Molecular arrangement of phosphatidylcholine and sphingomyelin in sarcoplasmic reticulum membranes

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 204, No. 1, October, pp. 148-152, 1980

Molecular

Arrangement of Phosphatidylcholine and Sphingomyelin in Sarcoplasmic Reticulum Membranes M. G. P. VALE

Center

for

Cell

Biology,

Department

of Zoology, Received

University

January

of Coimbra,

Coimbra,

Portugal

28, 1980

The distribution of phosphatidylcholine and of sphingomyelin in sarcoplasmic reticulum membranes was studied by using phospholipases. Treatment of intact membranes with phospholipase A from Vipera russeli, at 35”C, causes breakdown of about 50-55% of the total phosphatidylcholine present in the sarcoplasmic reticulum, whereas about 90-95% degradation is obtained under the same conditions in membranes disrupted by sodium deoxycholate. On the other hand, in intact membranes, sphingomyelinase hydrolyzes only 20% of the sphingomyelin, which is largely hydrolyzed by the enzyme after disrupting the membranes with deoxycholate. The results suggest that phosphatidylcholine is similarly distributed on both layers of the membrane (-50% on each side), whereas most of the sphingomyelin (-80%) is internally localized and, therefore, asymmetrically distributed in the sarcoplasmic reticulum membranes.

The structural organization of several types of biological membranes has been extensively studied by using chemical probes, phospholipases, exchange proteins, and NMR techniques. The results show that the membrane lipids and proteins are asymmetrically distributed in the biomembranes (1, 2). Utilizing chemical probes for amino compounds, 2,4,6-trinitrobenzenesulfonate and I-fluoro-2,4-dinitrobenzene (TNBS)’ (FDNB), we showed previously (3) that the amino phospholipids are differentially localized in the sarcoplasmic reticulum membranes. About 70% of the phosphatidylethanolamine (PE) is localized in the external layer of the membrane, whereas most of the phosphatidylserine (PS) is in the internal layer (3). Similar distribution has been reported by other investigators using the fluorescent probes, fluorescamine (4, 5) and the complex fluorescamine-cycloheptaamylose (6). All these probes react with 1 Abbreviations used: TNBS, 2,4,6-trinitrobenzenesulfonate; FDNB, l-fluoro-2,4-dinitrobenzene; PE, phosphatidylethanolamine; PS, phosphatidylserine; PC, phosphatidylcholine; Sph, sphingomyelin; DOC, deoxycholate; NL, neutral lipids; SR, sarcoplasmic reticulum. 0003-9861/80/110148-05$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

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primary amino groups (7-12) so that only the amino phospholipids from the lipid moiety of the sarcoplasmic reticulum membrane have been localized by these processes. In this work, we utilized phospholipases to investigate the molecular arrangement of the other major phospholipids of the reticulum membranes, phosphatidylcholine (PC) and sphingomyelin (Sph). Earlier studies showed that the action of phospholipases in intact erythrocytes is restricted to the phospholipids localized on the external surface of the membrane, whereas both sides are accessible to the enzymes after disrupting the membranes (13). MATERIALS

AND

METHODS

Isolation of sarcoplasmic reticulum. Sarcoplasmic reticulum vesicles were isolated from rabbit white skeletal muscle as previously described (14). The protein was measured by the biuret method (15) using bovine serum albumin as standard. Treatment of sarcoplasmic reticulum with phospholipases. Sarcoplasmic reticulum membranes (20 mg) were incubated with stirring, at 35”C, in a medium containing 0.1 M KCl, 10 mM Tris-maleate, 10 pg of phospholipase A (Vipera russeli)/mg of reticulum protein, and 0.1% of sodium deoxycholate (DOC) (if present) in a total volume of 10 ml at pH 7.0. As was

PHOSPHATIDYLCHOLINE

AND SPHINGOMYELIN

previously reported (16), enzymatic reaction occurs even in the absence of added Ca2+. In parallel experiments, the membranes were incubated as described above with 10 IU of sphingomyelinase. In both cases, aliquots of 5 ml were withdrawn from the medium at 10 and 30 min of incubation, and the reaction was inhibited by adding 2 mM EDTA and by chilling the samples in ice. Then, they were centrifuged at 56,OOOgfor 1 h, at 0°C. The pellets were rinsed with 0.1 M KCI-10 mM Tris-maleate (pH 7.0) and submitted to lipid extraction. In order to ensure complete recovery of the lipid moiety of the disrupted membranes, the supernatants from the DOC suspensions were also submitted to lipid extraction. Phospholipid analysis. After incubation as described above, the membrane lipids were extracted in CHClJCH,OH (l:l,v/v) mixtures (17). The extracts were centrifuged and the lipid fraction was taken to dryness in a rotatory evaporator. The residues were dissolved in CHCl&H,OH (2:l,v/v) mixtures for spotting on thin-layer chromatography plates (silica gel G, type 66, Merck). The phospholipids were separated by unidimensional chromatography in CHCl$ CH,OWNH,OWH,O (70:30:4:l,v/v). In the experiments where the membranes were treated with phospholipase A, two-dimensional chromatography was necessary to obtain perfect resolution of the different classes of phospholipids. The solvent described above was used in the first dimension and CHCl$CH,OH/ CH,COOH/H,O (90:40:12:2,v/v) was used in the second one. The lipids were located using iodine vapors, and quantitative analysis was determined by measuring the amount of Pi by the method of Bartlett (18) in the scraped spots previously digested in 70% HClO, at 196°C (19). Percentage degradation of phosphatidylcholine by phospholipase A was determined using sphingomyehn (which is not degraded) as an internal standard. For determination of sphingomyelin degradation by sphingomyelinase, the internal standard used was phosphatidylcholine. Reagents. All reagents were of analytical grade. Sphingomyelinase was a generous gift of Dr. B. Roelofsen (University of Utrecht) and was stored at -20°C in 56% glycerol, 5 mM CaCl,, and 50 mM ‘his, at pH 7.5. RESULTS

AND DISCUSSION

In sarcoplasmic reticulum membranes isolated from rabbit skeletal muscle, the phospholipids make up about 80% of the total lipid. As reported by several investigators (20-23), we found that the major phospholipid is phosphatidylcholine which accounts for about 70% of the total lipid phosphorus. The other phospholipids con-

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sist of about 20% phosphatidylethanolamine, 4% sphingomyelin, 3% phosphatidylserine, and 1% phosphatidylinositol. When intact sarcoplasmic reticulum was incubated 10 min, at 35”C, with phospholipase A from Vipera russeli, about 55% of the total phosphatidylcholine (lecithin) was degraded and this value increased only to 65% for 30 min of incubation with the enzyme. In contrast, phosphatidylcholine was almost completely degraded (90-95%) when digestion was performed under the same conditions but in the presence of 0.1% sodium deoxycholate (Fig. 1A). As the lecithin disappeared, the amount of lysolecithin increased as well as the amount of lysoproducts of other phospholipids attacked by phospholipase A. The phosphatidylethanolamine was greatly degraded, however, under the conditions reported here, it appears that phosphatidylcholine is the preferred substrate for the enzyme (Fig. 1B). Differences in the reactivity of lipids may be caused by several factors such as lipid-lipid or lipidprotein interactions, which restrict the use of phospholipases in studies of membrane sidedness (1). An intimate association of phosphatidylethanolamine with the ATPase enzyme has been suggested earlier (24). Thus, in sarcoplasmic reticulum membranes, the enzymatic degradation seems particularly suitable for localization of the phosphatidylcholine but not of the phosphatidylethanolamine. Since about 55% of the phosphatidylcholine is rapidly accessible to phospholipase A whereas for the same period of reaction about 95% is exposed by disruption of the membrane, it appears that phosphatidylcholine is similarly distributed on both sides of the sarcoplasmic reticulum membrane. Similar results were recently obtained by means of a 13C NMR technique (25). In contrast, when sarcoplasmic reticulum vesicles were incubated 10 or 30 min with sphingomyelinase from Staphylococcus aureus, about 17-20% of the total sphingomyelin was degraded, whereas the remaining SO-85% was hydrolyzed after disrupting the membranes with 0.1% sodium deoxycholate (Fig. 2A). Figure 2B shows the pattern of phospholipids resolved by unidimensional chromatography. Only sphin-

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M. G. P. VALE

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100

0

Intact

SR

ezla

Leaky

SR

Incubation

time

i min

)

FIG. 1. Hydrolysis of phosphatidylcholine by phospholipase A in intact and leaky sarcoplasmic reticulum membranes. Membranes, intact or disrupted by 0.1% sodium deoxycholate, were incubated for 10 or 30 min at 35”C, in a medium containing 0.1 M KCl, 10 mM Tris-maleate (pH 7.0), and enough phospholipase A to perform 10 pg of enzyme/mg of reticulum protein. The phospholipid degradation was determined by Pi analysis after chromatographic separation as described under Materials and Methods. (A) Percentage of phosphatidylcholine hydrolysis. (B) Bidimensional separation of phospholipids from intact vesicles treated 30 min with the enzyme. On the right, the separation was carried out only in the first solvent. 0, origin; Lys-PC, lysophosphatidylcholine; Lys-PE, lysophosphatidylethanolamine.

gomyelin was attacked by sphingomyelinase as we observed in the chromatogram c, which shows a negligible spot of sphingomyelin as compared with that in the sarcoplasmic reticulum not treated with the enzyme (chromatogram a). The results indicate that sphingomyelin is asymmetrically distributed between the

0 10

Incubation

30

30

time

( min. 1

two halves of the sarcoplasmic reticulum membranes. About 80% is located on the internal surface of the membrane, whereas only 20% is externally localized. Although with some restrictions, the phospholipases have been shown to be useful tools in studies of lipid topology in the biomembranes. Verkleij et al. (13) and

!

FIG. 2. Hydrolysis of sphingomyelin by sphingomyelinase in intact and leaky sarcoplasmic reticulum membranes. Membranes, intact or disrupted by 0.1% sodium deoxycholate, were incubated with 10 IU of sphingomyelinase as described in Fig. 1. (A) Percentage of sphingomyelin hydrolysis. (B) Unidimensional separation of phospholipids. (a) Normal sarcoplasmic reticulum. (b) Intact vesicles treated with sphingomyelinase. (c) Leaky vesicles (0.1% DOC) treated with sphingomyelinase. 0, origin; PI, phosphatidylinositol; LPC, lysophosphatidylcholine.

PHOSPHATIDYLCHOLINE

AND SPHINGOMYELIN

TPI

FIG. 3. Proposed distribution of phospholipids between the inner and the outer layer of the sarcoplasmic reticulum membrane. The figure was constructed with the present data and with that previously reported (3). TPL, total phospholipids.

Zwaal et al. (26) observed that in intact erythrocytes only the phospholipids of the outer surface of the membrane were degraded by phospholipase A and sphingomyelinase, whereas in nonsealed ghosts complete cleavage occurred due to exposure of both sides of the membrane to the enzymes. In sarcoplasmic reticulum, we also observed that these phospholipases cause a selective hydrolysis of the outer layer of the membrane in intact vesicles, since a new fraction of the membrane phospholipids was hydrolyzed by the enzymes after disrupting the membranes. Thus, we could differentiate the localization of the phosphatidylcholine and of the sphingomyelin in the sarcoplasmic reticulum membranes. In a previous report (3) we observed a strong asymmetry in the distribution of the amino phospholipids, phosphatidylethanolamine and phosphatidylserine. By using chemical probes for amino compounds, TNBS and FDNB, we determined that phosphatidylethanolamine is essentially localized on the external surface of the membrane, whereas most of the phosphatidylserine is internally localized. Identical topology of the amino phospholipids was also observed by other investigators using

IN SARCOPLASMIC

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151

fluorescent probes (4-6). These results together with those reported here give an indication about the organization of the lipid bilayer in sarcoplasmic reticulum. Figure 3 shows the probable distribution of the most important phospholipids of the reticulum membranes. The outer layer consists mainly of phosphatidylcholine and phosphatidylethanolamine, while the inner layer is essentially composed by sphingomyelin, phosphatidylserine, and also by phosphatidylcholine. This molecular arrangement supports a structural asymmetry of the sarcoplasmic reticulum membranes. The general concept of membrane lipid asymmetry has emerged from studies performed with several types of biological membranes; however, this conclusion has been questioned in liver microsomal vesicles and Golgi vesicles which were recently described as symmetric bilayers (27). The reasons for some contradictory results have been recently discussed in a review by Op den Kamp (1). In sarcoplasmic reticulum membranes, the lipid asymmetry appears to be evident. By using different chemical probes (3-6) and different phospholipases, the results indicate predominance of certain phospholipids in the outer or inner layer of the membrane, respectively. There is no doubt that phospholipids influence the mechanism of Ca2+ transport in sarcoplasmic reticulum (20, 2%32), however, the implications of the membrane asymmetry with respect to its function are not clear. ACKNOWLEDGMENTS I am grateful to Professor A. P. Carvalho for the stimulating and helpful discussions during the course of this work, and for the critical reading of the manuscript. This work was supported by grants from the National Institute for Scientific Research (INIC) of the Portuguese Ministry of Education. REFERENCES 1. OP DEN KAMP,

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