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Biochimica et Biophysics Acta, 488 (1977) 341-352 @ Elsevier/North-Holland Biomedical Press

BBA 57041

EFFECTS OF GROWTH TEMPERATURE AND SUPPLEMENTATION WITH EXOGENOUS FATTY ACIDS ON SOME PHYSICAL PROPERTIES OF CLOSTRZ~~~M R~TYRICUM PHOSPHOLIPIDS

HOWARD GOLDFINE a, G.K. KHULLER a*, REBECCA P. BORIE a, BERNARD SILVERMAN b, HAROLD SELICK IIb, NORAH C. JOHNSTON a, JANE M. VANDERKOOI b and A.F. HORWITZ b

a Department of Microbiology and b Department of Biochemistry and Biophysics, School of Medicine, University of Penttsylvania, Philadelphia, Pa. 19174 (U.S.A.)

(Received January 17th, 1977) (Revised manuscript received April 29th, 1977)

Summary

The effects of the compositional alterations induced in the plasmalogen-rich phospholipids of Clostridium butyricum by growth at different temperatures with biotin or at a given temperature on supplementation with exogenous fatty acids in the absence of biotin, have been studied in lipid dispersions with the spin probe 2,2,6,6-tetramethylpiperidine-l-oxyl (TEMPO) and with the fluores- cent probe diphenylhexatriene. Plots of TEMPO partitioning vs. temperature revealed deviations from linearity at 48” and 34.5”C for the phospholipids from cells grown on biotin at 37” and at 45” and 29°C for the phospholipids from cells grown on biotin at 25°C. Diphenylhexatriene fluorescence polari- zation showed a broad inflection beginning at 36°C for the lipids from 37” C- grown cells and at 32°C for the lipids from 25°Cgrown cells. Maximum polari- zation was reached at -5°C in each sample.

Phospholipids from cells grown on oleate at 37’C had 85% 18 : I plus Cig- cyclopropane acyl chains and 90% 18 : 1 plus Cab-cycloprop~e alk-l-enyl chains. The phospholipids had decreased proportions of ethanolamine plus N-methylethanolamine plasmalogens (Khuller, G.K. and Goldfine, H. (1975) Biochemistry 14, 3642) and markedly increased levels of the glycerol acetal derivatives of these plasmalogens. Deviations from linearity were seen in TEMPO partitioning plots at 44” and 28°C and diphenylhexatriene polariza- tion showed a broad transition beginning at 29°C and ending at -2°C. Polari- zation did not, however, reach a maximum, even at -15°C.

* Present address: Department of Biochemistry, Postgraduate Institute of Medical Education and Re-

search. Chandigarh, India. Abtkeviations: TEMP0,2.2,6,6-tetramethylpiperidiike-l-OxYl. TLC, thin-layer chromatopraphY.

The phospholipids from cells grown on elaidate at 3’7°C were greatly enriched in trans-18 : 1 in both the acyl chains (79%) and alk-1-enyl chains (86%). The phospholipids showed a relatively narrow melting transition begin- ning at 24°C with both probes and ending at 12” C with TEMPO partitioning and at 15” C with diphenylhexatriene fluorescence polarization. Membranes from elaidate-grown cells also gave a relatively narrow inflection in diphenyl- hexatriene polarization studies, but the mid-point was 5°C higher than that obtained with the phospholipids.

These studies indicate that the changes observed in the polar head group and glycerol acetal derivative composition of cells grown on oleate, partially compensate for the greatly increased proportions of cis unsaturated and cyclo- propane acyl and alk-1-enyl chains in the phospholipids.

-

Introduction

A determinant of some membrane functions is the physical state of the membrane lipids. For example, the temperature optima for certain membrane functions and for cell growth fall within a range of temperatures in which most of the membrane lipids are in a fluid state [l-3]. It appears that some cells regulate their lipid composition and hence physical properties to maintain these temperature optima. Much of the recent increase in our understanding of the interrelationship of cell membrane physical properties and function has been obtained using cells in which the fatty acyl composition can be altered experi- mentally. These include unsaturated fatty acid auxotrophs of Escherichia coli [2,4,5] and of yeast [6], natural fatty acid requirers such as Mycoplasma laid- lawii [ 7,8] and animal cells which can be made dependent on a supplemental source of fatty acids [g-12].

In these organisms diacylphosphatides are the major lipid constituents of the cellular membranes. Varying amounts of ether lipids containing either alkyl or alk-1-enyl chains are found in certain animal tissues [13--151 and they are undoubtedly important in tissues in which certain membranes have a prepon- derance of ether lipids. For example, myelin sheath has an alk-l-enyl group to lipid P molar ratio of 0.31-0.37, and in ox and sheep heart mitochondria and microsomes, this molar ratio is 0.39-0.48 [ 131.

Our studies have shown that the lipids of Clostridium butyricum are rich in alk-l-enyl ether lipids (plasmalogens) [16] and that the hydrocarbon chains as well as the lipid class composition of the phosphoglycerides undergo changes in response to variations in growth temperature providing a model system for their study [17]. We have recently shown that, when grown in the absence of biotin, this organism incorporates exogenous unsaturated fatty acids into both acyl and alk-1-enyl chains * [ 181. Under these conditions, cells grown on oleate had nearly 90% 18 : 1 plus the 19 : cyc fatty acid derived from it among the

* In this paper alk-l-enyl chains of total phospholipids include the glycerolsubstituted alk-l-cnyl

chains in the glycerol acetal derivatives of the ethanolamine and N-m~thylethanolamine plasmalo-

gem [171. For acvl groups and alk-l-my1 groups. the number beimp the colon is the chain length

and after. the number of double bonds. 19 : CY indicates a Cl <) cyclopropanr-containing chain.

343

acyl chains and even larger proportions of these structures in the alk-1-enyl chains of the phosphoglycerides. Similar results have also been seen with elaidic acid, the trans isomer of oleic acid, except that the corresponding cyclopropane acid is not formed from this compound. When cells were grown with oleic acid, marked changes in the phospholipid class composition were also seen [18]. These findings led us to study the effects of these temperature-induced lipid compositional changes and of enrichment with either cis- or truns-18 : 1 on the physical properties of the membrane lipids. The results of some physical studies designed to provide answers to this question are described.

Experimental Section

Materials The pure fatty acids used in growth media were obtained from Nu-check

Prep, Elysian, Minn. Vitamin-free Casamino acids were obtained as a 10% sterile solution from Nutritional Biochemicals, Cleveland, Ohio. Glass-distilled solvents used in all preparative and analytical work were obtained from the Burdick and Jackson Laboratories, Inc., Muskegon, Mich. 2,2,6,6_tetramethyl- piperidine-1-oxyl (TEMPO) was synthesized as described [ 191. l-phenyl-6- phenylhexatriene (diphenylhexatriene) was obtained from Aldrich Chemical Co., Inc., Milwaukee, Wis. 53233.

Methods Cells. Clostridium butyricum (ATCC 6015) strain 8 was obtained in this labora-

tory upon repeated culture of cells on oleic acid with either suboptimal or no biotin. It requires lower levels of unsaturated fatty acids for maximal growth than the culture from which it was derived and has lower levels of long-chain acyl-CoA reductase (Day, J.I.E. and Goldfine, H., unpublished). Cultures were grown as described [18] with the exception that most cultures were supplemented with 25 mg/l fatty acid rather than 10 mg/l. All cultures were harvested during expo- nential growth or within 8 h of entering stationary phase. All cultures were microscopically examined for spores.

Lipids. Methods for extraction, washing and separation of the phospholipids from the non-polar fraction are described elsewhere [ 16,201. Polar lipids were separated on silica gel G thin-layer plates as described [lS]. The spots were scraped into acid-washed conical centrifuge tubes, digested overnight at 160°C in 0.5 ml 10 N H2S04 and oxidized with 0.5 ml 30% Hz02 for 2 h at the same temperature. Phosphorus was determined by the method of Bartlett [21]. After color development, the tubes were centrifuged at 3500 rev./min for 15 min before removing portions for measurement in a spectrophotometer at 830 nm. Alk-1-enyl groups were analyzed as free aldehydes and acyl groups as methyl esters by gas chromatography as described [17,18].

Electron paramagnetic resonance spectroscopy The phospholipids in CS, were dried in a tube under vacuum, a glass bead

was added and the lipids were dispersed in 0.05 M KP04, pH 7.0 buffer con- taining 0.15 M KCl, 75 mg lipid per ml, by vortexing. The temperature was

maintained near 60°C. TEMPO was added at an effective concentration of 0.5 mM and the sample transferred to a capillary tube. Some reduction of the spin label was seen after addition to the lipid sample, but it was small and appeared to be complete before the experiment was begun. Spectra were taken on a Varian E-3 EPR spectrometer regulated with a variable temperature controller, which was calibrated after each use.

Fluorescence measuremcn ts. An aliquot of dipl~e~~yll~exatrie~~e in tetrahydro- furan was added to the ~~hospholip~ds in CS? solution. Solvents were removed under a stream of nitrogen or argon, and buffer (0.9% NaCl~O.05 N KP04, pH 7 .O, 1 : 1 v/v) was added to yield a suspension of 0.3 mg phospholipid/l~l and 0.2 PM diphenylhexatriene. The suspension was sonicated for 1 min on a Heat-Systems Ultrasonics Sonifer (Plainview, N.Y .). For measurements below O”C, glycerol was added after sonification to a final concentration of 30% (v/v). It was independently established that glycerol in this concentration and added in this manner did not shift or broaden the phase transition of dimyristoyl phosphatidylcholine. In the case of labeling of the intact membrane, an aliquot of diphenylhexatriene in tetra~lydrofuran was added to the membrane suspen- sion containing 0.025 ymol lipid P/ml in 0.9% NaCl~O.05 N KPO, to yield a final concentration of 0.2 PM diphenylhexat~ene. The sample was incubated for 1 h at room temperature (=22”C) prior to fluorescence measurement. Flr\orescence spectra were measured on an Hitachi MPF-2A spectrofluorimeter equipped with a thermostated cell compartment. For measurement of fluores- cence polarization a Polacoat 4-B polarizer was used to polarize the excitation and emission beams. Fluorescence polarization, P, is defined as: P = (ri ( - cl, )/ (I,, + cl, ) where Ii, and I, are the fluorescence intensities observed with anal- yzing polarizer parallel and perpendicular to the normal of the laboratory axis respectively and c is a factor used to correct for the inability of the instr~lnl~nt to detect the two polarized beams with equal facility. Excitation and emission wavelengths were at 360 and 430 nm. Half-maximal band pass was 6-8 nm.

Results

Lipid composition. The phospholipid acyl and alk-l-enyl chain composi- tions of the cells used in this study are shown in Table I. The results of these analyses are in very good agreement with those previously published [ 17,181. Cells grown at 25°C in the presence of biotin have a significantly higher propor- tion of unsaturated plus cyclopropane acyl chains than cells grown at 37”C, whereas the proportion of unsaturated plus cyclopropane alk-l-enyl chains does not change significantly. Cells grown in biotin-free medium supplemented with oleic acid have an average of 84.9% and 90.8% of 18 : 1 plus 19 : cy in the phospholipid acyl and alk-1-enyl chains, respectively. Cells grown in such media and supplemented with elaidic acid, had high levels of 18 : 1 in both the acyl and alk-l-enyl chains. The acyl and alk-1-enyl chains from one batch of these cells were analyzed on a l/8 in X20 ft column of Silar IOC, 10% on 100/120 GCQ (Applied Science Labs., Inc., State College, Pa.) which separated the cis and truns isomers. The acyl chain 18 : 1 was found to be 90% trans and the alk- I-enyl 18 : 1 was 95% trans. As noted previously, very little 19 : cy is found in elaidate-grown cells; indicative of the high truns-18 : 1 content.

345

TABLE I

PHOSPHOLH’ID ACYL AND ALK-l-ENYL CHAIN COMPOSITION (wt.‘%) OF (‘LOSTRIDIIJM BIITY- RICllAl GROWN AT TWO TEMPERATURES WITH BIOTIN OR ON OLEIC ACID OR ELAIDIC ACID WITHOUT BIOTIN

Cells were grown at the indicated temperatures. Thr number of srparatt~ batches of cells analyYcd IS given in sc~uare brackets. Values are means +S.E.M.

Growth conditions Biotin

37°C [41

Biotin

25°C [51 Oleate 37°C [61

Elaidatc 37°C [3]

Acvl chains 14 : 0 16 : 0 16 : 1

17 : CY

18 : 0

18 : 1 19 : CY Total unsaturated

+ CYcloProPane Total saturated

Alk-1-enyl chains 16 : 0 16 : 1 17 : cy 18 : 0 18 : 1 19 : cy Total unsaturated + cyclopropane Total saturated

2.8 + 0.8 53.5 f 2.7

14.5 + 2.9 13.6 + 1.3

1.3 * 0.4 4.4 + 1.1

10.0 ? 1.9

42.4 * 2.8 57.6 + 3.0

43.1 + 1.7 11.8 F 1.7 22.5 + 3.1

4.3 t 1.1 3.9 + 1.2

14.6 ?- 1.6

52.7 t 1.7 47.2 ?; 1.7

3.0 ? 0.4 46.6 ? 1.6 24.3 + 1.3 14.9 i 1.1

0.84 t 0.12 5.0 + 0.6 5.0 + 0.6

49.4 i 1.7

50.4 t 1.7

48.3 i 3.5 18.3 f 2.9 20.7 + 1.2

2.3 f 0.3 3.4 i 0.9 6.8 f 0.9

49.1 f 3.2 50.8 + 3.2

3.9 i 0.6 2.7 + 1.9 6.4 t 1.0 6.3 f 4.3 2.8 ?- 0.5 2.0 f 1.0

0.58 + 0.22 0.6 _t 0.6 1.4 f 0.6 0.6 f 0.4

63.6 t 3.6 87.5 t 7.7 21.3 _+ 2.5 0.5 ? 0.5

88.3 i 1.5

11.8 i 1.5

3.0 i 1.0 3.2 + 0.5 1.9 i 0.6 0.87 * 0.53

20.0 * 3.3 70.8 i 4.5

95.9 + 1.1

4.1 f 1.1

90.6 + 5.7 9.6 ? 5.7

3.1 f 1.0 2.8 + 1.1 3.0 ? 1.1 1.3 i 1.1

90.0 +_ 2.0 -

95.8 * 1.1 4.4 ? 1.1

TABLE II

COMPOSITION OF PHOSPHATIDES OF CLO.STRIDII!.II A~~T~‘RICI:N. EFFECT OF GROWTH TEM- PERATURE AND SUPPLEMENTATION WITH EXOGENOUS FATTY ACIDS

Cells wert’ grown at the indicated temperatures. The number of separate batches of cells analyzed is given in square brackets. Values are means ?S.E.M. Numbers in parenthesis represent the percent of the phos- pholipid in the alk-1-enyl-acyl form. Plasmalogen data for biotin-grown cells are from Khuller and Goldfinc [ 171 and for the ethanolamine pIusN-methylethanolamine phosphoglycerides of oleic acid-grown cells from Khuller and Goldfine [ 181. The other values are from this study and were obtained with representative sam- ples. a This fraction contains: (1) Mainly phosphatidylglycerol in log-phase biotin-grown cells 1161. (II) Mainly phosphatidylglycerol. but scnne cardiolipin was visible on TLC in oleate-grown cells. This was not

analyzed. (III) Phosphatidylglycerol and cardiolipin (57 : 43) in elaidate-grown cells.

Growth conditions Biotin 37OC [41 Biotin 25’C [51 Oleic 37OC 161 Elaidic 37’C 131

Lipid Classes % of lipid phosphorus

Ethanolamine and N-

methylethanolamine phosphoglycerides

46.3 ? 3.0(71) 45.0 + 2.1(63) 30.1 f 3.0(54) 32.5 + 0.4(80)

Glycerol acetals of ethanol- 29.2 I 1.6 22.6 f 0.8 49.0 + 2.0 25.9 1 2.0

amine and N-methyletha- nolaminc plasmalogens

Glycerol phosphoglycerides a 22.1 t 1.3(38) 27.8 + 0.7(60) 17.1 f 2.1(14) 27.3 + 1.7(63)

Unknown 2.5 + 1.1 4.6 + 1.5 3.8 f 2.2 14.4 + 0.9

The phospholipid class compositions are given in Table II. In cells grown in biotin medium at 25°C there is an increase in the glycerol phosphoglycerides, which we had previously shown to be largely in alk-l-enyl-acyl glycerylphos- phorylglycerol [17]. The concomitant decrease in the ethanolamine plus N- methylethanolamine phosphoglycerides is less marked, but there is a decrease in the glycerol acetal derivatives of the plasmalogen form of these phospho- glycerides [ 17’1.

In oleate-grown cells there is a large increase in the glycerol acetal derivatives of the ethanolamine plus N-methylethanolamine plasmalogens, as previously reported [ 181. The decrease in the other ethanolamine plus N-methylethanol- amine phosphoglycerides, however, was not as marked, as noted previously nor were large increases in less polar unknown lipids observed. In cells grown in biotin-free medium supplemented with elaidic acid the most noticeable change is a decrease in the ethanolamine plus N-methylethanolamine phosphogly- cerides and increases in unidentified phospholipids, two of which represented 6 and ‘7% of the total lipid phosphorus. The increase in the glycerol phospho- glyceride fraction is equal to that seen in biotin-grown cells at 25°C. Two- dimensional TLC, with HCl vapor treatment between the first and second runs, was used to determine the proportions of diacyl and alk-l-enyl-acyl forms of the ethanolamine plus N-methylethanolamine phosphoglycerides and the gly- cerolphosphoglycerides [22]. The ethanolamine plus N-methylethanolamine phosphoglycerides were 80% alk-1-enyl-acyl and the glycerol phosphoglycerides were 63% alk-1-enyl-acyl. These alk-l-enyl-acyl values are somewhat higher than those found in cells grown on biotin at 37°C [ 17). Indeed, we have recently observed that the glycerol phosphoglycerides in oleate-grown cells are only 14% alk-1-enyl-acyl. Thus elaidate-grown cells have high alk-l-enyl-acyl to diacyl ratios, whereas these ratios are low in oleate-grown cells [ 181.

It was previously shown that the proportions of the ethanolamine plus N- methylethanolamine phosphoglycerides in the alk-l-enyl-acyl (plasmalogen) form changed only slightly with growth temperature; 75% at 37°C and 64% at 25°C. We had also reported that the fraction containing the glycerol acetals of the ethanolamine plus N-methylethanolamine plasmalogens had higher amounts of alkali-stable lipid when isolated from cells grown at the lower temperatures [17]. We have since found that this fraction contains 95% of a single compo- nent by the use of two-dimensional TLC and this component is 94% alkali- stable and 90% labile in 90% acetic acid at 37”C, overnight *. Our previous results were obtained by the alkaline hydrolysis procedure of Tarlov and Ken- nedy [24], in which the alkali is neutralized with excess Dowex-50 H’, after completion of hydrolysis. It appears that the glycerol acetals are more labile than the plasmalogens and are extensively hydrolyzed in the presence of the cation exchange resin, whereas the percent alkali-stable obtained by this proce- dure with the plasmalogens agreed well with previous estimations of alkali- stable lipid [ 161 and with independent measurements of iodine uptake by the alk-lenyl bonds [ 171.

* Alkaline hydrolysis of the glycerol acetals. approximately 1 mg, was in 1 ml of 1 M NaOH at 37°C

for 20 min followed by careful neutralization with HCl. On partition against chloroformlisobuta-

no1 (3 : 1 v/v), 94% of the phosphate remained lipid-soluble. TLC showed that most of the original

compounds had been converted to a deacylated product. as reported by Matsumoto et al. 1231.

347

Physical studies EPR. Fig. 1 shows plots of the TEMPO spectral parameter cy (cu = B/F) [25]

vs. l/T for aqueous dispersions of the phospholipids. This parameter provides a measure of the partitioning of TEMPO between the fluid hydrophobic regions of the phospholipid bilayers and the aqueous phase [26,27]. Each curve shows at least two deviations from linearity. At one of these, T1, which occurs in a nar- row range of temperatures, the curves become essentially horizontal with increasing temperature (Table III). In the plots for phospholipids from the cells grown at 25 or 37” C with biotin, a second inflection was invariably seen and the average position of these was within two or four degrees of the growth tem- perature. The temperature (T2) of this break for the 25”Cgrown cells was more variable than that for the 37”Cgrown cells (Table III). This second deviation from linearity for oleate- and elaidate-grown cells did not occur close to the growth temperature of 37” C. Their average positions were 28 and 24” C, respec- tively. The TEMPO partitioning curves obtained with the phospholipids from elaidate-grown cells are unique in character and show a clear third break at 11.8” C (Fig. 1D). A third break appears to be present in the curve from the oleate-grown phospholipids, but this was not reproducible.

Fluorescence polarization. The steady-state de-polarization of diphenylhexa- triene reflects the probe’s rotational motion within the lipid bilayer. Polarization increases as the hydrocarbon chains become more ordered. The fluorescence polarization of the probe in dispersions of phospholipids from C. butyricum grown with biotin at 37°C showed a broad inflection beginning at 35.8 ? 1.4”C and ending at -5°C. No additional deviations from linearity were consistently seen between these two temperatures (Fig. 2A and B). Phospholipids from cells grown at 25°C in the presence of biotin produced a similar broad inflec- tion beginning at 32 f 2°C and ending at -5°C (Fig. 2C and D). Although several small deviations from linearity were seen between 10 and 30°C only those at 23 + 1°C were seen more than once. With the phospholipids from oleate-grown cells, a broad transition beginning at 29 and ending at -2°C can be seen (Fig. 2E and F). Below 0°C the curve did not flatten as much as those seen with lipids from biotin-grown cells. It should also be noted that the aver- age fluorescence polarization values for phospholipids from cells grown with biotin at both 25 and 37°C and with oleate at 37°C were within 0.02 units of each other over the range of measurements from 10 to 40°C. However, below 10°C the average polarization values of the phospholipids from oleate-grown

TABLE III

DEVIATIONS FROM LINEARITY OBSERVED IN PLOTS OF TEMPO PARTITIONING VS. T-’ FOR

PHOSPHOLIPIDS FROM CZ~OSTRZDZUMBLITYRZCUhl

Numbers in parenthesis indicate separate determinations. Values are means i- S.E.M. observed in at least

three runs. In earlier runs. measurements were not taken at high enough temperatures.

Growth condition t1 t2 t3

Biotin - 37”C(6) 48.3OC ? 0.3”C 34.5Oc + l.fC

Biotin - 25°C(5) 45 OC? 2 Oc 29.4OC f 1.8’C

Oleate - 37OC(7) 44 “Ck2 Oc 28 Oct2 Oc

Elaidate - 37’C(2) 46 Oc f 0.5Oc 24 Oc * 0.5Oc 12°C

348

+ 2 00000

CI 00 0 0

I

In* m “r 00 0 0 0

c3

349

Temrmtwe (T) --+

Fig. 2. Diphenylhexatriene fluorescence polarization in dispersions of phospholipids from Clostridium

but,vricum. (A and B) Cells grown with biotin at 37°C. (C and D) Cells grown with biotin at 25°C. (E and F) Cells grown without biotin plus oleate at 37°C. (G) Cells grown without biotin Phs elaidate at 37°C.

cells were lower and the difference increased to 0.05 units at 0°C. The phos- pholipids from elaidate-grown cells yielded a strong, narrower transition begin- ning at 24 + 1°C and ending at 15°C with its midpoint near 19°C (Fig. 2G).

The sharp inflection seen with ESR and fluorescence polarization in the lipids from elaidate-grown cells prompted a study of their membranes. Mem- branes were prepared by a previously published method [28] and the fluores- cence polarization of diphenylhexatriene was measured. A strong inflection beginning at 28 and ending at 20°C with a midpoint at 24°C was seen. This curve parallels that for the lipids, but is about 5” C higher (data not shown).

Discussion

Using both spin-labeled and fluorescent probes, we have observed significant differences and some similarities in the thermotropic behavior of phospholipids from C. butyricum grown with biotin at 25 and 37°C and with oleate or elai- date in the absence of biotin. These properties undoubtedly arise from the rela- tive compositions of the phospholipids and their acyl and alk-1-enyl chains.

Each of the phospholipid mixtures showed at least two deviations from linear- ity in Arrhenius plots of TEMPO partitioning vs. temperature. One break was seen with each sample well above the growth temperature, where the curve became horizontal. TEMPO solubility therefore had approached a maximum in lipid bilayers. Below this temperature, a second break, designated T1, was seen with all samples. This temperature was within 2-4°C of the growth tempera- tures in the samples from biotin-grown cells, but 9 and 13°C below the growth temperatures in the samples from oleate- and elaidate-grown cells, respectively.

Fluorescence polarization of diphenylhexatriene revealed some features in common with those seen with TEMPO and some distinct characteristics, espe- cially at low temperatures unprobed by TEMPO. At high temperatures diphenylhexatriene polarization in all samples was low and decreased very gradually as a function of temperature. The transition from a flatter to a rela- tively steep curve occurred within l-3°C of T, seen with TEMPO for the 4 mixtures examined. The lower end of these broad inflections was only studied with the fluorescent probe. In the lipids from biotin cells grown at both 25 and 37” C, it occurred at -5” C. In the lipids from oleate-grown cells it occurred at -2”C, but the relatively low polarization values below this temperature allow for the possibility that other breaks may occur. These low temperature proper- ties were not examined with TEMPO, owing to its low partition coefficient at these temperatures. Both the diphenylhexatriene polarization and TEMPO curves for phospholipids from elaidate-grown cells showed a narrow inflection at the same temperatures.

The probes used in this study have been well characterized in model systems and TEMPO has been used to study several biological systems [27,29]. In the pure lipid dispersions and binary mixtures studies, the partitioning of TEMPO between the fluid regions of the membrane and the aqueous phase has been found accurately to reflect the melting temperatures and the onset and comple- tion of phase separations [26,30]. Deviations from linearity can also arise in plots of TEMPO partitioning vs. temperature well above the melting transition of pure lipids such as dioleyoyl phosphatidylcholine [ 251, It has been suggested that they represent the disappearance of clusters of relatively ordered lipid molecules above the melting transition [25] or a general decrease in chain ordering throughout the lipid bilayer [ 301.

The fluorescent probe diphenylhexatriene resides in the hydrophobic regions of the lipid bilayer and its steady-state depolarization is sensitive to the melting transition of pure lipids in all systems studied including those with transitions below 0°C [ 31,321. In the binary mixtures studied it appears to monitor the onset and completion of phase separation.

Due to the complexity of the lipid mixtures from C. butyricum, the inter- pretation of our data is necessarily difficult. The narrow inflection seen with lipids and membranes from elaidate-grown cells with both probes most prob- ably reflects lipid melting. Model compounds such as dielaidoyl phosphatidyl- choline and natural membranes enriched with elaidate melt in this temperature range or higher. For example, in E. coli auxotrophs containing a large amount of dielaidoyl phosphatidylethanolamine, the melting transition occurs between approx. 32” and 38°C [3]. It follows that the influences of the lipids other than dielaidoyl phosphatidylethanolamine in C. butyricum serve to lower the

351

main transition. A 5°C lowering would be expected for N-methylethanol- amine as compared to ethanolamine phosphoglycerides [ 331, but the effects of the large amounts of alk-1-enyl-acyl lipids and their acetal derivatives is not known.

Considering the high content of cis-16 : 1 and 17 : cy in biotin-grown cells and of cis-18 : 1 and 19 : cy in oleate-grown cells, it is reasonable that the lowest temperature breaks observed with diphenylhexatriene also arise from melting phenomena. Therefore, it is probable that the highest temperature breaks, T,, seen by TEMPO, but not by diphenylhexatriene, do not arise from melting phenomena. They may represent an asymptote to a limiting fluid state. The interpretation of the intermediate breaks seen with TEMPO are not clear at this time, but since those with phospholipids from biotin37”C and oleate-37°C cells correspond to the high temperature of diphenylhexatriene polarization inflections, they probably correspond to the completion of melt- ing in these lipid mixtures. In order to interpret our observations more com- pletely it will be necessary to work with pure lipid components and their mix- tures. Preliminary examination of diphenylhexatriene fluorescence in disper- sions of the purified glycerol acetal derivatives of the ethanolamine and N- methylethanolamine plasmalogens from oleate-grown cells gave a plot remark- ably similar to that seen in Fig. 2E and F for the total phospholipids from these cells.

These results further indicate that the compositional alterations seen in C. butyricum lipids from cells grown at different temperatures are compensatory even though more complex than those seen in E. coli. That is, not only do the acyl chains become more unsaturated, but the alk-1-enyl chains of the plasma- logens become more saturated and the proportions of the glycerol phospho- glycerides increase at lower growth temperatures [ 171.

When C. butyricum cells grown at 37°C are forced to incorporate cis or truns-18 : 1 into their lipids, extensively, the temperatures of the TEMPO and fluorescence polarization inflections change. It appears that the cells are not completely able to overcome these perturbations from their normal composi- tions. We have pointed out the marked increase of the glycerol acetal deriva- tives of plasmalogens seen in oleate-grown cells. A molecular model of this lipid has been made and it is clear that the extra glycerol moiety serves as an addi- tional polar head group and is capable of hydrogen bonding to neighboring phospholipids through their phosphate and carboxyl esters. It is tempting to speculate that such hydrogen bonding helps to stabilize lipid-lipid interaction in the cell membrane and thus serves to compensate for the high degree of enrichment of oleate.

Verkley et al. [40] have observed lipid-phase transitions by freeze-etch elec- tron microscopy in two other anaerobes, Veillonella parvula and Anaerovibrio lipolytica, with high proportions of plasmalogens in their phospholipids. At 23°C they observed lateral redistribution of cell membrane particles into small aggregates, which became completely aggregated at 0°C. The extracted lipids of V. paruula also showed a significant enthalpy change between 5” and 20°C by differential scanning calorimetry. These organisms have simpler lipid compo- sitions than C. butyricum and may provide additional insights into the melting behavior of ethanolamine plasmalogens.

352

Acknowledgements

This investigation was supported by grants from the National Institute of Allergy and Infectious Diseases (AI 08903) and the National Institute of General Medical Sciences (GM 23244 and GM 12202). J.M.V. is the recipient of an NIH Career Development Award GM 00053 and A.F.H. is recipient of the Dr. W.D. Stroud Established Investigatorship of the American Heart Associa- tion.

References

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