Identification and partial characterization of phospholipid methylation in rat small-intestinal brush-border membranes

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BBA 50205

Identificationand partialcharacterizationof phospholipidmethylation in rat srn~l-intestio~ brush-bordermembranes Pradeep K. Dudeja and T~~rnas A. ~rasi~us ~e~r~ent~

~f~ed~~ne, Mickaei Reese ~~~#ai and U&.wrs2y of Chicugo Hospitals and Pritzker School ~f~edj~i~e of the University of Chicago, Chicago, IL (U.S.A.) (Rtived

5 January 1987)

An earlier study (Biochim. Biophys. Acta 46 (1961) 205-216) failed to detect the enzymatic synthesis of ~p~ti~l~line @T) from ~~ti~le~~~ine OpE) via a ~srne~y~a~on Amway in rat so-int~ti~ microsomal membranes. This pathway was therefore assumed to be absent from this organ. Reee&fy, however, in our b&oratory it has been debits that this pathway for the synthesis of ~ti~~rne is present in rat colonic bmh-border and basofateral membranes. ft was therefore of interest to examine whether phospholipid methylation activity was present in rat small-intestinal brush-border membranes. The results of the present experiments demonstrate for the first time that this pathway for the synthesii of ~~ti~l~ine exists in these plasma membranes. Evidence to support the enzymatic nature of this reaction include: (1) loss of activity by heat dena~tion and at 0 o C, (2) si~ifi~t ~~bi~o~ by ~-~~y~-L~~stei~e and (3) saturation kinetics. IIke pr~n~t product of this brush-border membrane ph~p~ipid rne~~~~e~ is phosphati~t-N-Maine. This enzymatic activity has BIIapparent K, for S-adenosyk-methionine of 40 PM, a V,, of 8.4 pmol/mg protein per 5 min, and a pH optimum of 8.0.

Previous studies have shown that phos~hatidylcholine is a major phospholipid of many plasma membranes [l], including rat small-intestinal brush-border membranes 121,Three pathways for the synthesis of PC have been identified and characterized in various cell types. The first, originally described by Lands f3], involves acylation of lysophosphatidylcholine to form PC. The second or so-called ‘de-nova’ pathway [4], involves synthesis of this phospho~pid via the reaction of di-

Correspond~ce: T.A. Bra&us, Department of Medicine and Gastroenterology, University of Chicago, Box 400, 5841 South Maryland Avenue, Chicago, IL 60637, U.S.A. Abbreviations: PC, ph~~ha~dyicho~e; anolamine.

PE, ph~phatidyle~-

acylglycerol with CIX choline. The third, involves enzymatic conversion of PE to PC by methylation reaction(s), utilizing S-adenosyl-r-methionine as the methyl donor 151. The presence of the first two of these synthetic pathways for PC is well established in rat small intestinal epithelial cells [6]. In 1951, Bremer and Greenberg [5], however, failed to detect phospholipid methylation activity in rat small-intestinal microsomes. Based on these observations, a tr~sme~ylation pathway for the synthesis of PC from PE has been assumed to be absent in this organ [5]. AIthougb initially reported in microsomal membranes [.5,7], it has become clear that the transmethylation pathway for the synthesis of PC is also, present in a number of plasma membranes [8-1.21. In this regard, in our laborato~ the

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existence of transmethylation pathways for the synthesis of PC in rat colonic brush-border [ll] and basoiateral [12] membranes has recently been demonstrated. Furthermore, these transmethylation reactions appeared to influence important transport and enzymatic functions in these plasma membranes [12,13]. It was therefore of interest to determine whether phospholipid methylation activity was present in rat sm~l-intes~n~ brushborder membranes. The results given below demonstrate for the first time that this pathway for the synthesis of PC exists in these small-intestinal plasma membranes of the rat. This finding, as well as a partial characterization of this enzymatic activity, serve as the basis for the present report. Albino male rats of the Sherman strain weighing 250-300 g were used throughout these studies. Small-intestinal brush-border membranes were isolated from the proximal half of the small intestine as described previously 1141. Purity of membrane preparation was assessed by estimating the specific activity of the marker enzymes, sucrase and p-nitrophenylphosphatase. Final ratios of the specific activities in the isolated membranes compared to the initial homogenates ranged from 15 to 20. Addition~y, membrane preparations were also examined for contamination by basolateral, mitochondrial and microsomal membranes, as assessed by the marker enzymes (Na+ + Kf)ATPase, succinic dehydrogenase and NADPHcytochrome-c reductase, respectively, and the specific activity ratios of membr~es/homogenates averaged 0.50-1.50 and did not exceed 2.00 [14]. Protein was estimated by the method of Lowry et al. [15], using bovine serum albumin as standard. The methylation of phospho~p~ds was measured by the incorporation of i3H]methyl groups from S-adenosyl+[ methyl- ’ Hlmethionine into phospholipids as described previously [ll]. The reaction mixture (500 ~1) contained 50 mM Trisacetate (pH 8.0), S-adenosyl-L-[ me&y!- 3Hjmethionine [lo0 PM, 4 @Zi] and brush-border membranes (100-200 pg protein). The assay was initiated by the addition of membranes and was incubated at 37” C for 5 min, unless otherwise indicated. The reaction was stopped by the addition of 3 ml chl&oform/methanol/2 N HCl (6 : 3 : 1, v/v), followed by the addition of 2 ml of

0.1 M KC1 in 50% methanol. The mixture was vigorously vortexed twice (120 s each) and centrifuged at 200 x g for 10 min. The aqueous phase was removed and the chloroform phase was washed twice with 2 ml of 0.1 M KC1 in 50% methanol. To identify the products of phospholipid methylation, the chloroform phase was evaporated to dryness under nitrogen and the residue dissolved in 100 ~1 of chloroform/methanol (2 : 1, v/v). The sample was applied on a Silica-Gel G plate and the chromatogram was developed in chloroform/propionic acid/n-propyl alcohol/ water (2 : 2 : 3 : 1, v/v). The phospholipid standards were simultaneously chromatographed and their positions were visuaiized using a saturated solution of iodine in chloroform. The areas corresponding to the standard phospholipids were each scraped individually, extracted with chloroform/methanol (2 : I, v/v), and their radioactivity was measured separately in a liquid scintillation counter as described [ll]. The values obtained for each phospholipid were then corrected as suggested by Audubert and Vance [16]. The chemical identify of the methylated products was further established by two-dimensional c~omato~aphy as described by Katyal and Lombardi 1171, and by hydrolysis of the phos-

I 5.0

8.0

7.0

8.0

9.0

PH

Fig. 1. Effect of pH on 13H]methyl incorporation into total phosphoIipids: Small intestinal brush-border membranes (100-200 pg protein) were incubated at 37 * C with 100 gM S-adenosyl-L-[ me&$- 3Hlmethionine in the presence of various buffers including: 50 mM sodium acetate (pH 5.0 and 6.0), 50 mM T&acetate (pH 7.0-9.0). Values expressed as pmol [3H]methyl groups incorporated into phospholipids per mg protein. Incubation time, 5 min. A representative plot of three separate experiments is shown.

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pholipids and identification of their free bases as described by Schneider and Vance [18]. Additionally, both labelled and unlabelled S-adenosyl+ methionine were routinely purified by ion-exchange chromatography [16]. To determine the optimal pH for phosolipid methylation, rat small-intestinal brush-border membranes were incubated with 100 PM Sadenosyl+[ methyl- 3H]methionine in the presence of various buffers including 50 mM sodium acetate (pH 5.0 and 6.0) and 50 mM T&-acetate (pH 7.0-9.0). The amount of [ 3H]methyl incorporation was highest using 100 PM S-adenosyl-L-methionine at pH 8.0 (Fig. 1). The incorporation of [3H]methyl groups into phospholipids was linear up to 500 pg of membrane protein. The [ 3H]methyl incorporation into phospholipids was not observed at 0 o C or with boiled membrane preparation (data not shown). At 100 PM S-adenosyl+methionine concentration, S-adenosyl+homocysteine (1 mM) inhibited the incorporation of methyl groups into phospholipids by approx. 60% (data not shown). Identification of [ 3H]methylated phospholipids formed after small-intestinal brush-border membranes were incubated with S-adenosyl-L-[ methyl‘H]methionine was determined by thin-layer chromatography. When brush-border membranes were incubated with 100 PM S-adenosyl+methionine (pH 8.0), three major peaks were detected with R, values corresponding to phosphatidyl-N-monomethylethanolamine, phosphatidyl-N, N-dimethylethanolamine and PC. The relative percentages for these three products formed were 61.5 f 2.2, 16.3 + 1.9 and 22.3 & 1.9% (N = 4), respectively. The incorporation of [3H]methyl groups into membrane phospholipids was found to be linear for at least 5 min (data not shown). To analyze the kinetic parameters of phospholipid methylation, brush-border membranes were incubated with various concentrations of Sadenosyl-r_.-methionine (between 5-50 PM) and the extent of [3H]methyl incorporation into phospholipids was measured at pH 8.0. Incorporation of [ “HImethyl incorporation into phospholipids exhibited saturation kinetics. A double-reciprocal plot [19] of the initial linear velocity for the formation of [3H]methylated phospholipids and Sadenosyl+methionine concentrations yielded a straight line with an apparent K, of 40 f 2.1 I_LM

and a V,, of 8.4 f 1.1 pmol/mg protein per 5 mm (N = 3). Compared to microsomal, mitochondrial, nuclear or original homogenate, the highest specific activity of phospholipid methylation was seen in brush-border membranes. The activity of phospholipid methylation in subcellular fractions ranged from 20-60% of plasma membranes (data not shown). The foregoing results demonstrate for the first time that the enzymatic synthesis of PC from PE via a transmethylation pathway exists in rat small-intestinal brush-border membranes. The evidence for the enzymatic nature of this transmethylation includes: (i) loss of activity by heat denaturation and at 0” C; (ii) inhibition by Sadenosyl+homocysteine; and (iii) saturation kinetics. Although the specific activity of this membrane enzymatic reaction is low compared to that seen in other plasma membranes [8-121, it is definitely present in brush-border membranes as well as in other intracellular membranes, including microsomal membranes of the enterocyte. The reason(s) why this enzymatic activity was not detected in the earlier studies of Bremer and Greenberg [S] is, therefore, unclear at this time. In rat colonic brush-border membranes several lines of evidence support the existence of at least two distinct methyltransferases involved in the synthesis of PC from PE [ll]. In contrast to these findings, preliminary characterization of phospholipid methylation in rat small intestinal brush-border membranes in the present experiments suggest that a single methyltransferase may exist in this membrane. Further characterization of this activity, however, will be necessary to clarify this issue. Finally it should be noted, that although the importance of phospholipid methylation in the regulation of important membrane processes has been questioned [9,16,20], recent studies have suggested that such transmethylation reactions may influence transepithelial sodium transport [10,21]. In this regard our laboratory has shown that alterations in transmethylation reactions in rat colonic brush-border [13] and basolateral [12] membranes may modulate Na+-H+ exchange and (Na+ + K+)-ATPase activities, respectively. Further stud-

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ies of phospholipid methylation activity in rat small-intestinal brush-border membranes will therefore be of interest. The authors are grateful to Ms. Kimberli Coleman for her technical assistance and Ms. Dolores Gordon for her secretarial support. This investigation was supported by PHS grant number CA36745 awarded by the National Cancer Institute, DHHS. T.A.B. is the recipient of a Merit Award from the N.C.L/N.I.H. References 1 Pelech, S.L. and Vance, D.E. (1984) B&him. Biophys. Acta 119,217-251 2 Brasitus, T.A. and Schachter, D. (1982) Biochemistry 21, 2241-2246 3 Lands, W.E.M. (1960) J. Biol. Chem. 235,2233-2237 4 Kennedy, E.P. and Weiss, S.B. (1956) J. Biol. Chem. 222, 193-214 5 Bremer, J. and Greenberg, D.M. (1961) B&him. Biophys. Acta 46,205-216 6 Mansbach, C.M., II and Parathasarthy, S. (1979) J. Biol Chem. 19,9688-9694 7 Hirata, F., Viveros, O.H., Diliberto, E.M. and Axelrod, J. (1978) Proc. Natl. Acad. Sci. USA 751718-1721

8 Hirata, F. and Axelrod, J. (1978) Nature (London) 275, 219-220 9 Chauhan, V.P.S., Sikka, S.C. and Kalra, V.K. (1982) Biochim. Biophys. Acta 688, 357-368 10 Sariban-Sohraby, S., Burg, M., Weissman, W.P., Chiang, P.K. and Johnson, P.J. (1984) Science 225, 745-746 11 Dudeja, P.K., Foster, E.S. and Brasitus, T.A. (1986) Biochim. Biophys. Acta 875,493-500 12 Brown, M.D., Dudeja, P.K. and Brasitus, T.A. (1986) Gastroenterology 90, 1359(A) 13 Dudeja, P.K., Foster, ES. and Brasitus, T.A. (1986) Biochim. Biophys. Acta 859, 61-68 14 Brasitus, T.A., Schachter, D. and Mamouneas, T.G. (1979) Biochemistry 18, 4136-4144 15 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 16 Audubert, F. and Vance, D.E. (1983) J. Biol. Chem. 258. 10695-10701 17 Katyal, S.L. and Lombardi, B. (1974) Lipids 9, 81-86 18 Schneider, W. and Vance, D.E. (1979) J. Biol. Chem. 254, 3886-3891 19 Lineweaver, H. and Burk, D. (1934) J. Am. Chem. Sot. 56, 658-666. 20 Moore, J.P., Johannsson, A., Hesketch, T.A., Smith, G.A. and Metcalfe, J.C. (1984) B&hem. J. 221, 675-684. 21 Weismann, W.P., Johnson, J.P., Miura, G.A. and Chiang, P.K. (1984) Am. J. Physiol. 248, F43-F47