonic a~l; PAPS, 3 ' - ~ n e 5'-phosphosulfate; PBM, peripheral blood mononuclear cells; PBS, phosphate-buffered saline: Pl-LA_ phytohemagglutinin; PPt, inorganic pyrophosphate; PRPPo 5-phosphonbosyl-I -pyrophosphate. Cor'reslxmdence: N.M. Kredich, p.o. Box 3100, Duke University Medical center, Durham, NC 27710, U.S.A.

and serves as the methyl donor for virtually all biologic transmethylation reactions [2,3]. in studies on the regulation of AdoMet metabolism in human lymphocytes, we found that the total utilization rate and cellular concentration of this important metabolite begin to increase in peripheral blood mononuclear cells (PBM) within 2 to 3 h after stimulation with phytohemagglutinin (PHA), and after 8 h reach levels that are 3- to 5-fold higher than in unstimulated cells [4]. These rapid increases in both the utilization and concentration of AdoMet present an interesting problem in regulation, since human lymphocyte AdoMet synthetase is inhibited by AdoMet at concentrations that are found in PBM [4-6]. Therefore, in the absence of other factors that might influence AdoMet synthetase activity, any increase in the steady-state concentration of AdoMet should be, accompanied by a decrease in the rate of synthesis. This apparent paradox might be explained by several different ns~hanisms, including changes in the levels of other metabolites that alter kinetic activity, activation of pre-existing enzyme, and synthesis of new enzyme. A detailed kinetic analysis of human lymphocyte AdoMet synthetase has shown significant product in-

226 hibition not only by AdoMet but also by inorganic phosphate (P,) and inorganic pyrophosphate (PPi) at concentrations that might be expected in cells [6]. Inhibition by these three products is markedly synergistic, particularly that between Pl and PP,, which have the effect of decreasing the apparent K i for AdoMet. These findings suggest a model for the early effects of PHAstimulation on PBM, in which a postulated lowering of high Pi and PP, levels would decrease sensitivity of the enzyme to inhibition by AdoMet and thereby increase both the rate of synthesis and the steady-state level of AdoMet [6]. In this paper we report that measurements of intracellular metabolites do not support this model of kinetic regulation by product inhibition, and that the early changes in AdoMet metabolism in PHA-stimulated PBM appear to be due to activation of pre-existing enzyme. Materials and Methods Materials Adenosine 5'-phosphosulfate (AlaS), 3'-phosphoadenosine 5'-phosphosulfate (PAPS), 5-aminosalicylate, the iodide salt of AdoMet, aprotinin, bovine serum albumin (BSA), yeast ATP sulfurylase, yeast inorganic pyrophosphatase and nuclease P1 were obtained from Sigma Chemicals. 1 U of activity for these enzymes is defined as the amount catalyzing product formation at a rate of 1/tmol/min, and refers to: formation of ATP from APS and PPi for ATP sulfurylase; generation of Pi from PPt for inorganic pyrophosphatase; and formation of P~ from 3'-AMP for nuclease PI. 5-Aminosalicylate was purified as described by Ellens and Gielkens [7], and AdoMet was purified by HPLC [8]. Tissue culture media and fetal bovine serum were purchased from Gibeo. PHA was from Wellcome Research Laboratories, Research Triangle Park, NC. [35S]Sulfuric acid (1200 Ci/mmol) was obtained from Du Pont-New England Nuc!ear. Cells and cell culture The WI-L2 lymphoblast cell line [9-11] was cultured in Eagle's minimal essential medium with Earle's salts, non-essential amino acids and 1070 fetal bovine serum. For the preparation of PBM, human venous blood from normal healthy adult donors was defibrinated under sterile conditions by gentle rocking in a glass bottle with glass beads, in some studies the blood was heparinized instead of defibrinated. In either case PBM were then purified by isopycnic centrifugation on a cushion of lymphocyte separation medium (Organon Teknika) as described by the manufacturer. The cells were washed twice in RPM! 1640 containing 170 fetal bovine serum, and resuspended at a concentration of 1.5-2.106 ceils/ml in the same medium containing 107o fetal bovine serum. After incubation at 37°C for 2-2.5 h,

one portion of the cell culture was treated with 1 ~tg/ml PHA; untreated cells served as a control. Cells were cultured in plastic flasks at 3"7°C in an atmosphere of air containing 570 CO 2, and were counted with a Coulter Counter. Where required cell counts were obtained before and after lysis of erythrocytes with the stromatolysing reagent Zap-Oglobin II from Coulter Electronics. Cellular concentrations of metabolites were calculated by using the cell volume estimates of 1.65 ml/10 9 cells for WI-L2 cells, and 0.42 ml/10 9 cells for PBM [41.

Cell extracts All procedures were performed at 4 ° C. For analyses of AdoMet, ATP and Pi, 11 ml of cell culture was centrifuged for 5 rain at 1200 x g, and the cell pellet was suspended in 1 ml of buffer consisting of 23 mM potassium N-2-hydroxyethylpiperazine-N "-2-ethanesulfonic acid (Hepes) (pH 7.4), 120 mM NaCl, 3.2 mM KCI, and 1 mg/ml BSA. The suspension was transferred to a microfuge tube, and after centrifugation for 1 min at 1000G x g, the cell pellet was extracted with 0.1 ml of 0.5 M perchloric acid for no more than 5 rain. Following removal of precipitated material by centrifugation, the superuatant was partially neutralized with 0.03 mi of 1 M KOH, adjusted to p H I with 1 M KHCO 3, and kept on ice for at least 5 rain. The potassium perchlorate precipitate was removed by centrifogation, and_ the supernatant was assayed for metabolites. For PPi analyses, cells from 2 ml of cell culture were collected by centrifugation for 1 rain at 10000 × g , washed twice with 2 ml portions of Hepes buffer containing 2 mM Na2EDTA, and re-collected by centrifugation. The final pellet was suspended in 0.1 ml of 50 mM Tris-HCl (pH 7.4), 2 mM NaEEDTA, heated in a boiling water bath for 0.5 rain (unless otherwise noted) and immediately cooled on ice. Precipitated material was removed by centrifugation for 1 min at 15000 × g, and the supernatant was assayed for PPiFor assays of AdoMet synthetase, cells from 10 ml of cell culture were collected by centrifugation, washed with 1.5 ml of Hepes buffer, and re-collected by centrifugation for 1 min at 10000 × g. The pellet was suspended in 0.07 ml of 50 mM potassium N-tris(hydroxymethyl)methyl-2-amino-ethanesulfonic acid (pH 7.4) containing 50 mM KCI, 15 mM MgCI 2. 0.3 mM Na2EDTA, 4 mM dithiothreitol, and 0.1 mg/ml BSA, and cells were disrupted by three freeze-thaw cycles in liquid nitrogen. Following removal of cell debris by centrifugation for 10 min at 15000 x g, the supernatant was frozen at - 70 ° C until assayed for enzyme activity and immuno-reactivity. The anti-proteolytic agents aprotinin and phenylmethylsuifonylfluoride were found to inhibit AdoMet synthetase activity slightly and were not included in the extract buffer.

227

Preparation of [35SLAPS A crude extract of sulfur-limited wild-tvpe Salmonella typhlmurium LT2 [12] was used as a source of ATPsulfurylase (ATP: sulfate adenylyltransferase, EC 2.7.7.4) and APS kinase (ATP: adenosine-5'-phosphosulfate 3'-phosphotransferase, EC 2.7.1.25), the two enzymes catalyzing synthesis of PAPS from ATP and sulfate. PAPS was synthesized in a 0.3 ml reaction mixture containing 50 mM Tris-HCl (pH 8.5), 20 mM MgCI 2, 10 mM ATP, 1 mCi [35S]sulfuric acid (1200 Ci/mmol), 0.6 U of inorganic pyrophosphatase, and 0.8 mg of protein from the S. typhimurium crude extract. The mixture was incubated at 30 ° C and monitored for charcoal-adsorbable radiolabel as described by Daley et al. [13]. The reaction was terminated after 30 rain by the addition of 0.05 ml of 2 M perchloric acid, and precipitated protein was removed by centrifugation. The supernatant was neutralized with 1 M KOH and after chilling on ice for 5 min, the potassium perchlorate precipitate was removed by centrifugation. Radiolabeled PAPS was purified by HPLC on a Whatman SAX anion-exchange column (0.46 × 25 cm) perfused with a mobile phase of 0.4 M NH4PO4, pH 2.9 containing 2~ acetonitrile at a flow rate of 1 ml/min for the first 15 rain and at 2 mi/min for the next 10 min. The eluate was monitored continuously for absorbanee at 254 nm and a splitstream portion was analyzed for radiolabel on a Flow One Beta radioactive flow detector from Radiomatic Instruments and Chemicals. Retention times for authentic compounds were: APS, 4.5 rain; PAPS, 8.5 rain; sulfate, 11 rain; and ATP, 17 rain. Fractions containing [35S]PAPS were pooled, and the PAPS was separated from the mobile phase by charcoal adsorption-desorption [13], lyophilized to dryness, and dissolved in 0.4 ml of 50 mM Tris-HC! (pH 8.0). Nuclease Pl was added to a final concentration of 10 U / m l to dephosphorylate [35S]PAPS to [35SLAPS, which was subsequently isolated by charcoal adsorption-dc~rption. Small portions were distributed into vials, lyophilized to dryness, and stored at - 7 0 ° C . The final [35S]APS product was estimated to be 99.5~ pure as judged by HPLC analysis.

Pyrophosphate assay PPi was assayed by a modification of the method of Daley e t a l . [13], which involves the determination of [35S]sulfate produced by the ATP sulfurylase catalyzed reaction of APS and PPi (Scheme I1). Though reversible, this reaction has an equilibrium constant of 109 in the 35S[APS]+ PPi 4-,ATP+35SlSulfateI Scheme!1 direction of APS pyrophosphorolysis [14]. The 0.2 ml assay mixture contained 50 mM Tris-HCl (pH 8.5), 7

mM MgCI,. 50 to 100 nM [3~S]APS diluted with unlabeled APS to give a total of approx. 1 - 104 dpm per assay tube, 10 to 60 nM PP,, and 0.04 U / m l of ATP sulfurylase. After incubation at 37 ° C for 6 min, 0.5 ml of a 20 mg/ml suspension of acid washed charcoal in 50 mM Tns (pH 8.5), 0.1 M Na2SO4 was added to stop the reaction and adsorb [35S]APS. The sample was vortexed for 5-10 s and centrifuged for 1.5 min at 15000 × g, and a 0.3 ml portion of supernatant was analyzed for [35S]sulfate by liquid scintillation spectrometry. Studies of the time course of reaction showed that the conversion of [3sS]APS to [3SS]suifate was 90~ complete after 1 rain of incubation and did not change appreciably between 5-15 min. At 100 nM [3sS]APS, the assay was linear with respect to PP, from 0 to 80 nM (reaction mixture concentration) with a blank value equivalent to approx. 20 nM.

Phosphate assays Pi was assayed by two different methods. In assay A, a ph~phomolybdate complex was reduced by ascorbate [15] in the presence of 5 vM CuSO4 [16]. In assay B, unreduced phosphomolybdate complex was measured directly by the method of Heinonen and Lahti [17] as modified by Kotb and Kredich [5].

ELISA for AdoMet synthetase Immunoreactive AdoMet synthetase was measured by an enzyme-linked immunosorbent assay (ELISA) with a monoclonal antibody to the a/a" subunit [18], which was purified by capryfic acid and ammonium sulfate precipitation [19]. Antibodies were diluted in phosphate-buffered saline (PBS; 0.14 M NaCl, 2.7 mM KCI, 8.1 mM Na2HPO4, 1.5 mM KH2PO4 (pH 7.2)) containing 1~ BSA and 0.005~ Tween-20. Cell extracts were thawed on ice, diluted in 0.05 M Na2CO3 (pH 9.4) and distributed in 0.1 ml portions into 96-well micra tiler plates (lmmulon-2 from Dynatech Labs), which were incubated at 37 ° C for 4 h. The wells were emptied by gentle shaking and then subjected to a cycle consisting of three washes with PBS containing 0.005~ Tween20 and one wash with deionized water. The plates were briefly air dried, and further non-specific adsorption was blocked by the addition of 0.25 ml of PBS containing 1~ BSA (but no Tween-20) and incubation at 37°C for 30 rain. After another wash cycle and air drying, 0.2 ml of a 0.2 mg/ml dilution of monoclonal antibody to AdoMet synthetase was added, and the plates were incubated at 37°C for 1 h. The plates were again washed, air dried and incubated at 37°C for 30 rain with 0.2 nd of a 1:1000 dilution of horseradish peroxidase-conjugated goat anti-mouse polyclonai antibody (Bio-Rad Labs). After another wash cycle and air drying, each well received 0.2 ml of a solution containing 10 mM Na2HPO4 (pH 6.0), 0.1 mM Na2EDTA, 6.5 mM 5-aminosalicylate and 0.005~$ H20 2. After 1 to 2 h

228 at 23°C, absorbance at 495 nm was determined with a microtiter plate reader. Other methods AdoMet and ATP were analyzed by HPLC [4,20]. AdoMet synthetase was assayed by a modification [5] of the method of Chou and Lombardini [21], and lactate dehydrogenase was assayed as described by Bergmeyer and Bernt [22]. Protein was measured by the method of Bradford [23]. Human platclets were prepared by mixing venous blood with 0.11 voi. of 0.13 M trisodium citrate (pH 7.4) and centrifuging for 15 min at 180 x g. Platelets in the supernatant were estimated with a Coulter Counter, collected by centrifugation for 15 rain at 400 × g, and extracted by the boiling method for PPi analyses.

TABLE I

Effects of A TP and sulfate on the assay for inorganic pyrophosphate Assays were carried out as described in Materials and Methods with 100 nM [35S]APS and 0.04 U of ATP sulforylase per ml. Complete conversion of [3sS]APS to [35SIsulfate would have given a value of about 3600 cpm. Additions

[ 3SS]sulfate ! 35S]sulfate after 6 rain after 20 min

cpm" (~) 30 nM 30riM 30nM 30nM

PP, PP, + 2 0 p M ATP PP, +20I~M A T P + 2 0 I ~ M sulfate PP, + 2 0 p M A T P + I 0 0 I ~ M sulfate

1059 1250 1282 1453

cpm°(To)

(100) b 1034 (100) b (118) 1253 (121) (121) 2008 (194) (137) 2338 (226)

Results

" All cpm have been corrected for blank values of 531 a n d 607 cpm obtained for the 6 rain and 20 min time points, respectively, in assays containing no additions. b The cpm obtained with 30 nM Pp. alone are arbitrarily set at 100~$ for the two time points.

Factors influencing the assay of pyrophosphate in cell extracts The pyrophosphate assay used in these studies is based on conversion of PPi and [ 3sS]APS to ATP and [35S]sulfate by ATP sulfurylase. Although the Kcq of this reversible reaction very much favors APS pyrophosphorolysis, the assay is still sensitive to ATP and sulfate, both of which are present in crude cell extracts. In theory, ATP should decrease estimates of PPi by decreasing net conversion of [35SLAPS to [3SS]sulfate, and unlabeled sulfate should increase estimates of PP, by exchanging with the radiolabel of the [3sSIAPS. From analyses of cell extracts, we calculated that the ATP concentration in a typical PP, assay mixture would be no more than 20/tM. The addition of this amount of ATP to an assay containing 30 nM PP, increased [35S]sulfate release by 1870 after a 6 min incubation and by 2170 after 20 min, indicating the presence of a PPi contaminant rather than any significant degradation of ATP during the assay (Table I). The failure of 20/~M ATP to decrease [35Slsulfate release is not surprising since the Keq of 109 for the pyrophosphorolysis reaction predicts that a 170 decrease in sulfate formation would require an ATP concentration of 0.7 M. At 20/tM ATP, the addition of 100 ~M unlabeled sulfate increased PPi estimates by 1970 over that with 20/~M ATP alone after a 6 min incubation and by 10570 after 20 rain (Table i). The time dependency of this effect and the finding of similar increases in [3SS]sulfate release were observed in blank reactions containing no added PPi (not shown), indicating that unlabeled sulfate was exchanging with [3sS]APS. Therefore, the sensitivity of the assay to sulfate is a funclion of enzyme concentration and time of incubation. Under our standard conditions of 0.04 U / m l of ATP sulfurylase per assay and a 6 rain incubation, and a mid-range PP~ concentration of 30 nM, the addition of 20 ttM sulfate and 20/AM ATP increased the

estimate for PPi by only 370 over that with 20 tiM ATP alone (Table I). This concentration of sulfate in our assay would require an intraceUular sulfate concentration of 1-2 raM. To our knowledge, lymphocyte sulfate levels have not been reported, but concentrations in human and mouse liver are 0.6 mM and 1.2 mM, respectively [24]. We believe that the concentrations of sulfate and ATP in our cell extracts are unlikely to cause appreciable errors in our estimates of PPi. Another possible source of error in the measurement of PPi is the release of PPi from labile compounds, such as 5-phosphoribosyl-l-pyrophosphate (PRPP), during the preparation and assay of cell extracts. Although perchloric acid extracts are suitable for ATP and AdoMet assays, PRPP is very acid labile [25]. Therefore, cell extracts for PPi analyses were prepared by heating a 0.1 ml cell suspension in boiling water for 0.5 rain [26]. When a 0.1 ml volume of 1.2 ttM PRPP in 50 mM Tris-HCI (pH 7.4) and 2 mM Na2EDTA was heated in this way, our PPi assay showed that PRPP was hydrolyzed at a rate of 670 per min between 1-2 min, but at less than 170 per min during the first minute, because of the lag time required to heat the contents of the tube from the starting temperature of approx. 4°C. PRPP stability was also determined in assay mixtures by measuring the appearance of [35S]sulfate as a function of incubation time. With free PPi alone, the release of [35S]sulfate is more than 9970 complete after a 5 rain incubation with ATP sulfurylase. With a mixture conraining 0.24 /~M PRPP, the continued release of [35S]sulfate between 5-15 min was equivalent to a hydrolysis rate for PRPP of 0.1670 per min (not shown) for a total of about 170 for a 6 min assay. From an extrapolation of this rate to zero time we estimated that our PRPP preparation contained a 4~$ contaminant of PP,. Taken together, these results indicate that hydroly-

229 sis of PRPP during the preparation and assay of extracts will cause us to over-estimate cellular PP, by an amount that is less than 2~ of the cellular PRPP concentration. WI-L2 cells were used as a convenient source of lymphocytes to determine the sensitivity and practical limitations of our PPi assay. With our standard conditions PP~ levels were diminished by less than 5~ in cell suspensions that were kept at 4 ° C for 15 rain prior to harvesting. Recovery of an internal PP~ standard equivalent to approx. 20 nmol/109 cells was 95c$ when added to a cell pellet prior to boiling. Cell extracts that had been prepared by boiling for 0.5 rain and for 15 min had PPi levels of 11 and 83 nmol/109 cells, respectively, in one experiment; and 19 and 137 nmol/109 cells in another. These differences are most likely due to hydrolysis of PRPP. From our estimated hydrolysis rate of 6~ per min during heating, these values would correspond to cellular PRPP concentrations of approx. 135 and 221 nmol/109, which are in the range of levels reported by Hershfield and Seegmiller [26]. if approx. 2~ of cellular PRPP were hydrolyzed during preparation and assay of extracts by our standard method, this amount of PRPP would have caused us to overestimate cellular PPi by approx. 30~ it~ WI-L2 cells Factors influencing the assay of phosphate in cell extracts Two different P~ assays were evaluated for hydrolysis of acid-labile compounds such as phosphocreatine, which would result in over-estimates of cellular PiUsing assay A in which phosphomolybdate is reduced by ascorhate at pH 4.0 [15], we found that 9¢~ of input phosphocreatine was hydrolyzed to Pi during our standard 7 rain reduction time. in assay B, the phosphomolybdate complex is measured directly by its absorbance at 335 nm in a solution of acetone and strong acid [17], and hydrolysis of acid-labile phosphate compounds is prevented by the rapid addition of citrate, which binds excess molybdate. By adding citrate within 12 s of

mixing the sample with the acetone-acid-molybdate solution, hydrolysis of phosphocreatine was limited to 1.6~ of input. To determine the effects of perchioric acid in the prepa:ation of cell extracts, we incubated phosphocreatine in ice-cold 0.25 M perchloric acid. Using assay A, we found no appreciable hydrolysis ( < 1~ of input) after 5 rain of incubation. These studies indicate that at very high levels of acid-labile compounds, hydrolysis in assay A could cause over-estimates of Pi- Such errors would be far less with assay B. Comparison of the two Pi assays with WI-L2 extracts gave values of 9.5 and 10.1 /~mol/10* cells by assay A and B, respectively, suggesting that acid-labile compounds are unlikely to cause appreciable interference in our estimates of cellular Pt in lymphocytes. Platelef and erythrocyte contamination of PBM Extracts of PBM that had been isolated from heparinized blood contained large amounts of PPi, ranging from 20 to 90 nmol/109 cells. These high values were attributed to contamination by platelets, which were present in a 10- to 20-fold excess over PBM. Platelets are known to store large amounts of PPi in granules [27] and with our assay were found to contain 6 to 8 nmol PP,/10 * platelets. Using blood that had been defibrinated with glass beads, we obtained PBM contaimng less than 1 platelet/10 PBM. This method resulted in an erythrocyte contaminant of 5 to 40¢$ of total cells. The effect of this contamination on PPi estimates was judged to be insignificant, since analyses of erylhrocyte extracts showed a PP~ content of less than 0.02 nmol/109 cells. Effects of PHA stimulation on metabolite levels in PBM A detailed kinetic analysis of human lymphocyte AdoMet synthetase demonstrated marked product inhibition by AdoMet, Pi and PPi, raising the possibility that under certain metabolic states, changes in intra-

T A B L E 11 Effects of P H A stimulation on cellularlevelsof AdoMet, PP,, P,, and A T P in P B M Cells were cultured with and without the addition of PHA at 1 t t g / m l at zero time, and cell extracts were prepared and assayed for metabolites as described in Materials and Methods. Concentrations have been estimated from cell counts and an estimate for cell volume o f 0.42 m l / 1 0 * c~ils [4]. Experiment

Time * (h)

AdoMet

PPi

P,

(/zM)

(/~M)

(raM)

ATP (mM)

- PHA

+ PHA

- PHA

+ PHA

- PHA

+ PHA

- PHA

+ PHA

1 1 1

0 5 8

15.5

4.3

13.6

23.3 36.0

3.8

4.8 6.7

4.0 3.6

4.5 4.3

1.26 !.00

1.26 0.93 1.21

2 2 2

0 5 8

11.9 10.7 I 1.4

15 31.0

6.7 5.0 5.0

4.8 5.7

6.2 6.9 6.4

7.9 8.3

1.31 0.88 0.95

0.69 !.31

• Time zero refers to the start o f the experiment after a 2.5 h preiacubation.

230 cellular levels of AdoMet, PP,, and Pi could regulate the activity of this enzyme in intact cells [6]. We chose cultured PBM to test this hypothesis because previous studies had shown that both the concentration and fractional turnover of AdoMet increases within a few hours after stimulation by PHA, prior to the onset of appreciable inc~ases in protein and D N A synthesis [4]. T o explain these changes in AdoMet metabolism by effects of product inhibition, PPi and Pi levels would have to be relatively high in unstimulated cells and fall within hours of treatment with PHA. Measurements of AdoMet in cultured PBM confirmed previously reported results [4] and showed approx. 2.5-fold increases in AdoMet 8 h after PHAstimulation and either a slight fall or no change in unstimulated cells (Table II). Pi a n d PPi levels, however, did not decrease as predicted from our ~odel. PPi levels in two separate experiments were actually 14 to 76% higher in PHA-stimulated cells thzat in unstimulated cells after 8 h, and Pi levels were 19 and 30% lfigher. A T P levels did not change markedly with time, and after 8 h were 21 and 38% higher in stimulated cells (Table ! I ) These findings do not support the notion that increased turnover and steady-state levels of AdoMet in PHA-stimulated cells are caused by decreases in P, and PPiEffects of PHA stimulation on AdoMet synthetase activity in P B M extracts The specific activity of AdoMet synthetase in crude extracts of stimulated PBM increased 2.5- a n d 3.3-fold after 8 and 24 of culture, respectively (Table III). Only a n 11% increase in specific activity occurred in unstimulated cells during the same period of culture, and lactate dehydrogenase levels in stimulated cells increased only 28% after 24 h. Assay of AdoMet synthetase in a 5 0 : 5 0 mixture of extracts from cells cultured for 8 h with and without PHA gave a value almost exactly mid-way between those of the individual extracts, thus providing no evidence for an in vitro inhibi-

t.O o

o.s-

~0.8"

o.6-

~

0.6"

O i

0.4-

O.4-

• ' ..... q O.~ O.I

" " ...... I 0.3 1.O

Protei~

" ' 3.0

Oag/mumyl

• i ..... 'l

0.3

I.O

'

| ..... 3.0

] IO

" ~lO

ademet tTatJmtue

Fig. l. Effects of PHA stimulation on levels of immunoreaetive AdoMet synthetase in crude extracts of PBM. lmmunoreactivity was determined by ELISA with use of a monoclonal antibody to the a/a" subunits of human lymphocyte AdoMet synthetase [18] and is proportional to the production of a chromophore absorbing at 495 nm, which was produced by a second antibody consisting of horseradish pc~oxidase-conjugated, goal anti-mouse polyclonal antibody. Immunoreactivit> is plotted vs. total protein assayed in panel (A). and vs. AdoMet synthetase catalytic activity in panel (B). Note the logarithmic scale for both abscissas. tor or activator. To determine whether these i n c r e a ~ s in A d o M e t synthetase activity were due to new protein synthesis or in vivo activation of previously synthesized enzyme, crude extracts were also analyzed for immunoreactive A d o M e t synthetase by a n ELISA assay employing a monoclonal antibody specific for the a / a " subunits of A d o M e t synthetase [18]. W h e n immunoreactivity was plotted as a function of the a m o u n t of protein assayed, virtually identical results were obtained with extracts of stimulated and unstimulated PBM, indicating that these cells contain almost identical amounts of immunoreactive a / a ' subunit even though enzyme specific activities varied as much as 3-fold (Fig 1A). A similar quantity of imm~noreactive a / a " subunit was found in a n extract of WI-L2 cells. In plots of immunoreactivity vs. catalytic activity, extracts of PBM harvested at 0 time and after 8 h of culture without PHA gave almost identical curves, while the extract from cells incubated for 8 h with P H A had approx. 4-fold ;ante A d o M e t synthetase catalytic activity per

TABLE 111 Effects of PHA stimulationon AdoMet synthetaseand lactate dehydrogenaseactwities in PBM Cells were cultured with and without the addition of PHA at 1 #g/ml at veto time. and extracts were prepared and analyzed as described in Materials and Methods. I U of enzyme activity catalyzes the reaction of I nmol/h for AdoMet synthetase and l #mol/h for lactate dehydrogenase. Time (h) 0 a 8

24 WI-L2

AdoMet synthetase (U/m8 protein) - PHA 3.5+0.54 (n = 5) b 3.9±0.67 (n ffi5)

3.9±0.79 (n ffi3) 12.2 and 17

+ PHA -

Lactate dehydrogenase (U/rag protein) - PHA 58(nffil)

+ PHA

8.9± 2.55 (n = 5)

59 (n = I)

53 (n = I)

11.5ffil.86(n=3)

65 (nffil)

74(n =l)

a Time zero refers to the start of the experiment after a 2.5 h preincubation period. b Values are given as the menn+ SE. The number of observations (n) is given in parentheses.

231 immunoreactive protein (Fig. 1B). The WI-L2 extract resembled that from stimulated PBM and contained approx. 3-fold more AdoMet syntbetase catalytic activity per immunoreactive protein than unstimulated PBM. Discussion

The data presented here indicate that a simple kinetic mechanism involving multi-product inhibition cannot explain the changes in AdoMet metabolism observed in PHA-stimulated PBM. Our kinetic data [6] predict that in order to double the rate of AdoMet synthesis and to counter the increased inhibition caused by increasing AdoMet from approx. 14 to 34/~M at 8 h (Table I!), Pi and PPi levels would have to be at least several-fold higher than actually found in unstimulated cells (e.g. 50 pM PPi and 10 mM Pi: or 20 pM PP, and 14 mM P,) and much lower than observed in stimulated cells. Yet the only changes observed in PP, and Pi were minor and in the wrong direction. ATP levels, which might also influence kinetic activity, did not change appreciably after PHA-stimulation. We did not measure intracellular methionine in these experiments, but it is unlikely that this substrate can account for increases in AdoMet synthesis either, since it is present in the medium a~ 100 pM and the reaction rate is about 80% of V,,~ at only 20 pM [6]. Our enzymatic analyses of crude PBM extracts show that increased cellular turnover of AdoMet after PHAstimulation is associated with a similar degree of increased AdoMet synthetase activity in vitro. Furthermore, these changes in catalytic activity were not accompanied by changes in immunoreactive a/a' subunit, suggesting that the increase in AdoMet synthetase activity noted in PBM within the first 8 h of PHA stimulation is due to an increase in the catalytic activity of pre-existing protein rather than synthesis of new enzyme. Comparison of the immunoassay data obtained for PBM and WI-L2 show that PHA-stimulated PBM resemble rapidly growing WI-L2 cells and have a high level of catalytic activity per immunoreactive a/a' subunit (Fig. 1B); in contrast, unstimulated PBM at zero time ana at 8 h have similar low levels of catalytic activity per immunoreactive a/a" subunit. Our findings suggest a model in which quiescent lymphocytes contain substantial quantities of a form of AdoMet synthetase with reduced or absent catalytic activity, which is activated when methylation requirements increase in response to lectin stimulation. Presumably, a similar response occurs to physiologic stimuli in vivo. This type of arrangement would allow cells to respond to stimuli quickly without the need for new RNA and protein synthesis, an obvious advantage for an enzyme that is feedback inhibited by a product required for RNA synthesis. The activation process could involve phosphorylation/dephosphorylationof an

AdoMet synthetase subunit, proteolytic cleavage or some other form of modification. It is of interest in this regard that AdeMet synthetase isozymes have been described in a number of organisms and cell types. including yeast 128,29], rat liver [30-321, and human erythrocytes, fibroblasts, lymphoid cells [33] and liver |34]. Furthermore, the human lymphocyte enzyme has been shown to have the subunit structure aa'/J2, where the 53 kDa a-subunit and the 51-kDa a'-subunit are immunologicallycross-reactive [18] and appear to differ by a post-translational modification 151. It is also possible that the early increases in activity following PHA-stimulation could be due to synthesis of an AdoMet synthetase isozyme that is not recognized by our monoclonal antibody. In addition, since our immunochemical assays measured only a- and a'-subunits, we cannot eliminate the possibility that differences in AdoMet synthetase catalytic activity arz related to the availability of ~subunit and that "activation' requires synthesis of new ~-subunit. Admowledgemeat This study was support by Grant AM12828 from the National Institutes of Health. References I Mudd. S.H. and Cantoni. G.L. (1958) J. Bid. Chem. 231,481-492. 2 Usdin. E.. BorchardL R.T. and Creveling. C.R. (1979)Transmethylation, pp. 1-631, Elsevier/North-Holland. New York. 3 Usdin. E.. Borchardt. R.T. and Creveling, C.R. (1982) Biochemistry of S-adenosylmethionine and Related Compounds. pp. ! -760. MacMillan Press, London. 4 German. D.C.. Bloch. C.A. and Kredich. N.M. (1983) J. Biol. Chem. 258. 10997-11003. 5 Kotb. M. and Kredich. N.M. (1985) J. Biol. Chem. 2~0, 3923-3930. 6 Kotb, M. and Kredich. N.M. (1990) Biochim. Biophys. Acta 1039. 253-260. 7 Ellens, D J . and Gielkens, A.L.J. (1980) J. lmmunol. Methods 37. 325-332. 8 Kredich. N.M. and Hershfield. M.S. (1979) Proc. Natl. Acad. Sci. USA 76, 2450-2454. 9 Levy, J.A., Virolainen. M. and Defendi, V. (1968) Cancer 22. 517-524. 10 Levy, S.A., Buell. D.N.. Creech. C.. Hirshaut, Y. ~ d Silverberg. H. (1971)J. Natl. Cancer Inst. 46. 647-654. 11 Lerner. R.A. and Hodge, L.D. (1971) J. Cell Physiol. 77, 265-275. 12 Kredich. N.M. (1971) J. Biol. Chem. 246. 3473-3484. 13 Daley. L.A.. Renosto. F. and Segel, I.H. (1986) Anal. Biochem. 157, 383-395. 14 Robbins, P.W. and Lipmann. F. (1958) J. Biol. Chem. 233, 6866~0. 15 Lowrey. O,H. and ~ J.A. (1946) J. Biol. Chem. 162. 421-428. 16 Lebo. R.V. and Kredich. N.M. (1978) J. Biol. Chem, 253. 26152623. 17 Heinonen, J.K. and Lahti. R J . (1981) Anal. Biochem. 113. 313317. 18 Kotb. M., Geiler. A. M.. Markham, G.D.. K_redich. N.M., De La Rosa. J. and Bcachy, E.H. (1990) Biochim. Biophys. Acta 1040. 137-144.

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