Role of sulfhydryl groups in phospholipid methylation reactions of cardiac sarcolemma

Molecular and Cellular Biochemistry 103: 85--96, 1991. © 1991 Kluwer Academic Publishers. Printed in the Netherlands.

Role of sulfhydryl groups in phospholipid methylation reactions of cardiac sarcolemma

Roland Vetter, Jian Dai, Nasrin Mesaeli, Vincenzo Panagia and Naranjan S. Dhalla Division of Cardiovascular Sciences St. Boniface General Hospital Research Centre and Departments of Anatomy and Physiology, Faculty of Medicine, University of Manitoba Winnipeg, Canada R2H 2A6

Received 27 September 1990; accepted 15 October 1990

Key words: rat heart, sarcolemma, S-adenosyl-L-methionine, phospholipid N-methylation, neutral lipid methylation, sulfhydryl groups

Abstract

The effect of reagents that modify sulfur-containing amino acid residues in the phosphatidylethanolamine N-methyltransferase was studied in the isolated rat cardiac sarcolemma by employing S-adenosyl-L-[methyl- 3H]methionine as a methyl donor. Dithiothreitol protected the sulfhydryl groups in the membrane and caused a concentration- and time-dependent increase of phospholipid N-methylation at three different catalytic sites. This stimulation was highest (9-fold) in the presence of 1 mM MgC12 and 0.1/.tM S-adenosyl- L-[methyl-3H]methionine at pH 8.0 (catalytic site I), and was associated with an enhancement of Vmax without changes in K~ for the methyl donor. Thiol glutathione was less stimulatory than dithiothreitol; glutathione disulfide inhibited the phosphatidylethanolamine N-methylation by 50%. The alkylating reagents, N-ethylmaleimide and methylmethanethiosulfonate, inhibited the N-methylation with IC50 of 6.9 and 14.1/zM, respectively; this inhibition was prevented by 1 mM dithiothreitol. These results indicate a critical role of sulfhydryl groups for the activity of the cardiac sarcolemmal phosphatidylethanolamine N-methyltransferase and suggest that this enzyme system in cardiac sarcolemma may be controlled by the glutathione/glutathione disulfide redox state in the cell.

Abbreviations: A d o M e t - S-Adenosyl-L-methionine, A d o H e y - S-adenosyl-L-homocysteine, DTNB - 5,5'- dithiobis (2-nitrobenzoate), NEM - N-ethylmaleimide, MMTS - methylmethanethiosulfonate, Dq"F - dithiothreitol, EDTA-Ethylenediaminetetraacetic acid, GSH-glutathione, GSSG-glutathione disulfide, PE - phosphatidylethanolamine, PMME - phosphatidyl-N-monomethylethamolamine, PDME - phosphatidyl-N-dimethylethanolamine, PC - phosphatidylcholine, NPL - nonpolar lipids, SL - sarcolemma

Introduction

The phospholipid composition of the membrane plays an important role in a variety of cellular functions and is controlled by an integrated network of enzymatic reactions for phospholipid synthesis and degradation. One of the reactions for a discrete phospholipid molecular remodelling is the biosyn-

thesis of phoshatidylcholine (PC) by three succes- sive N-methylations of phosphatidylethanolamine (PE) in which S-adenosyl-L-methionine (AdoMet) acts as a physiological methyl group donor. Inter- mediate products in this reaction are phosphatidyl- N-monomethylethanolamine (PMME) and phosphatidyl-N,N-dimethylethanolamine (PDME) [1, 2]. In cardiac sarcolemma (SL) three catalytic sites

86

(I, II, III) of intrinsic PE N-methyltransferase (EC 2.1.1.17) have been identified which are responsible for the stepwise conversion of a limited pool of PE into PC [3, 4]. Since N-methylation of the in- tramembranal PE has been shown to influence Ca 2+ transport across cardiac SL [5, 6] and membrane fluidity [7, 8], it is possible that changes in the N-methylation activities in cardiac SL may contribute to the regulation of Ca 2+ in the myocardium. Recently, we reported changes in the SL PE N- methylation activity in cardiac hypertrophy [9] as well as in diabetic cardiomyopathy [10] and catecholamine-induced cardiomyopathy [11]. How- ever, the underlying mechanisms for these changes are far from clear and very little is known about the cellular factors which can modulate PE N-methylation in cardiac membranes. There is general con- sent regarding the control of the transmethylating enzyme(s) by the cellular ratio of AdoMet/ AdoHcy (S-adenosylhomocysteine) as well as by the levels of phospholipid substrates and products [2, 4]. Recent studies of PE N-methylation in cardiac and liver membranes indicated no regulatory role for a cAMP-dependent reversible protein phosphorylation [12, 13].

Sulfhydryl groups are generally known to be of crucial importance for the catalytic activity of various enzymes [14, 15]. In fact, plasma membrane enzymes and other proteins like Na ÷, K+-ATPase [16], Ca2+-stimulated ATPase [17, 18] and the cardiac Na+-Ca 2+ exchanger [19, 20] are sulfhydryl- sensitive, and reagents such as N-ethylmaleimide (NEM), 5,5'-dithiobis(2-nitrobenzoate) (DTNB), and p-chloromercuriphenylsulfonic acid are inhibitory. However, the role of sulfhydryl groups for the function of the intrinsic phospholipid N-methyltransferase system in cardiac SL is presently unknown. Thus, the purpose of the present investigation was to examine the involvement of protein thiols in the enzymatic PE N-methylation of the isolated cardiac SL vesicles by using several sulfhydryl group-specific modifying reagents. We report that the chemical modification of sulfhydryl groups in cysteine residues of membrane proteins could significantly alter the cardiac SL PE N-methylation pathway for membrane PC biosynthesis.

Materials and methods

Materials

Adenosyl-L-[methyl-3H]methionine (spec. act. 72.4 Ci/mmol) was purchased from Amersham, Oakville, Ontario, Canada. (-)[ring, propyl- ~H(N)]dihydroalprenolol hydrochloride (96.8 Ci/ mmol) and (+)[7-methoxy-3H]prazosin (83Ci/ mmol) were from New England Nuclear, Missis- sauga, Canada. Phosphatidyl-N-monomethylethanolamine and phosphatidyl-N-dimethylethanolamine were from Calbiochem-Behring, San Diego, USA. Phosphatidylcholine, phosphatidylethanolamine, dithiothreitol, glutathione, glutha- thione disulfide, N-ethylmaleimide and methylmethanethiosulfonate were purchased from Sigma Chemical Co., St. Louis, USA. S-adenosyl-L-methionine was obtained from RBI, Natick, USA. Silica gel 60 F-254 thin-layer chromatography plates were purchased from E. Merck, Darmstadt, FRG. All other reagents were of analytical grade.

Experimental animals

Adult male Sprague-Dawley rats weighing 200- 250 g were used in this study. After decapitation of the animal, the heart was rapidly excised, im- mersed in ice-cold 250 mM sucrose, 10 mM histidine, pH 7.4; the atria and any large vessels were carefully trimmed and the ventricular muscle was processed for the isolation of SL-enriched membranes.

Isolation of sarcolemmal membranes

Sarcolemma-enriched membranes were prepared from the ventricular muscle by the procedure of Pitts [21]. The final pellet was resuspended in 250 mM sucrose, 10 mM histidine, pH 7.4. Samples were frozen and stored in liquid nitrogen until use. In these preparations the unmasked activity of the ouabain-sensitive K+-p-nitrophenylphosphatase, a well-known marker for SL, was 1.82 + 0.12 ~mol/

mg protein/h (n = 10). In agreement with earlier observations for the ouabain-sensitive Na +, K ÷- ATPase activity [6, 9], a 13-fold purification over homogenates was seen in these preparations. Puri- fication of plasma membranes was also indicated by detection of 0.35 + 0.05 (n = 18) and 0.21 + 0.03 pmol/mg protein (n + 18) maximum specific binding for the [3H]-labelled et- and ~-adrenergic antagonists, prazosin and dihydroalprenolol, respectively. Contamination of this preparation by mitochondria and sarcoplasmic reticular membranes did not exceed 5% as reported elsewhere [6, 9].

Membrane lipid methylation

Methyltransferase activities were assayed by measurement of the [3H]-methyl groups incorporation into membrane phospholipids and nonpolar membrane lipids (NPL) in the presence of S-adenosyl- L-[methyl-3H]methionine ([3H]AdoMet) according to the method described earlier [3, 9]. Different assay conditions were employed which have been reported to be optimal for three catalytic sites in the N-methylation reaction as well as typical for the formation of PMME, PDME and PC as the major N-methylated lipid products, respectively [3, 4, 9]. Unless otherwise indicated, the incubation of the membranes (0.3 mg protein) was carried out in the presence of 1 mM MgCI2 and 0.1 ~M [3H]AdoMet 72.4 Ci/mmol) in 50 mM Tris-glycylglycine buffer (pH 8.0) for catalytic site I. The incubation was performed without MgC12 at 10 txM [3H]AdoMet (200tzCi//xmol) in 50raM imidazole buffer (pH 7.0) for catalytic site II and at 150 ~M [3H]AdoMet (200 mCi/txmol) in 50 mM sodium hydroxide-gly- cine buffer (pH 10.0) for catalytic site III. After 10 min preincubation, the reaction was initiated by addition of [3H]AdoMet and terminated 5 to 30 min later (within the linear reaction period) by the addition of 3 ml chloroform/methanol/2N HC1 (6 : 3 : 1 vol/vol/vol). The chloroform phase was washed three times with 2 ml of 0.5 M KC1 in 50% methanol. The detailed procedures for the extraction and thin-layer chromatographic separation of mem-

87

brane lipids as well as quantification of methylated lipid products have been reported elsewhere [3]. Because chemical methylation of membrane lipids has been reported by Bansal & Kanfer [22], control experiments with heat-denaturated SL (5min, 90 ° C) were performed. No methylated products of PE or NPL were detected in these controls.

Treatment of membranes with dithiothreitol

Two different protocols for studying the effect of DTT on lipid methyltransferase activities were used. For the first protocol, the modifying reagent was present in the medium during preincubation period as well as after initiating the methylation reactions by addition of [3H]AdoMet (protocol I). To exclude any possible interaction between the modifier and the methyl group donor in the methylation medium, membranes were first preincubated with the modifier and then used for methylation in a medium which did not contain DTT (protocol II). For this purpose, membranes (2.0 mg protein/ ml) were first incubated with 5 mM DTT in 250 mM sucrose, 10raM histidine, pH 7.4 for 40rain at 37 ° C. Samples were then diluted 2-fold in ice-cold sucrose-histidine buffer and centrifuged for 20 rain at 54,000 g using a Beckman Type TLA 100.2 rotor. The pellet was resuspended in 1.0ml ice-cold sucrose-histidine buffer and centrifuged as before. The final pellet was resuspended in 250 mM sucrose, 10mM histidine, pH 7.4. Samples of this suspension were used immediately for determination of the phospholipid N-methyltransferase activity and/or for quantification of free membrane protein thiols; the protein concentration of the membrane suspension was determined afterwards. Control membranes that were incubated in the absence of the modifier were processed in the same way.

Determination of sulfhydryl group content

Sulflwdryl content of SL membranes pretreated according to protocol II with and without DTT was

88

determined with DTNB according to the procedure described by Boyne & Ellman [23]. The assay medium contained 250 mM sucrose, 10 mM histidine, pH 7.4, l mM ethylenediaminetetraacetic acid (EDTA) and 0.2 mM DTNB. For the determination of free sulfhydryl groups, 200/zg membrane protein was incubated for 3 rain in the above medium and the absorbance at 412 nM was measured. Calculation of the sulfhydryl group content was based on a molar extinction coefficient of 13,600 M/ cm at 412 nM for the thiophenol reaction product.

Miscellaneous procedures

The ouabain-sensitive K+-p-nitrophenylphospha - tase activity was determined in the presence of 1 mg alamethicin/mg membrane protein as described elsewhere [24]. The equilibrium binding experiments with (+)[7-methoxy-3H]prazosin and 1- [ring, propyl-3H(N)]-dihydroalprenolol hydrochloride were performed in duplicates according to Mukherjee et al. [25]. Membranes (20 to 50gg) were incubated in 0.5 ml binding medium containing 50mM Tris-HCl buffer, pH 7.4 and 10mM MgC12 with 0.02 to 0.47 nM (+)[7- methoxy-3H]prazosin and 0.04 to 2.27 nM 1-[ring, propyl-3H(N)]-dihydroalprenolol hydrochloride for 45 rain at 25 ° C. Nonspecific binding of the labelled a- and [3-antagonist was defined with 10/zM nonlabelled (+)phentolamine and (-)proprano- lol, respectively. Binding was terminated by rapid filtration through Whatman GF/B glass fiber filters using a filtration apparatus (Brandel M-24R). The filters were washed three times with 5 ml of 50 mM Tris-HC1, pH 7.4, 10mM MgClz and the radio- activity retained on the filters was measured by liquid scintillation counting. Membrane proteins were determined according to Lowry et al. [26], using serum albumin as a standard.

Statistics

Statistical analysis was done by the Student's t-test for unpaired observations and p < 0.05 was consid-

ered statistically significant. If not otherwise indicated, data are expressed as mean _ S.E.M.

Results

Modulatory influence of D TT on phospholopid N-methylation

When SL vesicles were exposed to DTT the rate of [3H]methyl group incorporation into total membrane N-methylated phospholipids (PMME+ PDME + PC) was significantly increased (Table 1). Stimulation was observed at three different concentrations of the [3H]methyl group donor ([3H]AdoMet) under assay conditions for catalytic sites I, II and II which, as described previously [3, 4, 9], allow the predominant formation of intrasar- colemmal PMME, PDME and PC, respectively. The synthesis of N-methylated phospholipids was stimulated about 9-fold at site I, which is the rate- limiting step of the phospholipid methylation pro- cess [2]; stimulation by DTT was considerably less under site II and III conditions. Table 1 also shows that the N-methylation of SL phospholipids at all three catalytic sites was accompanied by an addi- tional incorporation of [3H]methyl groups into a fraction of nonpolar lipids (NPL) which migrated as a radioactive peak with the solvent front [13, 27, 28]. However, DTT inhibited the [3H]methyl group incorporation into this membrane lipid fraction under optimal assay conditions for PE N-methyltransferase site I. This would indicate an opposite modulatory influence of DTT on two intrinsic membrane activities responsible for phospholipid (PL) methylation and NPL methylation. On the other hand, DTT caused about 2-fold stimulation of NPL methylation when experiments were performed under optimal conditions for site II and III (Ta- ble 1).

Due to the striking stimulatory effect of DTT on phospholipid N-methylation at the rate-limiting catalytic site I, as well as to its opposite inhibitory effect on the NPL methylation, we decided to study the effect of DTT at this site in more detail. Table 2 shows that both the stimulatory effect of DTT on

the N-methylation of PE and the inhibitory effect of the modifier on methyl group incorporation into NPL were dependent on its concentration. In the absence of DT-f, the rate of methyl group incorporation into NPL was approximately 10-fold higher than the PL N-methylation rate, and the phospholipid-to-nonpolar lipid ratio (PL/NPL) of formed methylated products was as low as 0.11. There occurred a steady increase of the PL/NPL ratio with increasing D T r concentrations. Maximum values for stimulation of N-methylation and for the depression of the synthesis of methylated NPL products occurred at approximately 5 mM DTI'; at this modifier concentration the PL/NPL ratio was found to exceed by 20-fold the control value in the absence of DTT. Determination of the thiol content in SL proteins revealed that the membrane sulfhydryl content was 27.1+ 1.63 (n= 3) and 39.2 + 3.32 (n = 3) nmol/mg membrane protein (p < 0.05) in the absence and presence of 5 mM DTT during a 40 min incubation period, respectively. Apparently, the DTT-induced protection of the reduced state of sulfur-containing residues in

Table 1_ Total N-methylated and nonpolar lipid products formed by rat heart sarcolemma at methyltransferase sites I, II, and III in the absence and presence of dithiothreitol

Catalytic site

I II III

- DTT + DTF

- DTF + DTT

N-methylated phospholipids 0.212+ 0.019 2.136+ 0.051 82.98+ 7.22 1.888+ 0.228* 5.997+ 0.616" 101.73+ 3.63*

nonpolar lipids 1.906 + 0.354 1.621 + 0.085 49.08 + 2.86 0.705 + 0.087* 3_362+ 0.309* 78.98+ 15.20"

Methylation of cardiac sarcolemmal membranes was carried out with and without 5 mM DTT as described in Materials and methods. The [3H]AdoMet concentrations for catalytic sites I, II, and III were 0.1, 10 and 150 tzM, respectively. Phospholipids were extracted, fractionated and quantified as indicated in Ma- terials and methods. Values are means + S.E.M. for 3 to 4 different experiments and are expressed as pmol [3H]methyl groups incorporated/rag/30 min. DDT = dithiothreitol_ * Significantly different (p < 0.05) from values in the absence 6f DTI'.

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SL proteins and/or in the PE N-transmethylating enzyme was responsible for the observed differences.

Influence of DTT on the formation of the single N-methylated phospholipids

Figure 1 shows that various concentrations of DTF changed the rate of PMME, PDME and PC synthesis in a different manner. The amount of [3H- methyl] group incorporation into PMME increased considerably at low DTT concentrations, being maximum at 0.25 mM DTT; thereafter, at higher DTT concentrations, it declined to a steady-state

Table 2. Effect of dithiothreitol on the AdoMet-induced [3H]methyl group incorporation into N-methylated phospholipids and nonpolar lipids of rat cardiac sarcolemma

Dq'T (mM) [3H]methyl group incorporation Ratio (pmol/mg protein/30 rain) A/B

N-methylated nonpolar lipids phospholipids (A) (B)

0 0_230+ 0.026 2.167+ 0.252 0.11 (100%) (100%)

0.05 0.812 + 0.117" 1.223 + 0.305* 0_67 (358%) (56%)

0.1 1.341 + 0.253* 1.462 + 0.345* 0.92 (583%) (68%)

0.5 1.540+ 0.216" 1.566+ 0.145" 0.98 (670%) (72%)

1.0 1.572+ 0.162" 1.268+ 0.165" 1.24 (683%) (58%)

3.0 1_944 + 0_364* 0.856 + 0.096* 2.27 (845%) (40%)

5.0 2.118 + 0.294* 0.818 + 0.213" 2.59 (921%) (38%)

10.0 2.259+ 0.063* 0.993+ 0.162" 2.27 (982%) (46%)

Preincubation of membranes with DTT was for 10 mm at 37 ° C. The methylation reactions were initiated in the same assay by addition of 0.1/zM [3H]AdoMet. Reactions were terminated after 30 min and the incorporation of [3H]methyl groups was determined as indicated in Materials and methods. Values are means _+ S.E.M. of 4 different experiments. * Significantly different (p < 0.05) when compared to values without DTT.

90

A ¢-

E 0 ¢0 r -

2

E 0 .

.o

0

0 U C

..C

E

1.0

0.5

0 I I I

o 2

dithiothreitol (raM)

Fig. 1. [3H]Methyl group incorporation into individual N-methylated phospholipids of cardiac sarcolemma at varying concentrations of dithlothreitol. Preincubation of membranes with DTT was for 10 min at 37°C in the assay medium for site I methyltranferase. Methylation was performed in the same assay medium by adding 0.1/zM [~H]AdoMet. Reactions were terminated after 30 min and the incorporation of [3H]methyl groups was determined as indicated in Materials and methods. Values for the N-methylated phospholipids, PMME (O), PDME (A) and PC (11) are means + S.E.M. for 3 to 4 experiments with different membrane preparations.

value exceeding 4-fold that of control (Fig. 1). In contrast, D T T increased the rate of P D M E and PC formation steadily in a concentration dependent manner. The increase in P D M E and PC formation was a saturable function of the D T T concentration. Both the reduced D T T stimulation of the PMME formation at modifier concentrations above 0.25 mM and the steady increase in the P D M E and PC formation resulted in striking changes with respect to the quantitative relation- ship between the single N-methylated phospholipid products. At D T T concentrations up to 0.5 mM the predominant [3H-methyl] incorporation was found in PMME whereas at higher concentrations PC was the predominant [3H-methyl] labelled N- methylated product; the ratio of methyl incorporation into PMME, PDME and PC was approximately 1: 1 : 2 at saturating D T T concentrations. It indicates that the redox state of sulfur-containing residues in membrane proteins and/or the transmethylating enzyme(s) affected the single N-methylation reactions with PE, PMME and P D M E as substrates differently.

Kinetics of N-methylation in the absence and presence of D T T

To further characterize the DTT-induced stimulation of PE N-methylation, we studied the kinetics of [3H]methyl group incorporation into N-methylated SL phospholipids in the absence and presence of D T T using different concentrations of [3H]Ado- Met as radiolabelled methyl donor (Table 3). At catalytic site I, the double reciprocal plots obtained in the concentration range 0.01-0.33/~M [3H]Ado- Met were linear in the absence and presence of D T T and indicate that the apparant affinities for the methyl donor AdoMet were unaltered by DTT. However , the maximum rate of incorporation (Vm~x) was higher in the presence of the modifier. Similar results were obtained in kinetic experiments performed under optimal conditions for catalytic site II and III (data not shown).

Effect of glutathione (GSH) and glutathione disulfide (GSSG) on the N-methyltransferase activity

Dithiothrei to l [29], which is capable of maintaining monoth io ls comple te ly in the reduced state be-

cause of its low redox potential , is a synthetic com-

p o u n d ( threo isomer of 2 ,3-dihydroxy-l ,4-di thiol-

butane) . The most prevalent cellular thiol is the

low molecular-weight pept ide glutathione (GSH) .

It acts as a reducing agent and keeps prote in thiol groups in a reduced state [30]. To obtain fur ther

informat ion regarding the role of the redox state of

prote in sulfur-containing residues on the SL P E

N-methyl t ransferase , we examined the effects of

G S H and its oxidized fo rm G S S G on the [3H]Ado-

Met - induced [3H]-methyl g roup incorpora t ion into

m e m b r a n e P E at catalytic site I. Addi t ion of G S H

into the methyla t ion react ion med ium caused a

3-fold s t imulat ion of N-methy la t ion (Table 4). Al-

though this s t imulat ion was less than that induced by equimolar concent ra t ions of D T T , it indicates

clearly that the physiological reducing thiol com- p o u n d G S H is capable of substituting D T T as an

act ivator of the t ransmethyla t ing enzyme. The de-

pendence of the N-methyla t ing enzyme activity on the redox state of pro te in sulfur containing residues

Table 3. Kinetic parameters of sarcolemmal site I phosphatidylethanolamine N-methylation in the absence or presence of dithiothreitol

Experimental Km (/~M) Vma. conditions (pmol/mg/10 min)

Control 0.35 0.255 5 mM DT1 ~ 0.25 1.005

Values of apparent Km (AdoMet) and Vma x (incorporation of [3H]methyl groups into total N-methylated phospholipids, i.e. PMME + PDME + PC) were obtained from Lineweaver-Burk plots and are representative of a typical experiment done in triplicate (variation < 8%). Membranes (0.3 mg) were preincubated at 37°C for 10 min in a medium containing 50 mM Tris- glycylglycine, pH 8.0, lmM MgC1 z (site I conditions) with or without 5 mM DTT. Methylation reaction was initiated in the same assay mixture by adding different amounts of [3H]methyl AdoMet (0.01-0.33/xM) and terminated 10 min later. For further details see Materials and methods.

91

is fur ther suppor ted by the finding that G S S G was

inhibitory (Table 4).

N-Methyltransferase activity of SL preactivated with DTT

In the exper iment described above, D T T was pre-

sent in the med ium before and during the methyla-

t ion reactions. This experimental approach en-

sured both the res torat ion of spontaneous ly oxi-

dized prote in thiol groups before initiating the PE

N-methyla t ion and the prevent ion of any fur ther

oxidat ion during the methyla t ion reaction. To ex-

amine whether a reduct ion of oxidized sulfur-con-

taining residues of SL proteins before per forming

the A d o M e t - i n d u c e d N-methyla t ion react ion was

sufficient to enhance the cardiac SL N-methyla t ing

activity, we measured the [3H]methyl incorpora-

t ion into PE in DTT-pre t r ea t ed membranes using a

methyla t ion assay which did not contain DTT. The

results in Table 5 show that the phosphol ip id N-

methyl t ransferase activity of DTT-p re t r ea t ed

membranes was approximate ly 3-fold higher than that of the un t rea ted membranes . The lower de-

gree of st imulation under these assay condit ions

Table 4. Comparison of the effect of dithiothreitol, reduced glutathione and glutathione disulfide on the [3H]methyl incorporation into N-methylated phospholipids in rat cardiac sarcolemma

Experimental conditions

[3H]methyl group incorporation Change (pmol/mg protein/30 min) (%)

no addition 0.199 + 0.025 5 mM DTI" 3.215 + 0.357* + 1516 5mM GSH 0.598 + 0.041" + 200 5 mM GSSG 0.108 + 0.009* - 46

Preincubation of membranes with and without the respective modifier was for 10 min at 37 ° C. Methylation was performed in the same assay medium (site I conditions) in the presence of 0.1/xM [~H]AdoMet. Reactions were terminated after 30min and the incorporation of [3H]methyl groups into N-methylated phospholipids (sum of [3H-methyl] labelled PMME, PDME and PC) was determined as indicated in Materials and methods. Values are means + S.E.M. for 6 to 8 determinations with three different membrane preparations. * Significantly different (p < 0.05) from control values.

92

i I00 o

4'*

o u

o

so t . "

o_

o e~

U

i -

E

0 m - f |

6 5

0,--.0 NEM

H MMTS

4 3

-log [modifier] (M)

Fig. 2. Inhibition of intrinsic N-methylation of cardiac sarcolem- real phospholipids by varying concentrations of the thiol reagents N-ethylmaleimide and methylmethanethiosulfonate. The methyltransferase system was first activated by preincubation of membranes at 25°C for 30min in 10mM histidine, pH 7.4,250 mM sucrose and 5 mM DTT. Membranes were washed free of DT]? and subsequently preincubated with 0 to 1raM NEM (O) or MMTS (0) for 10 rain at 37 ° C in standard site I methylation medium. Methylation was initiated in the same mixture by addition of 0.1/~M [3H]AdoMet and terminated after 30 rain_ The [3H]methyl group incorporation in the absence of NEM and MMTS was 0_684 + 0.091 pmol/mg/30min. Each point represents the average of triplicate determinations with an experimental error of less than 8%. N-methylated lipids repre- sent the sum of PMME, PDME and PC. For further details see Materials and methods.

could be attributed to a reoxidation of reduced thiols during the PE-N-methyltransferase reaction and indicates a high sulfhydryl-sensitivity of the N-methylating SL enzyme(s). DTT-pretreated membranes exhibited the typical sensitivity of the N-methylating enzyme system to exogenous N-methylated phospholipid intermediate substrates [4]. Table 5 shows that exogenously added PMME, the substrate for the second PE N-methylation reaction, significantly stimulated the [3H]methyl group

incorporation into the N-methylated phospholipids. This shows that the conversion of PE to PMME in DTT-pretreated membranes remained the rate limiting step of the PC synthesis by the N-methylating SL enzyme [2, 4, 31]. Both the basal and the DTT-stimulated part of the N-methylating SL activity were completely abolished in heat denaturated membranes (Table 5).

Effects of sulfhydryl reagents on the phospholipid N-methylation

In order to further support the conclusion that protein thiols play an important role in the mani- festation of the N-methylating activity of the car-

Table 5. Effect of dithiothreitol pretreatment of cardiac sarcolemma on the intrinsic phospholipid N-methyltransferase activity assayed in the absence or presence of the modifier

Assay DTT pH]methyl group Change conditions pretreatment incorporation (%)

(pmol/mg protein/30 min)

SL no 0.195 + 0.018 - (n : 3)

SL yes 0.704 + 0.016" + 261 (n = 6)

SL + yes 0.909 + 0.021" + 366 50/.~g PMME (n = 3) SL yes 0.010 + 0.001" - 95 (denaturated) (n = 3)

Membranes were incubated according to Protocol II for 40 min in 10raM histidine, pH 7.4, 250mM sucrose with and without 5 mM DTT and centrifuged afterwards for 20 min at 54 000 x g using a Beckman Type TLA 100.2 rotor. Pellets were resuspended in DTT-free histidine-sucrose medium and recentri- fuged as before. The final pellet was resuspended in the same medium at a protein concentration of 3 mg/ml. Aliquots of this suspension were either directly used (SL) for determination of intrinsic phospholipid N-methyltransferase activity or assayed after heating for 5 rain at 90°C (denatured SL). The reaction was performed under standard methyltransferase site I assay conditions with 0.1/~M [3H]AdoMet in the absence of DqT. PMME was added from a suspension prepared by sonicating 0.2mg/ml H20 for 10rain in a Branson 2000 sonicator. For further details see Materials and methods. Each value is the mean + S.E.M. for n determinations. * Significantly different (p < 0.05) from control values.

diac SL membrane, the effects of N-ethylmaleimide (NEM) and methyl methanethiosulfonate (MMTS), which differ significantly in their structure and mode of modification of the sulfhydryl groups [32, 33] were investigated on the incorporation of [3H]methyl groups. Fig. 2 shows that the N-methylating activity of DTT-preactivated membranes was inhibited by both alkylating reagents in a concentration-dependent manner. Half-maximum inhibition occurred at approximately 10-fold higher concentrations of the alkylating reagents. The NEM inhibition of N-methylation was irreversible, but could be prevented by simultaneous addition of 1 mM DTT. By contrast, the inhibitory effect of MMTS was readily reversible by addition of DTT after treatment with this inhibitor (data not shown).

Discussion

Conversion of PE to PC in the membrane through AdoMet-dependent N-methylation is catalyzed by the intrinsic PE-N-methyltransferase(s). Previous findings have indicated the PE N-methylation may be involved in modifying cardiac Ca z÷ transport systems such as SL Na+-Ca 2÷ exchange [6] and Ca/+-pump [5] and thereby in modulating the contractile performance of the heart [34]. This study shows that treatment of the cardiac SL with DTT, which is capable of maintaining protein sulfhydryl groups in the reduced state [29], increased significantly the intrinsic PE N-methyltransferase activity. The stimulating effect was detected at catalytic site I, II and III, but was highest at site I, the rate-limiting reaction of the transmethylation pro- cess [2]. The data also show that DTT affected the AdoMet-induced enzymatic methylation of nonpolar lipids, which has been reported previously to occur in membranes from heart [9, 13] and other tissues [27, 28]. The nature of the methylated nonpolar SL lipids is not defined yet; however, enzymatic methylation of fatty acids has been described for various tissues and the formed fatty acid methyl esters have been identified as a major com- ponent of the membrane NPL fraction [27]. Al- though the role of NPL methylation for the mem-

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brane function is presently unknown, it should be mentioned that DTT induced (at catalytic site I) a large change in the methylated phospholipid-to- methylated nonpolar lipid ratio. Zatz et al. [27] have reported considerable variations of this ratio in microsomes from different types of tissues. Al- though a number of factors may be responsible for this experimental finding, we speculate that differences in the reduced state of membrane thiols might contribute to it. Moreover, this finding indicates that the inclusion of DTT into the methylation medium can improve the assay conditions for the study of PE N-methylation at site I due to both the stimulation of the PE N-methylating enzyme and the depression of the unwanted NPL methylation.

The effect of DTI" seems to be due to the protection of the membrane sulfhydryl groups and was not due to an artificial interference in the reaction media. This conclusion is supported by the obser- vation that membranes pretreated with Dq-T, centrifuged washed, resuspended and assayed for N- methylation in media without DTT exhibited a significantly higher incorporation of [3H]methyl groups into N-methylated phospholipids. Al- though Bansal & Kanfer [22] have reported a chemical methylation of PE, it should be mentioned that we were unable to detect any lipid methylation in heat-denatured SL preparations which were pretreated either in the absence or in the presence of 5 mM DTT. This may suggest that both the basal and DTT-stimulated part of the observed PE N-methyltransferase activity cannot be attributed to any nonenzymatic type of transmethylation. In the presence of transition metal ions like Fe 3+ and oxygen, DTT can spontaneously autoxidize to generate reactive reduced oxygen species such 02- and -OH [35-38]; these oxygen free radicals are strong oxidants and thus promote lipid peroxidation. Since transitional metal ions in commercial preparations of reagents cannot be ruled out, it could be possible that the observed stimulatory effects of DTT were related to the formation of lipid peroxides or their products which may stimulate the PE N-methyltransferase activity. However, this possibility appears unlikely since data reported recently by Kaneko et al. [39] in-

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dicate an inhibitory effect of oxygen free radicals on the PE N-methyltransferase activity in cardiac membranes.

The analysis of the stimulatory effect of DTT on the [3H]methyl incorporation into the single intrinsic phospholipid substrates PE, PMME and PDME revealed striking differences among them. From the results presented in this study, it is obvi- ous that the rate-limiting first methylation step which results in the conversion of PE to PMME [2, 4, 31] is highly sensitive to changes in the redox state of protein thiols. Such a view is further supported if the amounts of formed PMME, PDME and PC are estimated by taking into consideration not only the [3H]methyl incorporation into the individual phospholipid, but also the number of labelled methyl groups which can be incorporated per mole of the respective phospholipid molecule. These stoichiometric considerations reveal that even in the presence of DTT, PMME remains the predominantly formed N-methylated phospholipid at site I. This confirms previous data [3, 4] in- dicating that the predominantly formed phospholipid at site I is PMME. It should also be noted that the presence of DTT in the medium increased the Vma, of the PE N-methylation reactions but did not alter the K m for the methyl donor AdoMet. This indicates that the increase in the transmethylating activity induced by the thiol reagent was linked to the sulfhydryl groups of membrane-associated enzyme molecules which are essential for their catalytic activity. However, the unequivocal proof that the catalytic sites in PE N-methyltransferase contain critical sulfhydryl groups should be performed with the purified enzyme in the absence of membrane protein and lipid components that might be involved in the DTT-caused stimulatory phenom- enon. Although the cardiac PE N-methyltranfe- ruse has not been purified at present, it shares common properties with the purified liver enzyme [40]. In fact, in an unrelated study Ridgway & Vance [12] have reported that the purified liver PE N-methyltransferase was completely inactive in the absence of DTT, and full activity was restored when 5 mM DTT was included in the methylation assay. Apparently, the SH-sensitivity is a general

property of PE N-methylating enzyme from different mammalian tissues.

The dependence of the PE N-methyltransferase on free sulfhydryl groups was further supported by the results of the experiments in which the sulfhydryl-modifying reagents, NEM and MMTS, inhibited this enzyme activity. The inhibition pattern was qualitatively and quantitatively similar irre- spective of the different chemical structure and mode of modification of sulfhydryl groups by these reagents [32, 33, 41]. The inhibitory analysis of the PE N-methylation was performed at pH 8.0 (catalytic site I). The alkaline pH facilitates the formation of the mercaptide anion which participates in the irreversible alkylation of sulfhydryl groups by NEM [23]. It should be noted that NEM can react not only with sulfhydryl groups, but also with the imidazole group of histidine and a-amino groups [41]. However, the latter modifications of amino acid residues, if they occur in general, appear to have no functional relevance in our system since the specific sulfhydryl reagent, DTT, was found to protect against NEM inhibition. The relevance of protein thiols and not other amino acid residues for the activity of the PE N-methyltransferase(s) is further supported by the finding of an inhibitory effect of MMTS. This type of chemical modification of proteins occurs via the introduction of me- thanethio groups into sulfhydryl groups and leads to the formation of mixed disulfides on the proteins. In contrast to alkylation by NEM, the mixed disulfide can be easily reduced by thiol reagents such as DTT or mercaptoethanol [32]. In fact, the enzyme activity could be restored by DTT after treatment of membranes with MMTS. A subse- quent reactivation of the enzyme activity by DTT was found to be impossible if inactivation was induced by NEM (data not shown). These data suggest that sulfhydryl groups are intimately involved in the catalytic mechanism of the transmethylating enzyme(s). It also may indicate that the alteration of free sulfhydryl groups, but not the steric effects which are expected to be different for NEM and MMTS, is the reason for the observed enzyme inhibition. The data presented in this study also demonstrate that the thiol GSH activated the SL

PE N-methyltransferase whereas its oxidized form GSSG was inhibitory. The exact role which cyto- solic glutathione redox system may play in the physiological regulation of the SL PE N-methyltransferase system is unknown; however, the de- pletion of intracellular GSH and a decrease of the ratio GSH/GSSG ratio have been reported to occur in pathological situations such as myocardial ischemia-repeffusion injury [42, 43]. Whether this type of oxidative stress may modulate the discrete PC biosynthesis by the PE N-methylation pathway in cardiac membranes is currently under investigation in our laboratory.

Acknowledgements

This work was supported by a grant to V. Panagia from the Heart and Stroke Foundation of Manito- ba. R. Vetter was a Visiting Scientist from the Division of Cellular and Molecular Cardiology, Central Institute of Cardiovascular Research, GDR Academy of Sciences, Berlin-Buch, DDR- 1115. J. Dai was the recipient of a Studentship Award from the Manitoba Health Research Coun- cil. We would like to thank Dr T. Hata for the determination of membrane thiol groups.

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Address for offprints: V. Panagia, Division of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, 351 Tache Avenue, Winnipeg, Manitoba, Canada R2H 2A6

Role of sulfhydryl groups in phospholipid methylation reactions of cardiac sarcolemma

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