Evidence for two methyltransferases involved in the conversion of phosphatidylethanolamine to phosphatidylcholine in the rat liver

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 211, No. 2, October 15, pp. 762-773, 1981

Evidence for Two Methyltransferases Involved in the Conversion of Phosphatidylethanolamine to Phosphatidylcholine in the Rat Liver’ B. V. RAMA SASTRY,*P~‘~CHARLES N. STATHAM,+ JULIUS AXELROD,t FUSAO HIRATAt

AND

Vanderbilt University School of Medicine, Nash&, Tennessee 37232,and *Department of Phnm, tsection on Pharmacology, Laboratory of Clinical Science, Naticmal Institutes of Mental Health, and *Molecular Toxicology Section, Clinical Pharmacology Branch, Clinical Oncology Program, Division of Cancer Treatment, National Cancer Institute, Bethesda, Margland 20205 Received January

5, 1981

The stepwise methylation of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) occurs in the rat liver. The properties of two methyltransferases that are involved in this stepwise methylation of PE to PC in the rat liver are reported. Rat microsomal membranes were used as the source of the enzymes and S-adenosyl-L-[methyl3H]methionine as the methyl donor. The first methyltransferase converted PE to phosphatidyl-N-methylethanolamine (PME), and the second methyltransferase converted PME to PC. The products were characterized by thin-layer chromatography and highpressure liquid chromatography. After incubation of rat liver microsomes with the methyl donor, three peaks corresponding to PME, dimethyl-PME (PMME), and PC were found and quantified. The first methyltransferase had low K,,, (0.83 PM), pH optimum of 8, and was activated by Mg2f. The second methyltransferase had a high K, (-67 pM) and a pH optimum of 10. The proportion of the first methyltransferase in the microsomal membranes was increased by repeated washings in hypotonic medium containing EDTA. When the microsomal membranes were subjected to repeated mild sonication and centrifugation at 105,OOOga fraction of the second methyltransferase was solubilized (i.e., appeared in the nonparticulate fraction). The solubilized enzyme utilized dipalmitoyl-PME and -PMME as substrates. Both enzymes were also present in mitochondrial and nuclear membranes with highest specific activities occurring in the microsomal membranes.

Several investigations have indicated that phosphatidylethanolamine is methylated by stepwise transmethylation with

S-adenosylmethionine ( SAM)3 to form phosphatidylcholine in the mammalian liver (l-6). Bremer and Greenberg (1) assumed that there were three different enzymes, each incorporating one methyl group. They found that incorporation of the first methyl group was the rate-limiting step in the overall reaction leading to phosphatidylcholine. Rehbinder and Greenberg (4) solubilized the microsomal methyltransferase and concluded that there was only one enzyme. In two other studies (5, 6) using solubilized methyltransferase systems, no evidence was presented for the involvement of more than

i Part of this investigation was presented at the Meeting of the Federation of American Societies for Experimental Biology and Medicine at Dallas, Tex., in April, 1979. z On sabbatical leave from the Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tenn. 37232. This author was partially supported by a Faculty Fellowship from the Vanderbilt University Research Council; United States Public Health Service Research Grants HD10607, AG-02077, HL-25358, and United States Public Health Service General Research Support Grant RR05424, and a grant from The Council for Tobacco Research U. S. A., Inc. The authors wish to thank Dr. Michael R. Boyd of the Molecular Toxicology Section, NCI, for consultations on HPLC and Professors John G. Coniglio and Erwin J. Landon of Vanderbilt University for critical evaluation of the manuscript. 0003-9861/81/120762-12$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of repmduction in any form reserved.

z Abbreviations used: SAM, S-adenosylmethionine; TLC, thin-layer chromatography; HPLC, high-pressure liquid chromatography. 762

RAT LIVER

PHOSPHATIDYLETHANOLAMINE

one enzyme in the stepwise methylation of phosphatidylethanolamine. In neurospora, the stepwise methylation of phosphatidylethanolamine was found to be catalyzed by two membrane-bound N-methyltransferases (7, 8). The first methyltransferase incorporated the first methyl group, and the second methyltransferase catalyzed the addition of the remaining two methyl groups. Recently our laboratory reported the existence of two methyltransferases in bovine adrenal medulla (9), rat erythrocytes (10, ll), and rat brain (12). In view of these observations, the phosphatidylethanolamine Nmethyltransferase system of the rat liver, a prime source of phosphatidylcholine (1, 2), was investigated. This communication provides evidence for the existence of two methyltransferases in rat liver microsomes. The first methyltransferase converts phosphatidylethanolamine to phosphatidyl-N-methylethanolamine. The second methyltransferase converts phosphatidyl-N-methylethanolamine to phosphatidylcholine. MATERIALS

AND METHODS

Materials. Phosphatidylethanolamine, phosphatidyl-N-methylethanolamine, and phosphatidyl-N,Ndimethylethanolamine were obtained from Grand Island Biological Company (Grand Island, N. Y.). These phospholipids were derivatives of egg phosphatidylcholine by the exchange of bases in the presence of phospholipase D. Synthetic &y-dipalmitoylL-a-phosphatidylethanolamine and its N-methylated derivatives were purchased from Calbiochem-Behring Corporation (La Jolla, Calif.). Bovine brain phosphatidylcholine and bovine liver phosphatidylcholine were obtained from Sigma Corporation (St. Louis, MO.). N-Methylaminoethanol, N,N-dimethylaminoethanol, and choline iodine were supplied by Eastman Organic Chemicals (Rochester, N. Y.). S-Adenosyl-L,-[meth&zH]methionine (64 Ci/mmol) (SAM) was purchased from New England Nuclear Corporation (Boston, Mass.). All other reagents were of analytical grade. Assay for phospholipid N-methyltran&roses. The total volume of the reaction mixture was 50 ~1 and was composed of three solutions in Tris-glycylglycine buffer (50 mM) at pH 8. The first solution (20 ~1) consisted of the membrane preparation (200 ccg of protein) which contained endogenous substrate and a second solution (20 ~1) which was used for the ad-

N-METHYLTRANSFERASE

763

dition of various compounds such as activators (MgCls) and exogenous substrates. The third solution (10 ~1) contained [sH]SAM (specific activity: 64 Ci/ mmol for low SAM experiments and 0.32 Ci/mmol for high SAM experiments). Unless otherwise specified, the final reaction mixture contained 10 mM MgClz and 0.1 mM EDTA. The reaction was started by adding the third solution to the mixture of the first and second solutions, and the final reaction mixture was incubated at 37°C for a specified time. The reaction, was stopped by the addition of 3 ml of chloroform/methanol/hydrochloric acid (2/l/0.02, v/v) and the mixture was shaken for 10 min. The chloroform/methanol/HCl extract was washed twice by shaking (2 X 10 min) with 2 ml of 0.1 M KC1 in 50% aqueous methanol. The aqueous phase was aspirated each time. One milliliter of the chloroform phase was transferred to a counting vial and evaporated to dryness at 80°C. Ten milliliters of aquasol was added, and radioactivity was measured. The remaining chloroform phase was dried over anhydrous MgSO, and used for characterization and estimation of the radioactive metabolites. The amount of ‘H-methyl group incorporated into phosphatidyl-N-methylethanolamine, phosphatidylN,N-dimethylethanolamine, and phosphatidylcholine was determined by thin-layer chromatography (TLC) or high-pressure liquid chromatography (HPLC) as described in separate sections of TLC and HPLC. Unless otherwise specified, data were expressed in terms of the products formed. Preparation of the liver homogenate and separe

tion of subcell&r

fractions bp differential

centrif-

ugation. Male white rats (75-85 g) were decapitated and bled and their livers dissected. Livers were washed with isotonic sucrose (0.25 M) at 4°C and homogenized in isotonic sucrose (5 ml/g) using a Teflon hand homogenizer (size C) at 1200 rpm (six strokes). The homogenate was filtered through four layers of gauze, and the filtrate was centrifuged at 15009. The nuclear pellet was saved and the postnuclear supernatant was again centrifuged at 12,100~ for 20 min. The postmitochondrial supernatant was again centrifuged at 105,OOOgfor 60 min, and the microsomal pellet was saved. The nuclear, mitochondrial, and microsomal pellets were washed once by resuspension in isotonic sucrose and recentrifugation. The washed pellets were resuspended in Tris-glycylglytine buffer (100 mM) at pH 8.0 and stored at -2O“C. All operations were done at 2-4°C. Most of our studies on phospholipid methyltransferases were carried out using microsomes. The above procedure for the preparation of liver microsomes has been validated by determining the activities of several marker enzymes by methods routinely used in our laboratores (13-19). Cytochrome c oxidase activity (13, 16) in the microsomes was about 2.9% of

764

SASTRY

that in the mitochrondrial fraction. Succinic dehydrogenase activity (13, 17) in the microsomes was about 0.4% of that in the mitochrondrial fraction. No measurable amount of sodium-potassium-dependent ATPase, a marker for plasma membrane (13), was found in the microsomes. The microsomal fraction was high in activities of glucose-6-phosphatase (15, 18) (4 pmol phosphate/mg protein115 min) and cytochrome P-450 (19) (0.97 nmol/mg protein), which are markers for endoplasmic reticulum. All of these observations indicate that the microsomal fraction contains mainly membranes from endoplasmic reticulum. Thin-layer chromatography (TLC) of phospholipids. The chloroform extracts of phospholipids were concentrated under a stream of nitrogen, and lOO~1 samples were applied on l-in. strips of a Silica gel G plate (Uniplate, Analtech Inc., Newark, Del.). Chromatograms were developed in two solvent systems: (a) chloroform/propionic aeid/n-propylalcohoi/water (2/2/3/l, v/v) and (b) chloroform/methanol/water (65/25/4, v/v). The solvent was allowed to move 6.5 in., and 0.25-in. cuts were collected into counting vials. Aquasol was added and radioactivity was counted. Authentic samples of phospholipids from bovine liver or rat liver were chromatographed simultaneously and visualized with iodine vapor. High-pressure liquid chromatography (HPLC) of phospholipids. An HPLC method for separation and characterization of the three major phospholipids formed by methyltransferases, ‘H-labeled phosphatidyl-N-methylethanolamine, phosphatidyl-N,N-dimethylethanolamine, and phosphatidylcholine, was developed. One-milliliter samples of the chloroform phase (see under assay for phospholipid N-methyltransferases) were evaporated to dryness under a stream of nitrogen and reconstituted in chloroform/ methanol/water/ethanolamine (77.8/20/2/0.2, v/v) for HPLC analysis. All phospholipid samples were chromatographed using a Waters M6OOA pump, U6K loop injector, and an R401 differential refractometer. A 10 to 100 ~1 aliquot of each sample was injected onto a PPorasil column (30 X 0.39 cm), and eluted with a mobile phase of chloroform/methanol/water/ethanolamine (77.8/ 20/2/O/2, v/v) at a rate of 2 ml/min. Fractions containing the radiolabeled phospholipids were collected directly into scintillation vials, and 15 ml ACS counting fluid was added to each fraction. Vials were counted in a liquid scintillation counter. All counts were corrected for quench by automatic external standardization. Identification of bases frm phospholipids. ‘H-Labeled phospholipids, phosphatidylcholine, phosphatidyl-N-methylethanolamine, and phosphatidyl-N,Ndimethylethanolamine formed enzymatically were

ET AL. extracted into chloroform/methanol and separated by preparative HPLC. The separated samples of each phospholipid (equivalent to 2.4-4.4 x 10’ dpm) were combined; reduced to dryness under nitrogen in screw-cap centrifuge tubes; and hydrolyzed with 2 ml of aqueous 6 N HCl for 2 h. Bases were extracted from the reaction mixture with 1 ml of t-butanol as described by Lester and White (20). The base in an aliquot of the t-butanol extract was applied on a TLC plate and developed in n-propanol/NHIOH (4/l, v/ v). Simultaneously, the standard samples of choline, N,N-dimethylaminoethanol and N-methylaminoethanol were applied, developed, and visualized with iodine vapor. The bases were also identified by HPLC (20). Preparation of phospholipid vesicles. Liposomes of dipalmitoylphosphatidylethanolamine and its derivatives were prepared in Tris-glycylglycine buffer (25 mM, pH 8.0). The phospholipid was suspended in the buffer, warmed to 37°C and sonicated for 2 min at 50% intensity using a Branson Sonifier. These vesicles were used for increasing the substrate concentration with partial solubilized enzyme. RESULTS

Subcellular Distribution of Phospholim*d Methylation in Rat Liver The various subcellular fractions of rat liver were examined for their abilities to incorporate 3H-methyl groups into phospholipids after they were incubated with [methyl-3H]SAM. The highest incorporation of methyl-3H radioactivity in phospholipids was found in the microsomal fraction, though substantial amounts were present in the mitochondrial and nuclear fractions (Table I). Separation and Ident&ation Methylated Phospholipids

of

The identity of the 3H-methylated phospholipids formed after the microsomal fraction was incubated with [methyl3H]SAM was determined by TLC and HPLC. There were three distinct peaks of radioactivity cochromatographing with phosphatidyl - N - methylethanolamine, phosphatidyl - N,N - dimethylethanol amine, and phosphatidylcholine in the HPLC pattern (Fig. 1). Similarly, three distinct peaks were observed for the three methylated phospholipids when they were chromatographed by TLC. There were con-

RAT LIVER PHOSPHATIDYLETHANOLAMINE

N-METHYLTRANSFERASE

765

TABLE I DISTRIBUTIONOF PHOSPHOLIPIDN-MFXHYLTRANSFERASESIN RAT LIVER”

Membrane fraction Total homogenate Nuclear Mitochondrial Microsomal

sH-Methyl groups incorporated into phospholipids (fmol/m protein, mean + SE)b (200 PM SAM) 249 + 435 f 657 f 1266 f

21 33’ 18” 15e

Product formed (fmol/Ng, mean + SE) PME by methyltransferase (2 PM SAM) 40.1 26.2 45.7 83.3

f f + f

1.3 0.2 0.4 1.4

I”

PC by methyltransferase IId (200 PM SAM) 8.3* 7 145 f lie 219 f 6e 422 k 5e

’ Livers were obtained from young rats. All values are means from three to six determinations. b Reaction time: 40 min; pH: 8.0. The number of methyl groups incorporated into PME, PMME, and PC are 1, 2, and 3. ’ Reaction time: 10 min; pH: 8.0. Total PME formed was estimated quantitatively by TLC or HPLC. d Reaction time: 40 min; pH: 8.0. PC formed was estimated quantitatively by TLC or HPLC. “These values should be considered minimal. Fractions must be washed to obtain purity. Washing may solubilize part of the methyltransferase II, and some of it may be lost.

siderable amounts of phosphatidyl-Nmethylethanolamine and phosphatidylN,N-dimethylethanolamine in the methylated phospholipids when microsomal membranes were incubated with a low concentration of SAM (0.79 wM, Fig. 1A). Phosphatidylcholine was the major product when microsomal membranes and high concentrations of SAM (200 PM) were incubated (Fig. 1B). The bases for the three phospholipids were identified as described in the methods after separation of the phospholipids by HPLC.

Effects of Low SAM Concentrations on Phospholipid Methylation in Microsomes Rat liver microsomal preparations were incubated with various concentrations of SAM (between 0.03 and 1.6 FM), and the extent of the formation of 3H-methyl phospholipids was measured. These low concentrations of SAM were selected because the adrenal microsomal methyltransferase which converted phosphatidylethanolamine to phosphatidyl-Nmethylethanolamine has a K,,, of about 1.0 /IM (9). The rate of incorporation of 3Hmethyl groups into phospholipids was linear during the first 10 min of incubation of liver microsomes and low concentra-

tions of SAM. Therefore a reaction time of 10 min was chosen. The formation of phosphatidyl-N-methylethanolamine was linear up to an enzyme level of 310 pg of protein in each assay. Therefore, 200 pg of protein was used per assay. When the total radioactivity in the methylated phospholipids was plotted as a functisn of SAM concentration, the resulting curve showed two phases with a sharp inflection point at about 0.80 j.&M (Fig. 2). The various methylated phospholipids were separated by TLC and quantified. When the 3Hmethyl group incorporated into phosphatidylmonomethylethanolamine was measured at various SAM concentrations, there was no break in the curve. These observations suggest that at least two enzymes were involved in the methylation of phospholipids. Endogenous phosphatidylethanolamine was the substrate for the first enzyme. Negligible amounts of phosphatidyl-Nmethylethanolamine occur endogenously in the microsomes (2, 20, 21). It should be formed in the reaction to serve as a substrate for the second enzyme. At about 0.8 PM SAM, optimum levels of phosphatidylN-methylethanolamine were obtained so that the enzymatic incorporation of a second or a third 3H-methyl group into the phosphatidylethanolamine proceeded at a

766

SASTRY 12-

ET AL.

B

io-

? s

6-

3 8 PC

4-

I’ P 7 i--F ii: 2 4 6 6 10 mil n

2 t

,YlS I , I -I\, 2 4 6 6 10 min

FIG. 1. HPLC separation of ‘H-labeled phosphatidyleholine (PC), phosphatidyl-N,N-dimethylethanolamine (PMME), and phosphatidyl-N-methylethanolamine (PME) formed as products when rat liver microsomes were incubated with low and high concentrations of 4adenosyl-L-[methyl-aH]methionine (SAM). The peaks for PME, PMME, and PC were characterized by comparison with HPLC patterns of the authentic samples of PME, PMME, and PC. The bases were identified as described in the text. S, solvent front. 3H-methyl groups incorporated into PCPMMEPME = 3211. (A) SAM: 0.79 @d; reaction time: 10 min. The incorporation of ‘H-methyl groups was linear during this reaction time. (B) SAM 200 pM; reactiod time: 40 min. The incorporation of %Imethyl groups was linear up to 30-40 min and reached maximum in 40-60 min.

higher rate than that observed at SAM concentrations below 0.8 PM.

Effects of High SAM Concentrations on Phospholiw’d Methylation in Microsomes In order to evaluate whether more than two enzymes were involved in the stepwise methylation of phosphatidylethanolamine, rat liver microsomes were incubated with high concentrations of SAM (between 32 and 650 PM). These concentrations of SAM were selected because the K,,, of adrenal microsomal methyltransferase which converted phosphatidylN-methylethanolamine into phosphatidyl-N,N-dimethylethanolamine and phosphatidylcholine was about 100 PM. At these SAM concentrations, incorporation

I”

0

loo

zoo

300

400

PMWC

so0

(cl

ooo

700

SAM Irnl

FIG. 2. (A) Enzymatic methylation of liver microsomal phosphatidylethanolamine as a function of the concentration of S-adenosyl-L$H]methionine (SAM). Concentrations of SAM were low (between 0.026 and 1.6 &, pH 8.0. Reaction time: 10 min during which incorporation of aH-methyl groups into phospholipids was linear. The ordinate represents total radioactivity in the methylated phospholipids. The upper curve (0) indicates the radioactivity incorporated into all three methylated phospholipids as a function of SAM concentration. There was a split in this curve at 0.80 PM. The mean of the point at 0.80 gM SAM (235,493 + 10,395 dpm) was significantly different (P < 0.0001) from the mean (331,555 2 782 dpm) at 0.85 PM SAM. The lower curve (m) indicates radioactivity incorporated into phosphatidyl-Nmethylethanolamine (PME) as a function of SAM concentration. There was no split at 0.80 PM in the lower curve, which is approximately a rectangular hyperbola. (B) Enzymatic methylation of liver microsomal phosphatidylethanolamine at high eoncentrations of SAM. Concentrations of SAM: 35-650 CM; pH: 8.0; reaction time: 40 min during which incorporation of ‘H-methyl groups into phospholipids was linear. The ordinate represents the radioactivity incorporated into all phospholipids in curve a (0) into phosphatidylcholine (PC) in curve b (A) and into phosphatidyl-N,N-dimethylethanolamine (PMME) in curve c (w). The shape of each curve is approximately a rectangular hyperbola.

RAT LIVER

PHOSPHATIDYLETHANOLAMINE

of methyl groups into phospholipids was linear for more than 30 min and reached maximum at about 40 min. Therefore, a reaction time of 40 min was selected. About 85% of the total radioactivity incorporated :into methylated phospholipids was found in phosphatidylcholine and the remaining radioactivity was found in phosphatidyl - N,N - dimethylethanol amine. The formation of phosphatidylcholine was linear up to an enzyme level of 310 pg of protein per assay. Therefore, 200 pg of protein was used per assay. When 3H-methyl groups incorporated into phospholipids were plotted as a function of SAM concentration, a typical rectangular hyperbola was obtained (Fig. 2, curve a). No significant inflection points were observed in the curves. Similar curves were obtained when 3H-methyl groups incorporated into phosphatidylcholine (Fig. 2, curve b) and phosphatidyl-N,N-dimetheylenthaolamine (Fig. 2, curve c) were plotted as a function of SAM concentration. Properties for Two Putative Methyltrunsferases Involved Methylation of Microsomal Phospho@ids

in the

The experiments using varying concentrations of SAM suggested the presence of at least two methyltransferase enzymes. To further explore this possibility, microsomal membranes were incubated with low concentrations of SAM (0.61 PM) and the methylated products examined at various time intervals by TLC. At low SAM concentrations, all three methylated phospholipids were formed (Fig. 1A). As there was no evidence for the endogenous occurrence of phosphatidyl-N-methylethanolamine, it was first formed, and part of it was further methylated to phosphatidyl-N,N-dimethylethanolamine. Similary, there was no evidence for the endogenous occurrence of phosphatidyl - N,N - dimethylethanol amine. Therefore, part of it was further methylated to phosphatidylcholine. In Fig. 3, the total amount of each of the 3H-labeled methylated phospholipids formed

767

N-METHYLTRANSFERASE

was plotted as a function of time. According to Fig. 3,75% of phosphatidyl-N-methylethanolamine, which was formed from phosphatidylethanolamine, was further methylated to phosphatidyl-N,N-dimethylethanolamine or phosphatidylcholine. When the SAM concentration was increased to 200 PM, phosphatidylcholine was the major product (Fig. 1B). Phosphatidyl-N,N-dimethylethanolamine was found to be formed as a minor product at 200 /.LM SAM. A double-reciprocal plot between the initial linear velocity for the formation of phosphatidyl - N,N - methylethanolamine and low SAM concentration was a straight line with a K, of 0.83 pM (Fig. 4A). It is not possible to determine the exact initial linear velocity for the formation of phosphatidylcholine by microsomal membranes at low SAM concentrations. In stepwise methylation of phosphatidylethanolamine, phosphatidyl-N-methylethanolamine must be formed by the first enzyme before it can serve as a substrate for enzymatic methylation in the succeeding step. At high SAM concentrations, a curve approximating a rectangular hyperbola was obtained for the formation of phosphatidylcholine (or phosphatidyl-N,Ndimethylethanolamine). A double-reciprocal plot between the linear velocity for

0

10

l%c

30

(min)

40

50

FIG. 3. Enzymatic formation of phosphatidyl-Nmethylethanolamine (PME), phosphatidyl-N,N-dimethylethanolamine (PMME) and phosphatidylcholine (PC) at low concentrations of SAM as a function of time in liver microsomes. SAM concentration: 0.61 pM; protein: 240 pg/assay; pH: 8.0; reaction time: 10 min.

768

SASTRY

-1

0

1

2

3

4

5

6

ET AL.

7

-20

-10

0 [l/W]

10

20

30

x I#

[l/N]

FIG. 4. (A) Double-reciprocal plot between low concentrations of S-adenosyl-L-[3H]methionine (SAM) and initial linear velocities for the formation of phosphatidyl-N-methylethanolamine (PME). The initial linear velocities were determined from the reaction period, O-10 min, during which PME formation was linear. SAM concentrations: 0.16 to 1.53 PM; pH: 8.0; Km: 0.83 PM. (B) Double-reciprocal plot between high concentrations of SAM and linear velocity for the formation of phosphatidylcholine (PC, 0). The points for the formation of phosphatidyl-N,N-dimethylethanolamine (PMME, 0) fall on the same line as those for the formation of PC. K,,,: 67 PM. SAM of methyl concentrations: 35-340 pM; pH: 8.0; reaction time: 40 min during which incorporation groups into phospholipids was linear.

the formation of phosphatidylcholine (or mation occurred at about 8.0 (Fig. 5A, upphosphatidyl - N,N - dimethylethanol - per curve). Between pH 5.8 and 9.0, the amine and high SAM concentrations (be- major product was the monomethyl derivtween 32 and 340 PM) was a straight line ative. At the low concentration of SAM, the formation of phosphatidyl-N,N-diwith a Km of 67 PM (Fig. 4B). and phosphatidylThe effect of Mgz+ on the rate of for- methylethanolamine mation of methylated phospholipids at choline exhibited a minor peak at 8.0 and various SAM concentrations was exam- a prominent peak at 10.0 (Fig. 5A, lower ined. In the presence of Mg2f, there was curve). The first peak (pH 8.0) in this lower a stimulation of phosphatidyl-N-methylcurve was due to increased formation of ethanolamine formation at low concentra- phosphatidyl - N - methylethanolamine, tions of SAM. Mgz+ increased phosphatiwhich served as a substrate for the second dyl-N-methylethanolamine formation in enzyme. The second peak represented the pH optimum for the formation of phoshomogenates, and nuclear, mitochondrial, and microsomal membranes by 50,35, 30, phatidyl-N,N-dimethylethanolamine and and 32%, respectively. At high SAM con- phosphatidylcholine. centrations, Mgz+ increased the formation At the high SAM concentration (200 of phosphatidylcholine by about the same p&f), the major product formed at every amount. pH was phosphatidylcholine (Fig. 5B). To provide further evidence for the pres- There was no accumulation of phosphaence of two phospholipid methyltransfertidyl-N-methylethanolamine at any pH. ases in the microsomes, the pH optimum The optimal pH for phosphatidylcholine formation was 10. These observations for the formation of phosphatidyl-Nmethylethanolamine at a low SAM con- again support the presence of two phoscentration (0.28 PM), and phosphatidylpholipid methylating enzymes, one formcholine at a high concentration of the ing phosphatidyl-N-methylethanolamine methyl donor (200 FM) were studied. At with a pH optimum of 8.0 (methyltranslow SAM concentration, the peak for ferase I) and the other forming phosphosphatidyl-N-methylethanolamine for- phatidyl-N,N-dimethylethanolamine and

RAT LIVER

PHOSPHATIDYLETHANOLAMINE

769

N-METHYLTRANSFERASE

A?7 , il:~s as

as

7.5

as

9s

I

10.5 1l.S

PH

5.5

95

1.s

8.S

9.5

10.5

11s

PH

FIG. 5. (A) Enzymatic formation of [‘H]phosphatidyl-N-methylethanolamine (PME) from the liver microsomal phosphatidylethanolamine as a function of pH. Reaction time: 10 min during which PME formation was linear. SAM concentration: 0.29 FM. The major product (about 90%) formed was PME at all pH values below 9.5 (upper curve, 0). The pH optimum for the formation of PME was 8.0. At all pHs, some minor amounts of phosphatidyl-NJGdimethylethanolamine (PMME) and phosphatidylcholine (PC) were formed. PMME + PC formed was plotted in the lower curve (w). The first peak (pH 8.0) in this curve was due to increased formation of PME which served as a substrate to the second enzyme. The second peak at pH 10.0 represented the pH optimum for the formation of PMME + PC. (B) Enzymatic formation of [cH]phosphatidylcholine (PC) from liver microsomal phosphatidylethanolamine (PE) as a function of pH (upper curve, 0). pH optimum for the formation of PC was 10.0 which was the pH optimum of the second enzyme. SAM concentration: 200 MM. Reaction time: 40 min during which incorporation of %-methyl groups into phospholipids was linear. At pH values between 7.5 and 11.0, some minor amounts of phosphatidyl-N,N-dimethylethanolamine (PMME, lower curve, n ) was formed. PMME formation also exhibited a pH optimum of 10.0. Buffers: Sodium phosphate buffer (50 mM), pH 5.8-8.0; glycylglycine-NaOH buffer, pH 8.011.0.

phosphatidylcholine with a pH optimum of 10.0 (methyltransferase II). Partial Separation of Two Phospholipid &fethylating Enzymes To examine whether the two phospholipid methyltransferases could be separated, the effect of repeated washings of microsomal membranes and mild sonication of microsomal membranes on the incorporation of 3H-methyl groups at low and high concentrations of SAM were studied. Microsomes (10 mg protein) were suspended in 5 ml Tris-glycylclycine buffer (25 mM; pH 8.0) containing 2 mM EDTA and vigorously shaken for 5 min at 4°C and centrifuged at 105,000g.The pellet was washed three times with the buffer and was analyzed for phospholipid methyltransferases using low (0.79 PM) and high

(200 PM) concentrations of SAM. The proportion of phosphatidyl-N-methylethanolamine and phosphatidylcholine in the reaction products was determined. With both washed and unwashed microsomes, 96.8% of the total radioactivity extracted with chloroform-methanol was found in the three phospholipids after separation with TLC. At the low SAM concentration, phosphatidyl-N-methylethanolamine represented 70.4 and 27.2% of the radioactivity in the products of washed and unwashed pellet preparations, respectively. This indicates that successive washings increased the proportion of the first methyltransferase relative to the second methyltransferase in the pellet. At high SAM concentration, phosphatidylcholine contained 21 and 97% of the total radioactivity in the methylated products obtained from the washed and unwashed preparations, respectively. These obser-

770

SASTRY

ET AL.

TABLE II vations suggest that methyltransferase I was more tightly bound and methyltransINFLUENCEOFSONICATIONONTHEUTILIZATIONOF ferase II was more easily solubilized. DIPALMITOYLPHOSPHATIDYLETHANOLAMINE(PE), The objective of the following experiDIPALMITOYLPHOSPHATIDYL-Nment was to partially solubilize methylMETHYLETHANOLAMINE(PME),AND transferase II by a mild sonication DIPALMI~~YLPHOSPHATXDYL-N,NDIMETHYLPHOSPHATIDYLETHANOLAMINE procedure and to examine the effects (PMME) BYLIVERMICROSOMES of exogenous substrates. A microsomal membrane preparation (5 mg/ml) from 3H-Methyl groups incorporated the rat liver was subjected to mild soni(fmol/pg protein) cation at 4°C for 1% min in Tris-glycylglycine buffer (25 mM, pH 8.0) and cen- Exogenous Unsonicated Combined Pellet microsomes substrate supernatant trifuged for 1 h at 105,OOOg. The pellet was subjected to sonication twice more. The 448 567 3271 resulting pellet and the three superna- None 231 269 2306 PE” tants were analyzed for enzymatic incor2401 1874 491 PME” poration of 3H-methyl groups into the var1815 1223 PMME” 2594 ious phospholipids at the high SAM concentration (200 PM). The first superNote. SAM concentration: 201 FM. All values are natant did not contain significant enzyme means from three to six values. activity. Equal amounts of enzyme activa 500 rg/ml added as liposomes. ity were present in supernatants 1 and 2 with no exogenous substrate added. They were combined for further experiments poration in the presence of dipalmitoylusing exogenous substrate. phophatidyl-N,N-dimethylethanolamine. In the absence of added exogenous phos- There is no explanation for this increase pholipid, there was a markedly reduced in methyl group incorporation in the presmethylation of the phospholipid in the ence of dipalmitoylphosphatidyl-N,N-dipellet and the combined supernatant as methylethanolamine. compared to unsonicated microsomes (TaThe various products, after the addition This indicates that the of exogenous substrates, were separated ble II). methyltransferases were inactivated dur- and quantified by both TLC and HPLC. ing sonication. The addition of dipalmiAfter the addition of dipalmitoylphosphatoylphosphatidylethanolamine resulted in tidyl-N-methylethanolamine, almost all the decreased incorporation of 3H-methyl of the enzymatically formed product was groups in unsonicated microsomes, pellet, the corresponding N,N-dimethylphosphaand supernatant fractions. The possible tidylethanolamine; negligible amounts explanation for this might be substrate of phosphatidylcholine were present. Afinhibition of methyltransferase I. ter the addition of dipalmitoylphosphaThe addition of exogenous dipalmitoylphostidyl-N,N-dimethylethanolamine, phosphatidyl-N-methylethanolamine and phatidylcholine was the main product dipalmitoylphosphatidyl - N,N- dimethyl - formed. ethanolamine resulted in a fourfold increase in the incorporation of 3H-methyl Distribution of Phospholipid groups in the supernatant fraction, again Methyltramfwases in Liver Fractions indicating that methyltransferase II is Methyltransferase I could be estimated more easily soluble. In the case of the pellet, there was no change in the incorpo- by measuring PME formation during the ration of 3H-methyl groups in the presence first 10 min at a low SAM concentration of 2.0 PM (2.5 Km of methyltransferase I), of exogenous dipalmitoylphosphatidyl-Nmethylethanolamine, but there was a two- when the velocity of PME formation was fold increase in the methyl group incor- linear as a function of time (Table I, Col-

RAT LIVER

PHOSPHATIDYLETHANOLAMINE

umn 3). For example, liver microsomes formed about 83 fmol of phosphatidyl-Nmethylethanolamine/pg protein in 10 min. In about 40 min, the rate of formation of phosphatidyl - N - methylethanolamine reached maximum. This means that the maximal rate of formation of phosphatidyl-N-methylethanolamine by the liver microsomes is 322 fmol/pg protein/40 min. The order of the activities of methyltransferase I in the liver fractions could be arranged in the following order: microsomes > mitochondria > nuclei. Methyltransferase II could be estimated by measuring phosphatidylcholine formation during the first 40 min at a high SAM concentration (3 Km of methyltransferase II), when the incorporation of aHmethyl groups into phosphatidylcholine reached maximum. Under these conditions, liver microsomes formed about 422 fmol/pg protein. The activities of methyltransferase II could be arranged in the following order: microsomes > mitochondria > nuclei. In general, the activity of methyltransferase II was higher in these fractions than that of methyltransferase I. The values for the amount of methyltransferase II should be considered minimal because this enzyme was less tightly bound to the phospholipid matrix than methyltransferase I. The values decreased depending upon the method of preparing microsomes and the washing of microsomes. At saturating concentrations of SAM for methyltransferase I (2 PM), 322 fmol/pg protein/40 min of phosphatidylN-methylethanolamine is formed. This should serve as a substrate for methyltransferase II. At saturating concentrations of SAM for methyltransferase II (200 PM), 422 pmol/pg protein/40 min of phosphatidylcholine was formed. Since both products were formed from the same molecules of phosphatidylethanolamine in the stepwise methylation at high SAM concentrations, these results suggest that the first step was considerably accelerated. At low SAM concentrations, the product of methyltransferase I, phosphatidyl-hrmethylethanolamine, accumulates and

N-METHYLTRANSFERASE

771

causes product inhibition. At high SAM concentrations, the product of methyltransferase I does not accumulate, but is further methylated as soon as it is formed. Hence, stepwise methylation is considerably accelerated. The only limiting factor seems to be the availability of SAM. DISCUSSION

This report describes the occurrence and properties of two phosphatidyl-Nmethyltransferases in rat liver microsomes. The first enzyme, phosphatidylethanolamine-N-methyltransferase (methyltransferase I), converted membrane-bound phosphatidylethanolamine to phosphatidyl-N-methylethanolamine. It had a low Km of 0.81 NM, a pH optimum of about 8.0, and its activity could be increased in the presence of Me. The enzyme seems to be firmly bound to membranes. It did not utilize the exogenous substrate dipalmitoylphosphatidylethanolamine, presumably because there is excess substrate present. The proportion of methyltransferase I relative to methyltransferase II could be increased by repeated washings of the microsomal membranes in hypotonic medium containing EDTA. Methyltransferase II, phosphatidyl-Nmethylethanolamine N-methyltransferase, converted phosphatidyl-N-methylethanolamine to phosphatidyl-N,N-dimethylethanolamine and phosphatidylcholine. It had a high apparent Km of about 67 PM, and a pH optimum of 10. It could be washed away from the membrane in hypotonic medium and solubilized by mild sonication. Unlike the firmly bound methyltransferase I, methyltransferase II can utilize exogenous substrates. The solubilized enzyme formed dipalmitoylphosphatidylcholine from dipalmitoylphosphatidyl-N,N-dimethylethanolamine, and dipalmitoylphosphatidyl-N,N-dimethylethanolamine from dipalmitoylphosphatidylN- methylethan olamine. The properties of methyltransferase II are similar to the properties of Nmethyltransferase systems described by other investigators (l-6) who used reaction conditions closer to the optimum properties of methyltransferase II.

772

SASTRYETAL.

Depending upon the SAM concentration, one or more products were formed by the liver N-methyltransferase system. At SAM concentrations of thrice the Km of methyltransferase II (~200 PM), the major product formed was phosphatidylcholine. At SAM concentrations below one-half of the Km of methyltransferase (~0.4 PM), the major product formed was phosphatidylN-methylethanolamine. Between 0.4 and 200 PM SAM, the proportion of the three products varied. At very low SAM concentrations (e.g., 0.28 FM, Fig. 5A), the product of methyltransferase I, phosphatidyl-Nmethyl-ethanolamine, accumulates within the membrane in measurable amounts, and the conditions of its formation could be studied with reasonable accuracy. This accumulation of the product also causes a certain degree of product inhibition, because exogenous phosphatidyl-N-methylethanolamine inhibited the phospholipid methylation. At a high SAM concentration (e.g., 200 FM), phosphatidyl-N-methylethanolamine does not accumulate at any pH (Fig. 5B). It is further methylated by methyltransferase II, and the whole process of stepwise methylation is considerably accelerated. This acceleration of stepwise methylation at high concentrations of SAM indicates that the two methyltransferases are located in the membrane in a definite juxtaposition to one another. In stepwise methylation of phospholipids, the limiting factor is the availability of SAM. Although properties of liver microsomal methyltransferase I have not been previously described, two other groups have reported low values for K,,, (l-3 PM) for initial methylation of phosphatidylethanolamine (6, 22). These agree with Km of SAM for the formation of phosphatidylN-methylethanolamine. Similarly, a low apparent Km for SAM was reported for methyltransferase I in human spermatozoa (23), plasma .membrane of human placental villus (24), rat brain synaptosomes (12), and adrenal microsomes (9). The conditions (pH, Km for SAM) for the formation of phosphatidyl-N,N-dimethylethanolamine and phosphatidylcholine

are the same. Therefore, the formation of these two phospholipids may be catalyzed by the same enzyme, methyltransferase II. The possibility that these two methylated phospholipids are formed by two different enzymes has not been definitely excluded by the present studies. The rat liver microsomal fraction in isotonic medium consists largely of sealed fragments from the endoplasmic reticulum (21, 25). The exterior of the microsomal vesicles corresponds to the cytoplasmic surface of the endoplasmic reticulum, whereas the luminal surface becomes the interior of the microsomes. In this configuration, phosphatidylethanolamine (96%) faces the outside surface of the microsomes, and phosphatidylcholine (55% of total) is located on the inside surface (21). Incubation of microsomes with trypsin for 1 min decreased methylation of phosphatidylethanolamine by 95% (25). There is also some indirect evidence that the enzyme which catalyzes formation of phosphatidyl-N-methylethanolamine from phosphatidylethanolamine is assymmetrically distributed in microsomes (26). These observations suggest that both phosphatidylethanolamine and methyltransferase I are asymmetrically distributed and possibly localized toward the outside surface of microsomes. Although high specific activities of the two methyltransferases are present in mammalian liver microsomes, the occurrence of these enzymes is not restricted to liver. Their presence was demonstrated in microsomes prepared from bovine adrenal medulla (9), rat aorta and islet cells of rat pancreas (27), rat brain synaptosomes (12), human placental microvilli (24), and spermatozoa from several species (23). The wide occurrence of methyltransferase in several types of membranes indicates an important regulatory role for these enzymes in the cell membrane function. REFERENCES 1. BREMER, J., FIGARD, P. H., AND GREENBERG, D. M. (1960) Biochim. Biophys. Acta 43,477. 2. BREMER, J., AND GREENBERG, D. M. (1961) Biochim. Biophys. Acta 46, 205.

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3. GIBSON, K. D., WILSON, J. D., AND UDENFRIEND, S. (1961) J. Biol. Chem. 236, 673. 4. REHBINDEK, R. AND GREENBERG, D. M. (1965) Arch. Biochem Biophys, 109,110. 5. TANAKA, Y., DOI, O., AND AKAMATSU, Y. (1979) Biochem. Biophys. Res. Commun 87.1109. 6. SCHNEIDER, W. J., AND VANCE, D. E. (1979) J.

A’-METHYLTRANSFERASE

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Biol. Ch.em. 254, 3886. 7. CROCKEN, B. J., AND NYC, J. F. (1964) J. BioZ.

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Chem. 239.1727. 8. SCARBOROIJGH, G. A., AND NYC, J. F. (1967) J.

Biol. Ch,em. 242, 238. 9. HIRATA, F., VIVEROS, 0. H., DILIBERTO, E. J., JR., AND AXELROD, J. (1978) Proc. Nat. Acad Sci.

USA 75,1718. 10. HIRATA, F., AND AXELROD, J. (1978) Proc. Nat. Acad Sci. USA 75, 2348. 11. HIRATA, F., STRIITMATTER, W. J., AND AXELROD, J. (1979) Proc. Nat. Acad. Sci. USA 76,368. 12. CREWS, F. T., HIRATA, F., AND AXELROD, J. (1979) Fed. Proc. 38, 517. 13. MOORE, L., CHEN, T., KNAPP, H. R., JR., ANDLANDON, E. J. (1975) J. Biol. Chem. 250,4562. 14. MOORE, L., DAVENPORT, G. R., AND LANDON, E. J. (1976) J. Biol. Ch.tm. 251.1197. 15. MOORE, L. (1980) Biochem. Pharmacol. 29,2505. 16. WHARTON, D. C., AND TZAGOMFF, A. (1967) in

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Methods in Enzymology (Estabrook, R. W., and Pullman, M. E., eds.), Vol. 10, p. 245, Academic Press, New York. PENNINGTON, R. J., (1961) Biochem. J. 80,649. ARONSON, N. N., ANDTOUSTER, 0. (1974) in Methods in Enzymology (Fleischer, S., and Packer, L., eds), Vol. 31, p. 90, Academic Press, New York. SASTRY, B. V. R., STATHAM, C. N., ANDAXELROD, J. (1979) Pharmacologist 21, 260. LESTER, R. L., AND WHITE, D. C. (1967) J. Lipid

Res. 8, 565. 21. DEPIERRE, J. W., AND DALLNER, G. (1975) Biochim Biophys. Acta 415.411. 22. CASTANO, J. G., ALEMANY, S., NIETO, A., AND MATO, J. M. (1980) .I. Biol. Chem. 255,9041. 23. JANSON, V. E., AND SASTRY, B. V. R. (1981) Fed. Proc. 40, 717. 24. BARNWELL, S. N., AND SASTRY, B. V. R. (1981)

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ILondon) 270.268. 26. HIGGINS, J. A. (1981) B&him. Biophys. Acta 640, 1. 27. LANDON, E. J., SLONIM, A., AND SASTRY, B. V. R. (1980) Fed. Prod. 39, 636.