THE JOURNAL OP BIOLOGICAL CHEMISTIIY Vol. 252, No. 24, Issue of December 25, pp. 8191-8803, 1977

Prmted in U.S.A.

Modification of Membrane Lipid FUNCTIONAL PROPERTIES OF MEMBRANE IN RELATION TO FATTY ACID STRUCTURE*

(Received for publication, August 22, 1977)

JOSEPH J. BALDASSARE,$ GEORGE M. BRENCKLE,§ MICHAEL HOFFMAN,~~ AND DAVID F. SILBERT~~

From the Department of Biological Chemistry, Division of Biology and Biomedical Sciences, Washington University School of Medicine, St. Louis, Missouri 63110

At a given growth temperature, Escherichia coli has approximately six major molecular species with respect to the arrangement of fatty acyl groups on membrane phos- pholipids. Changes in the relative proportions of various molecular species and the concomitant effects on membrane function were explored in lipid mutants by in uiuo nutri- tional adjustments and by in vitro lipid transfer to isolated membranes. In particular, as the mole fraction of phospho- lipids acylated with cis-An-l&k1 on the 1 and 2 positions exceeded that found in normal cells grown under similar conditions, there was a progressive inactivation of mem- brane-bound NADH, L-cu-glycerol-3-phosphate, succinate, and n-lactate oxidases. However, the dehydrogenase activi- ties associated with these oxidases were not affected. Iden- tical loss in activity was produced in vitro using the transfer of lipids from liposomes to isolated membranes to produce the same membrane lipid modifications. Furthermore, ac- tivity was restored to near normal levels when the mem- branes were subsequently incubated with liposomes con- taining a normal mixture of lipid molecules. These observa- tions demonstrate that the presence in the membrane of phospholipid molecules with two different acyl chains has an important role in conserving some membrane functions. The relationship between lipid composition and membrane function can be rationalized in terms of composition-in- duced changes in the physical properties and possibly in the topographical distribution of the membrane lipids.

Naturally occurring membranes generally have a heteroge- neous lipid composition. Presently, the relationship between lipid composition and membrane structure and function is not well understood. To approach this problem, the fatty acid

* This investigation was supported by Research Grant GM-16292 from the United States Public Health Service. The costs of publica- tion of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertise- men? in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Recipient of United States Public Health Service Fellowship Award lF-32-GM-05157.

8 Supported by United States Public Health Service Grant l-T32- GM 07067-03.

?I Supported by United States Public Health Service Grants RR00954-03 and RR05389A.

11 Recipient of United States Public Health Service Research Ca- reer Development Award l-K4-GM 70,654.

composition and the physical properties of the lipids of unsat- urated fatty acid auxotrophs have been modified and the effect of these modifications on membrane-associated activi- ties has been determined (l-5). Since these auxotrophs still synthesize saturated fatty acids, they can regulate in part the properties of their membrane lipid. There are now Escherichia coli mutants defective in total fatty acid synthesis and fatty acid degradation in which the fatty acid composition of the phospholipids is determined almost exclusively by the fatty acid supplement in the growth medium (6, 7). A particularly interesting membrane modification was produced by growing these strains with cis-A”-l&l, one of the three major fatty acids synthesized by E. coli. As the mole per cent of c&A”- 18:l exceeded that found in a control strain capable of synthe- sizing its own fatty acids, growth slowed, passive permeability to some small molecules increased, and membrane-associated NADH oxidase activity diminished (6). The mutant strain cultivated with a combination of naturally occurring saturated and unsaturated fatty acids or of different unsaturated fatty acids grew at a normal rate and the cytoplasmic membranes from these cells gave normal NADH oxidase activities. The loss of membrane function which arose with relatively high content of cis-A”-18:1 in the cells correlated with distinct changes in the physical properties of the phospholipids and of the isolated membranes (7). For example, calorimetric studies revealed that the thermotropic transition of phospholipids in the membranes changed from the normal broad transition, to a broad plus a sharp one at lower temperature, and finally to just the sharp transition as the amounts of cis-An-l&l in the mutant strain increased from the normal range to values either just above or far in excess of those found in the control strain. The position and the width of the sharp transition suggested that it was due to the melting of the molecular species 18:1-18:l.l

In this paper, we quantitate the mole fraction of molecular species present in the membrane lipid of control and mutant strains grown with various fatty acid supplements. Inactiva- tion of several membrane-associated oxidase functions is shown to be correlated with changes in the content of 18:1- 18:1. Moreover, we show that the loss of NADH oxidase activity brought about by high content of 18:1-18:l can be

’ The abbreviations used are: short hand designations for molecu- lar species, e.g. l&O-16:l refers to a phospholipid with 16:0 and 16:l on the 1 and 2 positions, respectively; TLC, thin layer chromatogra- phy; GLC, gas-liquid chromatography.

8797

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8798 Membrane Function in Relation to Fatty Structure

reversed by transfer of lipids from liposomes to cytoplasmic density at 620 nm was measured. To determine the relative amounts of glycerophosphatides, the polar fraction from silica gel chromatog- raphy was further resolved by TLC and analyzed as described nreviouslv (131. For fatty acid analysis, the glycerophosphatide fractions from TLC were pooled and subjected to methanolysis, and the fatty acid esters resolved by GLC (13). To measure membrane linid comnosition in terms of the arraneement of acvl soups on the phosphohpid, monoacetyldiglycerides were prepared by the proce- dure of Renkonen (14). The phospholipids (1 to 10 mgl purified by the above TLC procedure were placed in lo-ml glass ampules with 2 ml of acetic acid:acetic anhydride (2:3, v/v). The ampules were sealed under a vacuum and heated to 150” for 5 h. After cooling, the ampules were opened and the solvent was evaporated. The monoac- etvldielvcerides were extracted in a mixture of chloroform:

membranes which restores to the latter a normal lipid mix- ture. These studies support the contention that the presence in the membrane of molecules containing two different acyl chains has an important role in conserving membrane func- tion. Furthermore, it is apparent that the acyl group structure of the phospholipids can influence not only the characteristics of the thermotropic phase change but also the behavior of the lipid molecules in the liquid-crystalline state at temperatures well above calorimetrically determined gel to liquid-crystal- line phase changes.

MATERIALS AND METHODS

Fatty acids were obtained from NuCheck Prep (Elysian, Minn.). Brij 58, Ficoll (400,000 or 70,000), ubiquinone-10, and Unisil were purchased from Sigma Chemical Co.

Strains and Media- Bacterial strains AB1623, L51, and La-2 are all Escherichia coli K12 derivatives and have been described previ- ously (6, 7). Strain L51 has a p oxidation defect (fidE) and was constructed by conjugation from strain AB1623 (fodE+). Strain La-2 was derived from strain AB1623 and contains a temperature-sensi- tive mutation (fabE1) affecting total fatty acid synthesis and the p oxidation block (fodE).

Bacteria were m-own overnight at 30” and subcultured at 37 f 0.3” in Medium 63 (8) containing 0.4% glycerol, 5 rnM glutamate, 1 pg/ ml of thiamin, 0.5 pg/ml of yeast extract, and 1 mg/ml of Brij 58. Fatty acids were supplemented at final concentration of 0.01%.

Preparation of Cell Envelopes-The method is a modification of that described by MacGregoret al. (9). Cells were harvested, washed twice at room temperature in Medium 63, and then suspended at 1 g of cells/ml in a 0.1 M Tris buffer (nH 8.0) containing 50 &ml of DNase and RNase, 5 rnM EDTA, 0.5 kg/ml of lysozyme, and 0.2 mM dithiothreitol. The suspended cells were rapidly frozen in an acetone/ dry ice bath, then thawed at 37”. The mixture was vortexed and poured into 10 volumes of a 1 rnM MgCl,, 0.2 mM dithiothreitol solution. Intact cells were removed by centrifugation in Tris buffer and the above procedure repeated. Approximately 85 to 90% of the cells were lysed after the third freeze-thaw process. The broken cells were centrifuged at 1,200 x g for 20 min to remove any whole cells, then centrifuged at 30,000 x g for 1 h. The pellet was washed with 10 ml of a 10 mM Tris/HCl buffer (pH 7.5), 0.2 mM dithiothreitol and then resuspended in 1 to 2 ml of the same buffer.

Prenaration of Cvtonlasmic Membranes - Cvtonlasmic membranes were prepared by isopyknic centrifugation of sonicated spheroplasts. Spheroplast formation was carried out as described by Osborn et al. (i0). Intact cells were removed by centrifugation at 1,200 x g for 20 min and then the crude membranes pelleted by centrifugation at 78,000 x g for 2 h. The pellet was homogenized in 20% (w/v) sucrose, 3 mM EDTA, 0.4 mM dithiothreitol (pH 7.5) using a 23- gauge needle, then layered on a 44% (w/w) sucrose, 3 mM EDTA (pH 7.5) solution and centrifuged at 78,000 x g for approximately 12 to 18 h at 4” (11). The upper layer was collected with an la-gauge needle, diluted with 3 volumes of 10 mM Tris/HCl buffer (pH 7.5), 0.4 rnM dithiothreitol, and spun at 78,000 x g for 2 h. The pellet (cytoplasmic membranes) was resuspended in 2 to 4 ml of the same buffer and stored in l-ml aliquots at -180” for not more than 3 weeks.

Lioid Extraction and Analvsis - Cells were harvested during log- arithmic growth and washed twice at room temperature with Me- dium 63 containing 800 pglml of Brij 58. Total lipid extracts were obtained by the method of Bligh and Dyer (12). Neutral lipids and phospholipids were obtained from the total lipid extracts by silica gel (U&l, 100 to 200 mesh) column chromatography using chloro- form to apply the extracts and elute the neutral lipids followed by methanol to remove the polar lipids. To quantitate the quinones, the neutral lipid fraction was chromatographed on Silica Gel G (Anal Tech) using chlorofornmientane (80:20). Chromatographic separation was monitored with ubiquinone-10, oleic acid, and phos- phatidylethanolamine standards. The Rp values for these standards were 0:59,0.23, and 0.0, respectively. Quinone content was measured using ubiquinone-10 as a standard, as follows. The appropriate silica eel fraction was scraped and extracted with chloroform. The sampli was dried with a &ream of nitrogen and redissolved in 0.1 ml of absolute ethanol. Then 0.8 ml of ethyl cyanoacetate plus 0.1 ml of 5% KOH in 95% ethanol were added and after 30 s the optical

methanol:water (8:4:3, v/v/v). The product, in the organic layer, was dried over sodium sulfate and spotted on Silica Gel G TLC plates impregnated with silver nitrate. The plate was developed with benzene:chloroform:methanol (98:2:0.1) (15). The monoacetyl- diglycerides migrated according to the degree of unsaturation and were visualized using Rhodamine 6G. The fractions were scraped off the plate and extracted with anhydrous ether. At the time of extraction, a constant amount of tricaprin was added to each fraction to serve as an internal standard. Further resolution of the monoac- etyldiglycerides based on chain length was obtained by GLC using a column packed with 3% OV-17 on 60 to 80 mesh chromosorb Q. The column temperature was 280”. The monoacetyldiglycerides were identified according to retention time by comparison with standards prepared from diglycerides and by using GLC-mass spectrometry system.

Assay of NADH, L-a-Glycerol Phosphate, Succinate and D-LaCtUte Oxidase, and Dehydrogenase Activities- Respiration rates were de- termined at 37” with a Beckman electrode that was calibrated by saturating a 50 mM potassium phosphate buffer (pH 7.51 with gas mixtures containing various proportions of nitrogen and oxygen. The assay mixtures (3 ml) containing 0.5 mg of envelope protein in 50 mM notassium nhosnhate (PH 7.51 were first eauilibrated with oxygen by passing air throughthe solution for 2 to 3 min. After the eauilibration neriod the reaction was initiated by adding 0.2 ml of a 1 M solution 01 the substrate.

L-a-Glycerol phosphate, succinate, and n-lactate dehydrogenase activities were measured spectrophotometrically by following the reduction of 5-diphenyl tetrazolium bromide coupled to phenazine methosulfate (16). The assay mixtures contained 0.1 M Tris/HCl (pH 7.81, 30 pg/ml of 5-diphenyl tetrazolium bromide, 60 pg/ml of phenazine methasulfate, 10 mM KCN, and membrane envelopes. The reaction was initiated by addition of substrate at a final concentration of 50 rnM and followed at 500 nm. NADH dehydrogen- ase was determined by measuring the reduction of potassium ferri- cyanide at 420 nm (17).

Preparation of Phospholipid Vesicles - Either total lipids or phos- pholipids isolated by column chromatography were dried with a stream of nitrogen and then suspended at a final concentration of 5 to 10 pmol/ml in 10 mM Tris buffer (pH 7.5). The mixture was dispersed at approximately 30-40” by sonication in a bath sonitier for 1 to 2 h. During the sonications. nitrogen was bubbled through the solution. Undiipersed lipids were removed by centrifugation at 40,000 X g for 1 h at 4”. The lipid vesicles were stored at 4” under nitrogen and used within 3 days after preparation.

Conditions for Transfer of Lipid from Liposomes to Cytoplasmic Membranes -Cytoplasmic membranes prepared as described above were incubated at approximately 1 mg/ml of protein with lipid vesicles at final concentration of 1 to 4 mg/ml. The incubations were carried out at 37” with gentle shaking and stopped by placing the mixture at 0”. In some cases, the cells were tested for NADH activity without any further treatment. However, if the lipid com- position was determined or if a second incubation was carried out the liposomes were separated from the membranes by sedimentation at 4” of the mixture through a layer of 10% Ficoll. The membrane pellet was then washed twice by centrifugation in 10 mM Tris buffer, 0.2 mM dithiothreitol (pH 7.5) and finally resuspended at a final concentration of 1 mg/ml in the same buffer.

Protein Content - Protein was determined by the method of Lowry et al. (18).

RESULTS

Molecular Species of Phospholipids of fadE Control Cells (Strain L51) and fabEfadE Cells (Strain L8-2) Grown with

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Membrane Function in Relation to Fatty Structure 8799

Various Fatty Acid Supplements - When mutant strains which were defective in total fatty acid synthesis and in fatty acid degradation (fabEfadE or fabEfabBfadE strains) were grown with cis-An-l&l, a sharp thermotropic phase transition was detected by differential scanning calorimetry when the content of l&l in the phospholipids exceeded approximately 70 mol % (7). Since this sharp transition was thought to be due to the melting of lipid molecules containing two c&-A”- l&l (see introduction and Ref. 7), we have quantitated the molecular species present in the membrane lipid of control and mutant strains grown under various conditions to obtain direct evidence in support of this contention. When the /3 oxidation-defective control strain (L51) was grown with cis- A”-18:l the mole fraction of molecular species 18:1-18:l reached but never exceeded 40% (Table I, lines 1 to 3) and the growth rate remained unchanged. Cultivation of strain L8-2 for several generations with cis-A”-18:1 led to a reduced exponential growth rate and progressive increase in the mole fraction of 18:1-18:l (Table I, lines 4 to 6). The onset of the altered growth rate occurred when the content of this molecu- lar species exceeded the amount found in strain L51 grown under similar conditions. Hence, the data are in agreement with our previous deduction that the functional changes in the mutant strain and the unusual melting properties of its phospholipids were associated with the accumulation of exces- sive amounts of 18:1-18:l.

When strain L8-2 is grown with two different fatty acids as supplements, one might anticipate the synthesis of three molecular species; e.g. supplementation with 16:0 and cis-As- 16:l could lead to formation of 16:0-16:0, 16:0-16:1, and 16:1- 16:1. As the data in Table I show for this case (line 7), the membrane accumulates only two of the species and particu- larly 16:0-16:l. In contrast to the findings with 18:1-18:1, high levels of 16:0-16:l are compatible with broad thermotropic phase changes and retention of normal function (Ref. 7 and see below). These results suggest that the diversity of types of molecular species (intermolecular heterogeneity) is not as critical to the physical and functional properties of membrane as is the retention of molecules containing two different acyl groups (intramolecular heterogeneity). When strain L8-2 is

cultivated for several generations with cis-As-16:l and cis-A”-

18:1, the relatively broad thermotropic phase can still be observed (7) and growth rate as well as some membrane functions (see below) remains normal. In the example shown in Table I (line 8), the mutant strain accumulated 18:1-16:1, 18:1-18:1, and 16:1-16:1 on shifting from a medium supple- mented with 16:0 and cis-A9-16:l to one containing the two unsaturated fatty acids. However, in this particular experi- ment the compositional changes were not extensive enough to show the relative importance of intramolecular as opposed to intermolecular heterogeneity as defined above.

Respiratory Activities of Membrane Envelopes of Cells from Strain L8-2 Grown with Either 16:0 plus cis-As-16:1 or cis- A”-18:1- Earlier findings demonstrated that growth rate and passive permeability to o-nitrophenyl thiogalactoside changed abruptly after growth of strain L8-2 with cis-A”-18:1 for 1 generation. Fig. 1 shows that the membrane NADH oxidase activity in this mutant also began to diminish progressively after a similar period of growth with this supplement. How- ever, cultivation of these cells with combinations of saturated plus unsaturated or of two unsaturated fatty acids did not change the NADH oxidase activity. To determine whether the progressive decrease in this activity was indicative of a general loss of respiratory functions, oxygen uptake studies were carried out using membrane envelopes from cells of strain L8-2 and various substrates as electron donors (Fig. 2). It is clear from the results that the effect was general. Although the onset and rate of inactivation were very similar for the different activities, the rate of loss of NADH oxidase as measured by NADH versus oxygen consumption (Fig. 1 versus 2) was not identical. Oxygen uptake decreases more rapidly than NADH consumption and this discrepancy may reflect the greater sensitivity of the former measurement to changes in any of the intermediate steps involved in the overall electron transfer reaction.

In Table II are shown NADH, L-a-glycerophosphate, succi- nate, and n-lactate oxidase and dehydrogenase activities for membrane envelopes prepared from cells of strain L8-2 grown for three generations with ci~-A~~-18:1 or with 16:0 plus cis-A9- 16:l. In contrast to the pronounced effect on oxidase activities

TABLE I

Fatty acid composition of phospholipids in terms of molecular species for cells of strains L51 and L8-2 grown with various fatty acid supplements

Monoacetyldiglycerides were prepared as described under “Materials and Methods.” All values are expressed as mole percentage of the total moles of phospholipid in the membrane. Any components that were ~0.5% were not included in the Table.

Molecular species Strain, supplement, extent of

growth Growth rate 16:0- l&O- 18:1- El l&l El y& ;; :‘-

hldoubling of culture mol %

L51 (fadE) cis-A”-18:1 1. 0.2 generation 1.9 21 15 28 32 3 2. 1.2 generation 1.9 14 16 26 40 2 3. 2.2 generation 1.9 13 15 28 40 2

LS-2 (fabE fadE) cis-A”-18:l 4. 0.4 generation 5. 1.5 generation 6. 2.4 generation

1.9 23 8 28 34 6 2.5 8 6 23 59 3 2.5 5 4 20 68 1

16:0 + cis-As-16:l 7. >lO generation 1.7 69 1 14 0 14

cis-A9-16:l + cis-A”-18:1 8. 2.5 generation 1.7 23 2 46 9 18

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8800 Membrane Function in Relation to Fatty Structure

L-- I F---y I

GROWTH ,I” *,n,r.,,on,l

FIG. 1. NADH oxidase activities of membrane envelopes from cells of strain L8-2. Aliquots were removed at the indicated times during growth of strain L8-2 and then membrane envelopes were prepared as described in the text. Activities were measured as described under “Materials and Methods” and are expressed as percentage of the control cells (i.e. membrane envelopes of strain L51 grown without supplement). The curves are for membrane envelopes (O--O, 0-O) from cells of strain L8-2 grown with cis-A”-18:l; the open and closed circles represent values determined from different experiments. O---XI, from cells of strain L8-2 grown with 16:0 plus cis-A9-16:l; m---W, from cells of strain LB-2 grown with cis-As-16:l plus cis-An-18:l.

(column 21, adjustments in the fatty acid composition of the membrane lipid did not alter the dehydrogenase activities for any of the electron donors (column 3).

Transfer of Phospholipid from Lipid Vesicles to Cytoplas- mic Membranes - In the data presented above, we have shown that there is a correlation between the increase of species l&1-18:1 and loss of several oxidase activities. If the loss of these activities was due solely to the increase of lipids enriched in species 18:1-18:1, one might expect these effects to be reversed by readjustment of the lipid composition. Since protein turnover could be an important factor in any in vivo manipulations, the lipid composition was changed in vitro by transfer of lipid from single compartment lipid vesicles to isolated cytoplasmic membranes.

Fig. 3 demonstrates that when cytoplasmic membranes isolated either from the wild type parent (strain AB1623) or from the double mutant (strain L8-2) grown for approximately one generation on cis-A”-18:1 were incubated with 3H-labeled lipid vesicles, a net transfer of phospholipids from the lipid vesicles to the cytoplasmic membranes occurred. Similar rates of transfer were observed for vesicles prepared from total lipids or phospholipids. The net transfer for any fixed time period was dependent on the initial ratio of membrane protein to vesicle phospholipid. I f the initial ratio was 1:2, the amount of phospholipid transferred after 60 min of incubation was equivalent to 25% of the total membrane phospholipid. Back transfer of membrane phospholipid to vesicles was measured by incubation of W-labeled membranes prepared from cells of strain AB1623 grown with [Wlacetate. Under incubation conditions (data not shown) in which 20 nmol of 3H-phospho- lipids were transferred to the membranes, less than 1 to 2 nmol of W-phospholipid were recovered in the remaining lipid vesicle fraction.

Effect of Lipid Transfer on NADH Oxidase Activity-The effect of transfer of the total lipids from cells of strain AB1623 and from cells of strain L8-2 enriched with cis-A”-18:1 are

I 16~0+16~l-envel ape 5 -__-_

1s~1-envelopas -

L I I , - I 2 3

GROWTH (1” gsneratmrl

FIG. 2. Respiratory activities of membrane envelopes from cells of strain L8-2 grown with cis-Ah”-18:l. Aliquots were removed at the indicated times during growth of strain L8-2 with cis-A”-18:l and then membrane envelopes were prepared as described in the text. Activities were measured as described under “Materials and Meth- ods” and are expressed as percentage of the control cells (i.e. membrane envelopes of strain L8-2 grown with 16:0 plus cis-As- 16:l). The curves are for membrane envelopes incubated with NADH (m----W, n-lactate ([7---O), succinate (O-O), and n-a-glycerol- P (O-O). The activities of membrane envelopes from cells of strain L8-2 grown with 16:0 plus cis-A9-16:1 remained constant throughout the growth period.

TABLE II

Oxidase and dehydrogenase activities in envelopes isolated from cells of strain L8-2 grown with various fatty acid supplements

Cells of strain L8-2 were grown with the indicated fatty acid supplements. Envelopes were prepared from these cells and then oxygen uptake and dehydrogenase activities were measured as described under “Materials and Methods.”

Electron donor and sample Oxidase Dehydrogenase

patom. Otlminlmg pm01 acceptorlminlmg NADH

16:0 + 16:l 0.495 0.523 18:l 0.110 0.554

n-o-Glycerol-P 16:0 + 16:l 0.450 0.372 18:l 0.105 0.340

Succinate 16:0 + 16:l 0.498 0.352 18:l 0.135 0.361

n-Lactate 16:0 + 16:l 0.045 0.129 18:l 0.013 0.126

shown in Fig. 4. Transfer of total lipids from liposomes prepared from cells of strain AB1623 to these membranes had no effect on NADH oxidase activity. In contrast to these results, incubation of these membranes with total lipids

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Membrane Function in Relation to Fatty Structure 8801

r

P I

h- 20 30 40 50 60

Time lin minutes)

FIG. 3. Time course of transfer of vesicle lipid to cytoplasmic membranes. Lipid transfer was carried out at a membrane protein to vesicle phospholipid ratio of 1:2 (w/w) as described under “Mate- rials and Methods.” The data are for cytoplasmic membranes (A, 0) from lipids of cells of strain AB1623 grown at 37” incubated with lipid vesicles prepared from 3H total lipids and 3H phospholipids, respectively, and (A, 0) from lipids of cells of strain L8-2 grown at 37” for one generation with cis-A”-18:l incubated with lipid vesicles prepared from 3H total lipids and 3H phospholipids, respectively.

‘p 0 N

! i 0.6

0”

*, I - I I

IO 20 30 40 50 60

Time ( minutes )

FIG. 4. Effect of lipid transfer on NADH oxidase activity. Trans- fer was carried out at a membrane protein to vesicle phospholipid ratio of 1:2 as described in the text. Samples were withdrawn at the indicated times and assayed for NADH oxidase activity. Data for membranes prepared from cells of strain LB-2 grown for one genera- tion with cis-A”-l&l incubated with liposomes prepared (A) from lipids of cells of strain AB1623; A, from lipids of cells of strain LS-2 grown for three generations with cis-A”-18:l.

enriched with cis-A”-18:1 led to a loss of NADH oxidase activity (Fig. 4).

Table III summarizes the fatty acid compositions and rela- tive oxidase activities for the in uivo and in vitro modifications of fatty acid composition. A close correlation exists between the two sets of data. A reduction in activity was observed in each case when the content of cis-All-lb:1 exceeded approxi- mately 64%.

branes of strain L8-2 grown with cis-A”-18:1 might be due to a change in the ratio of quinones to phospholipid. Table IV demonstrates that the ratio of ubiquinone, the major neutral lipid present under these growth conditions, to phospholipid for cells of strain L8-2 grown for several generations with cis- A”-18:l was equivalent to this ratio for cells of the control strain L51 and for cells of L8-2 grown with 16:0 plus cis-As- 18:l. Furthermore, analysis of the major glycerophospholipids showed that there were no significant compositional differ- ences between the phospholipids from the control strain and those from strain L8-2 grown with cis-A**-18:l (data not shown).

Quantitative Analysis of Quinones and Glycerophospha- Restoration of NADH Oxidase Activity in Vitro Accompa- tides- In the course of the lipid transfer experiments, we nying Restoration of the Lipid Heterogeneity Characteristic found that incubation of cytoplasmic membranes with phos- of Normal Membranes - Since there was no correlation be- pholipids isolated from the total lipids of cells of strain AB1623 tween the reductions in activity and any change in the ratio led to a decrease in NADH oxidase activity (data not shown). of ubiquinone to phospholipid or any change in the content of George-Nascimento et al. (4) had previously shown that fusion the glycerophosphatides, it seems probable that the loss of of Escherichia coli membrane vesicles with phospholipids respiratory activities was dependent on the presence of an lowered the NADH oxidase activity. These results suggested excess of molecular species 18:1-18:l. To determine whether that the loss of the oxidase activities observed with mem- the inactivation of respiratory function was reversible and,

TABLE 111

Fatty acid composition and relative NADH oxidase activities

In vitro lipid transfer was carried out by incubation of cytoplasmic membranes from cells of LS-2 grown for one generation with cis-A”- 18:l with liposomes at a membrane protein to vesicle phospholipid ratio of 1:2, w/w (see “Materials and Methods”). The liposomes were prepared from total lipid extracts from either cells of strain AB1623 or from total lipid extracts from cells of strain L8-2 grown for several generations with cis-A”-18:1 and were incubated with mem- branes for 0, 30, and 60 min, respectively. Cells of strains L51 and L8-2 were grown with cis-A”-18:1 at 37”. The fatty acid analogues were prepared as described under “Materials and Methods”. All values are expressed as weight percentage of the total fatty acids in the phospholipids. Any components that were ~0.5% were not included in the table. NADH oxidase activities are expressed as percentage of the activity of membranes from cells of L8-2 grown with fatty acids 16:0 plus cis-A9-16:l.

16:0

In vitro lipid transfer to mutant membranes (in min) from lipo- somes of normal composition

0 30 60

From liposomes enriched with 18:l

0 30 60

In uiuo growth with 18:l (in gen- erations)

Control cells 0.2 1.2 2.2

Mutant cells 0.4 1.5 2.4

12 14 19

12 10 10

18 15 14

16 7 4

Time course for lipid 16:l l&l

Relative NADH oxi- dase activ-

ity 9%

24 64 90 30 56 90 27 51 90

24 63 90 21 68 65 21 70 60

28 54 100 22 61 100 22 62 100

32 52 100 18 74 60 14 80 30

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8802 Membrane Function in Relation to Fatty Structure

TABLE IV

Analysts of ubiquinone content

Cells of the strains in the table were grown with the indicated fatty acid supplements. The ubiquinone content was determined for each samole as described under “Materials and Methods.”

1. AB1623, none 3 33

2. L51, none 3 38

3. L8-2, crs-A”-18:l ‘I2 39 4. L8-2, cis-A”-18:1 3 42

5. L8-2, 16:0 plus czs-A!‘-16:1 3 39

* Refers to the number of generations of growth before harvest.

1 I 1 1 I I I 30 60 90 120

TIME (mln)

FIG. 5. In vitro restoration of NADH oxidase activity. Cytoplas- mic membranes from cells of strain L8-2 grown for one generation with cis-A”-18:l were initially incubated with liposomes at a mem- brane protein to vesicle nhosnholinid ratio of 1:2 nrenared (A-A) from lipids of cells of strain AB1623; 0- - -0, from lipids of cells of strain L8-2 grown for several generations with cis-A”-18:l. After 60 min, the membranes were separated from the remaining liposomes by centrifugation through ficoll (see “Materials and Methods”) and then reincubated with liposomes at a membrane protein to vesicle phospholipid ratio of 1:16 prepared (C)-.0) from lipids of cells of strain AB1623. At the indicated times, the membranes were assayed for NADH oxidase activity.

therefore, solely related to this compositional change in mem- brane lipid, the content of species 18:1-18:l was first increased in excess of 60 mol % (Table I, line 6) and then decreased by incubation with lipids from cells of strain AB1623 (Fig. 5). During the first lipid transfer the NADH oxidase activity of membranes incubated with liposomes which have amounts of cis-A”-18:1 in excess of 90% decreased to 60% of its initial value. Further incubation with these lipid vesicles led to a progressive decrease of this activity for up to 90 min. In contrast to this result, the NADH oxidase activity was re- stored to within 10% of its original value when the liposomes enriched with species lB:l-18:l were removed by centrifugation (see “Materials and Methods”) and the membranes reincu- bated with vesicles prepared from lipids of strain AB1623.

DISCUSSION

Our approach to the problem of why membranes have a complex lipid composition has been to change the relative amounts of the various naturally occurring molecular species of the phospholipids and then to determine the effects of these

alterations on membrane-associated functions. Since it is difficult to determine the physiological significance of Arrhen- ius plots which have discontinuities below the growth temper- ature, we have studied lipid modifications that alter mem- brane functions at 37”. Both in uivo modification of the fatty acid composition of Escherichia coli mutants defective in total fatty acid biosynthesis and in vitro adjustments of the lipid composition ofE. coli cytoplasmic membranes by transfer of lipid molecules from liposomes of various composition were combined to change the phospholipid compositions. Recently, we have focused on a specific modification, namely enrichment of the membrane lipids with phospholipid molecules contain- ing two cis-A”-18:l. Growth of the fatty acid mutants with this supplement continued for several generations provided the external potassium levels were high enough to compensate for permeability changes occurring after approximately one generation of growth. Under these conditions, macromolecular synthesis continued at near normal rates and the loss of certain respiratory capacities could be followed before they became growth-limiting. In contrast, growth of these E. coli lipid mutants did not persist much beyond one generation when the other major fatty acids synthesized by E. coli (namely, 16:0 and cis-Ag-16:l) were provided individually as growth supplements. These fatty acids were, in fact, taken up and incorporated into the glycerophosphatides, but in the case of supplementation with 16:0, the change in composition led to cell lysis (6).

In this study, we demonstrate that several oxidase activities were reduced when the membrane lipids contained excessive amounts of molecular species 18:1-18:l. Enrichment of the membrane with respect to molecular species 16:0-16:l or 18:1- 16:1 did not alter these functions. Furthermore, the oxidase activities were unaffected when the content of species 18:1- 18:1 was less than approximately 40 mol %. These observations suggest that the loss of function was associated specifically with loss of molecules possessing two different acyl groups rather than simply with a reduction in the types of molecular species present. To firmly establish that the inactivity of these oxidases was due to changes in the acyl groups, in vitro adjustments of the lipid composition were studied. Identical loss of NADH oxidase was found at compositions strikingly similar to those found in uivo and the activity was restored to near normal levels by in vitro modifications which reduced the relative content of cis-A”-18:l. Under these in vitro conditions, the loss of NADH oxidase cannot be attributed to loss of any protein components but must be due to changes in lipid composition. Moreover, since no differences were found in the amounts of the major quinones produced under these growth conditions nor in the relative amounts of the major glycerophosphatides between cells with normal or reduced functions, the inactivation observed must have resulted from excessive levels of molecular species 18:1-18:l (Table III and Fig. 5).

Previously we had shown that when the membrane lipids contained relatively high contents of cis-A”-18:1, discontinui- ties in the plots of partitioning of 2,2,6,6-tetramethylpeperidi- nyl-1-oxy (Tempo) between the aqueous and lipid phases versus l/T were observed above the calorimetrically deter- mined gel to liquid crystalline phase transition (7). Moreover, calculations of the size of the cooperative unit taken from the differential scanning calorimetric measurements on lipid mix- tures consisting of excessive amounts of molecular species 18:1-18:l demonstrated that these discontinuities correlated with increased lipid-lipid interactions. On the basis of similar

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Membrane Function in Relation to Fatty Structure 8803

discontinuities observed with aqueous dispersions of dioleoyl- lecithin and in the membrane lipids from sarcoplasmic reticu- lum, Lee et al. (19) suggested the presence of short lived quasicrystalline structures in the liquid-crystalline phase of the bulk lipid. The presence of quasicrystalline clusters could increase the viscosity of the bulk lipid decreasing the rota- tional or lateral diffusion of small molecules such as quinone, thereby lowering their effective concentration. This interpre- tation is consistent with the observation that individual cata- lytic activities, i.e. the dehydrogenases, were not altered. We have also observed2 that whereas the overall activity of the anaerobically induced nitrate reductase (20) was lost, the individual activities were unaffected. In addition to changes in the bulk properties of the lipids, changes in the relative amounts of the molecular species of the phospholipids could influence the boundary lipid of membrane proteins. Jost et al. (21, 22) have postulated, based on spin labeling data, that hydrophobic surfaces of integral proteins are solvated by a constant amount of an immobilized layer of lipid and that the boundary lipid is in equilibrium with the adjacent bulk lipid. Although the notion of a lipid annulus surrounding individual membrane proteins remains at present a working hypothesis, several laboratories (23, 24) have reported results consistent with this idea. Lipid modifications such as those reported in this study which change significantly the relative amounts of molecular species and influence lipid-lipid interactions could affect either the kinds of lipid molecules which form the annulus or the exchange between the boundary and bulk lipid. Changes in either of these could in turn alter integral membrane protein conformations or protein-protein associa- tions.

We had previously concluded that E. coli maintains a heterogeneous mixture of lipid molecules (7). In this study, we demonstrate a direct correlation between loss of this heterogeneity by a modification that leads to excessive amounts of a naturally occurring molecular species 18:1-18:l and loss of several oxidase activities. We have also observed (7)3 that enrichment of membrane lipids with molecular spe- cies 16:0-16:0 led to cell lysis and have correlated these changes in lipid composition with altered membrane structure. In prokaryotes, the placement of different acyl groups on the same phospholipid molecule, e.g. 16:0-16:1, seems to be impor- tant for maintaining weak interactions between phospholip- ids. While synthesis of lipid molecules that have identical acyl groups on both positions occurs and is advantageous to the cell for rapidly adjusting the membrane to changes in environmental temperature (25), an excessive amount of these

2 J. J. Baldassare and D. F. Silbert, unpublished observations. 3 C. Lau, C. Freter, N. Neungton, and D. F. Silbert, unpublished

results.

molecules leads to strong lipid-lipid interactions, potentially resulting in formation of clusters which are deleterious to various cellular functions.

Acknowledgments-We thank Drs. L. Glaser and M. Bauza for reviewing this manuscript.

REFERENCES

1.

2.

3.

4.

5.

Beacham, I. R., and Silbert, D. F. (1973) J. Biol. Chem. 248, 5310-5318

6.

7.

8.

9.

10.

11.

12.

13.

14. 15.

16.

17.

18.

19.

Trauble, H., and Overath, P. (1973) Biochim. Biophys. Acta 307, 491-512

Linden, C. D., Wright, K. L., McConnell, H. M., and Fox, C. F. (1973) Proc. Natl. Acad. Sci. U. S’. A. 70, 2271-2275

George-Nascimento, C., Zehner, 2. E., and Wakil, S. J. (1974) J. Su~ramol. Struct. 2. 646-669

Morrisett, J. D., PownalI, H. J., Plumlee, R. T., Smith, L. C., Zehner. Z. E.. Esfahani. M., and Wakil, S. J. (1975) J. Biol. Chem. i50, 6669-6976

Davis, M. B., and Silbert, D. F. (1974) Biochim. Biophys. Acta 373, 224-241

Baldassare, J. J., Rhinehart, K. B., and Silbert, D. F. (1976) Biochemistry 15, 2986-2995

Pardee, A. B., Jacob, F., and Monod, J. (1959) J. Mol. Biol. 1, 165-178

MacGregor, C. H., Schnaitman, C. A., Normansell, D. E., and Hodgins, M. G. (1974) J. Biol. Chem. 249, 5321-5327

Osborn. M. J.. Gander. J. E.. and Parisi. E. (1972) J. Biol. Chem. 247, 3973-3986

Yamato, I., Anraku, Y., and Hirosawa, K. (1975) J. Biochem. (To&) i7, 705-718

Bligh, E. G., and Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37, 911-917

Silbert, D. F., Ladenson, R. C., and Honegger, J. L. (1973) Biochim. BioDhvs. Acta 311. 349-361

Renkonen, 0. i1965) J. Am. dil Chem. Sot. 42, 298-304 Kito, M., Ishinaga, M., Nishihara, M., Kito, M., Sawada, S.,

and Hata, T. (1974) Eur. J. Biochem. 54, 55-63 Kistler, W. S., and Lin, E. C. C. (1971) J. Bacterial. 108, 1224-

1234 Ruiz-Herrera, J., Showe, M. K., and DeMoss, J. A. (1969) J.

Bacterial. 97, 1291-1297 Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R.

J. (1951) J. Biol. Chem. 193, 265-275 Lee, A. G., Birdsall, N. J. M., Metcalfe, J. C., Toon, P. A., and

Warren, G. F. (1974) Biochemistry 13, 3699-3705 20. Garland. P. B.. Downie. J. A.. and Haddock. B. A. (1975)

Biochkm. J. lk?, 547-559 ’ 21. Jost. P. C.. Griffith. 0. H.. CaneIdi, R. A., and Vanderdaoi, G.

(1973) Pioc. Natl.Acad. &i.*lJ. S’. A. 70, 480-484 22. Jost, P. C., Nada Karen Karen, K. K., and Griffith, 0. H.

(1977) Biochemistrv 16. 3110-3114 23. Warren, G. B., Toon”, P. ‘A., Birdsall, M. J. M., Lee, A. G., and

Metcalfe. J. C. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 622 24. Nakamura; M., and Ohnishi, S. (1975) J. Biochem. (Tokyo) 78,

1039 25. Nishihara, M., Ishinaga, M., Kato, M., and Kito, M. (1976)

Biochim. Biophys. Acta 431, 54-61

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J J Baldassare, G M Breneckle, M Hoffman and D F Silbertfatty acid structure.

Modification of membrane lipid. Functional properties of membrane in relation to

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