38
Biochimica et Biophysica Acta, 552 (1979) 38--52 © Elsevier/North-Holland Biomedical Press
BBA 78300
MODIFICATION OF MEMBRANE LIPIDS P H E N E T H Y L ALCOHOL-INDUCED A L T E R A T I O N OF LIPID COMPOSITION IN TETRAHYMENA MEMBRANES
Y O S H I N O R I N O Z A W A , R E I K O KASAI and T A K A S H I S E K I Y A
Department of Biochemistry, Gifu UniversitySchool of Medicine, Tsukasamachi-40, Gifu (Japan) (Received August 16th, 1978)
Key words: Lipid composition; Phenethyl alcohol treatment; Membrane modification; (Tetrahymena pyriformis)
Summary
Tetrahymena pyriformis NT-I cells in the early-logarithmic phase were incubated with phenethyl alcohol (2-phenylethanol) and effects on the lipid composition were examined in various membranes. 1. There was a marked modification in phospholipid head, as well as fatty acyl group composition in pellicles, mitochondria and microsomes of the phenethyl alcohol-treated cells. Compared with membranes of the control cells, the membranes from phenethyl alcohol-treated cells were found to contain a higher level of phosphatidylcholine content with the compensating decrease in phosphatidylethanolamine, while 2-aminoethylphosphonolipid showed only a slight decrease in these membranes. The acyl group profile of membrane phospholipids in the presence of phenethyl alcohol was also modified so that a p r o f o u n d elevation of the c o n t e n t of polyunsaturated fatty acids, linoleic and ~/-linolenic acids. The major monounsaturate, palmitoleate decreased. Such lipid alteration is a reversible process, and therefore upon removal of phenethyl alcohol the modified lipid composition returned to normal. 2. By freeze-fracture electron microscopy in combination with temperature quenching, the outer alveolar membrane of the phenethyl alcohol-treated cell was observed to reveal less aggregation of intercalated-membrane particles, as compared with the control membrane. The quantitative analysis of the thermotropic lateral m o v e m e n t of membrane particles provided evidence that the membrane in the phenethyl alcohol-treated cell became more fluid. Such fluidizing effects m a y result from an increase in the acyl group unsaturation and also in the phosphatidylcholine content. 3. With regard to the mechanism responsible for the marked decrease in
39 palmitoleate in membrane phospholipids, there was found a depressed conversion of the palmitate to palmitoleate in the phenethyl alcohol-treated cells. It was further suggested that the drug may have an inhibitory effect on the synthesis of palmitoyl-CoA desaturase involving the (16 : 0 -~ 16 : 1) conversion. Also, it was demonstrated that the increase in a precursor-product fashion of phosphatidylcholine with the corresponding decrease in phosphatidylethanolamine was not due to transformation of phosphatidylethanolamine to phosphatidylcholine through stepwise methylation.
Introduction In order to shed light on the problem of the structure-function relationship in biomembranes, one useful approach has been to manipulate either quantitatively or qualitatively the membrane lipid composition of cells under the defined growth conditions, and to examine functions in the membranes with altered lipid composition. Although various methods for modifying membrane lipids have been available [1,2], one of the most commonly used procedures is to supplement cells with different substances. The majority of studies of membrane lipid manipulation has largely involved alterations in the phospholipid acyl chain composition in bacteria and mycoplasma [1,3,4]. However, these manipulation techniques have recently been extended to animal cells growing in tissue cultures for altering not only fatty acid [5--7] but also polar head group [8--12] composition of membrane phospholipids. Indeed, since Vagelos' group has developed techniques to manipulate the phospholipid composition of mouse fibroblasts in tissue culture with choline analogues [8], they have extensively investigated effects of lipid alterations upon physical properties and enzyme activities of membranes [13--16]. Since Tetrahymena cells have a proven ability to modify their membrane lipid composition in response to growth conditions, this cell also is a promising system for better understanding the roles which lipids play in membrane-associated activities in the cell [17]. Actually, supplementation with sterols [18,19], fatty acids [20,21], glyceryl ether [22], choline analogues (unpublished data) has been applied to Tetrahymena cells. Recently, Nunn and co-workers [23--25] have shown that phenethyl alcohol induces a drastic alteration in the lipid composition in Escherichia coli. We applied this drug to Tetrahymena cells for modifying the membrane lipid composition. In this communication, we describe the phenethyl alcoholinduced alteration in lipid composition of various membranes, and also the mechanisms for lipid alterations by the drug will be discussed. Materials and Methods
Cell growth and membrane isolation. Culture conditions for strain NT-I of Tetrahymena pyriformis were described in a previous paper [26]. Cells were grown at 39.5°C in an enriched proteose-peptone medium in the shaking incubator at 90 cycles per min. Cell growth was monitored by counting cell number by a hemocytometer. When cell density reaches the early logarithmic
40 phase, 20 • 104 cells/ml, phenethyl alcohol (2-phenylethanol, C6HsCH2CH:OH, Nakarai Kagaku Chemicals Co., K y o t o ) was added dropwise into the culture medium to a final concentration of 5.5 mM and then incubation was further continued. Isolation of various membrane fractions from control and phenethyl alcohol-treated cells was performed according to the procedure of Nozawa and Thompson [27]. Lipid extraction and analysis. Lipids were extracted from whole cells and various membrane fractions b y the m e t h o d of Bligh and Dyer [28], and individual phospholipids were separated on silica gel H plate. After charring the developed plate with 50% H2SO4, the bands corresponding to individual phospholipids were scraped o f f the plate and their phosphorus contents were determined by the Rouser's m e t h o d [29]. The phospholipid fraction was separated from neutral lipids by a silicic acid-Hyflo Super Cel column chromatography as previously described [ 26 ]. Total phosphorus in extracted lipids was determined by the method of Bartlett [30], as modified by Marinetti [31]. Quantitative analysis of fatty acids was carried o u t by gas-liquid chromatography as previously described [26]. Radioisotope labeling. For determination of lipid synthesis, 20-ml aliquots of the growth culture were withdrawn at several times during incubation with phenethyl alcohol and labeled with 5 pCi of [14C]acetate (NEN, 58.3 Ci/mol) for 30 min before extraction. The incorporation of [~4C]acetate into the individual lipid fractions was measured by counting the radioactivities in areas corresponding to lipid fractions separated on silica gel H plate using a Beckman LS-8000 scintillation counter. The desaturation rate of palmitate to palmitoleate was measrued according to the procedure described previously [32]. Samples (20 ml) were taken at desired intervals from the control and phenethyl alcohol-treated cultures, and pulse-labeled with 0.25 ~Ci of [~4C]palmitate (NEN, 50.2 Ci/mol) for 20 min. Furthermore, the rate of conversion of phosphatidylethanolamine into phosphatidylcholine was estimated b y incorporation of [~4C]ethanolamine into phosphatidylcholine fraction. The 200-ml culture was labeled with 5 ~Ci of [~4C]ethanolamine (The Radiochemical Centre, Amersham, 55 Ci/mol) for 165 min, and labeled cells were harvested by centrifugation at 164 × g for 5 min and the resuspended in the fresh medium. The culture was divided into two portions and to one of them phenethyl alcohol was added to a concentration of 5.5 mM. Lipids were extracted from both cultures at different intervals during labeling. The radioactivities in individual phospholipids separated on the thin-layer plate were measured as above. Freeze-fracture electron microscopy. Freeze-fracture electron microscopy has been proved to be an useful technique for the quantitative analysis of physical states of membrane lipids. Detailed procedures have been described in the previous paper [33]. The particle density index was used, which can be calculated from the equation (X --A/B - - A ) × 100, where A, B and X is the particle n u m b e r of 39.5°C -, 0.5°C-fixed membranes and membrane to be examined at a given temperature, respectively. Cells grown at 39.50C in the presence and absence of phenethyl alcohol were cooled to the desired temperatures over 4 min. After 5 min incubation at each temperature samples were fixed by glutaraldehyde at a final concentration of 1%. The fixed samples were freeze-fractured in Hitachi device and the obtained replicas were examined
41
with a JEM 100U electron microscope. The protoplasmic fracture face of the outer alveolar membrane was used to monitor distribution of membraneintercalated particles for determination of the particle density index. Results
Effects o f phenethyl alcohol on growth and lipid synthesis in T. pyriformis NT-I Phenethyl alcohol at different concentration (0.6--9.8 mM) was added to the culture in the early logarithmic phase. There was a somewhat dose
Biochimica et Biophysica Acta, 552 (1979) 38--52 © Elsevier/North-Holland Biomedical Press
BBA 78300
MODIFICATION OF MEMBRANE LIPIDS P H E N E T H Y L ALCOHOL-INDUCED A L T E R A T I O N OF LIPID COMPOSITION IN TETRAHYMENA MEMBRANES
Y O S H I N O R I N O Z A W A , R E I K O KASAI and T A K A S H I S E K I Y A
Department of Biochemistry, Gifu UniversitySchool of Medicine, Tsukasamachi-40, Gifu (Japan) (Received August 16th, 1978)
Key words: Lipid composition; Phenethyl alcohol treatment; Membrane modification; (Tetrahymena pyriformis)
Summary
Tetrahymena pyriformis NT-I cells in the early-logarithmic phase were incubated with phenethyl alcohol (2-phenylethanol) and effects on the lipid composition were examined in various membranes. 1. There was a marked modification in phospholipid head, as well as fatty acyl group composition in pellicles, mitochondria and microsomes of the phenethyl alcohol-treated cells. Compared with membranes of the control cells, the membranes from phenethyl alcohol-treated cells were found to contain a higher level of phosphatidylcholine content with the compensating decrease in phosphatidylethanolamine, while 2-aminoethylphosphonolipid showed only a slight decrease in these membranes. The acyl group profile of membrane phospholipids in the presence of phenethyl alcohol was also modified so that a p r o f o u n d elevation of the c o n t e n t of polyunsaturated fatty acids, linoleic and ~/-linolenic acids. The major monounsaturate, palmitoleate decreased. Such lipid alteration is a reversible process, and therefore upon removal of phenethyl alcohol the modified lipid composition returned to normal. 2. By freeze-fracture electron microscopy in combination with temperature quenching, the outer alveolar membrane of the phenethyl alcohol-treated cell was observed to reveal less aggregation of intercalated-membrane particles, as compared with the control membrane. The quantitative analysis of the thermotropic lateral m o v e m e n t of membrane particles provided evidence that the membrane in the phenethyl alcohol-treated cell became more fluid. Such fluidizing effects m a y result from an increase in the acyl group unsaturation and also in the phosphatidylcholine content. 3. With regard to the mechanism responsible for the marked decrease in
39 palmitoleate in membrane phospholipids, there was found a depressed conversion of the palmitate to palmitoleate in the phenethyl alcohol-treated cells. It was further suggested that the drug may have an inhibitory effect on the synthesis of palmitoyl-CoA desaturase involving the (16 : 0 -~ 16 : 1) conversion. Also, it was demonstrated that the increase in a precursor-product fashion of phosphatidylcholine with the corresponding decrease in phosphatidylethanolamine was not due to transformation of phosphatidylethanolamine to phosphatidylcholine through stepwise methylation.
Introduction In order to shed light on the problem of the structure-function relationship in biomembranes, one useful approach has been to manipulate either quantitatively or qualitatively the membrane lipid composition of cells under the defined growth conditions, and to examine functions in the membranes with altered lipid composition. Although various methods for modifying membrane lipids have been available [1,2], one of the most commonly used procedures is to supplement cells with different substances. The majority of studies of membrane lipid manipulation has largely involved alterations in the phospholipid acyl chain composition in bacteria and mycoplasma [1,3,4]. However, these manipulation techniques have recently been extended to animal cells growing in tissue cultures for altering not only fatty acid [5--7] but also polar head group [8--12] composition of membrane phospholipids. Indeed, since Vagelos' group has developed techniques to manipulate the phospholipid composition of mouse fibroblasts in tissue culture with choline analogues [8], they have extensively investigated effects of lipid alterations upon physical properties and enzyme activities of membranes [13--16]. Since Tetrahymena cells have a proven ability to modify their membrane lipid composition in response to growth conditions, this cell also is a promising system for better understanding the roles which lipids play in membrane-associated activities in the cell [17]. Actually, supplementation with sterols [18,19], fatty acids [20,21], glyceryl ether [22], choline analogues (unpublished data) has been applied to Tetrahymena cells. Recently, Nunn and co-workers [23--25] have shown that phenethyl alcohol induces a drastic alteration in the lipid composition in Escherichia coli. We applied this drug to Tetrahymena cells for modifying the membrane lipid composition. In this communication, we describe the phenethyl alcoholinduced alteration in lipid composition of various membranes, and also the mechanisms for lipid alterations by the drug will be discussed. Materials and Methods
Cell growth and membrane isolation. Culture conditions for strain NT-I of Tetrahymena pyriformis were described in a previous paper [26]. Cells were grown at 39.5°C in an enriched proteose-peptone medium in the shaking incubator at 90 cycles per min. Cell growth was monitored by counting cell number by a hemocytometer. When cell density reaches the early logarithmic
40 phase, 20 • 104 cells/ml, phenethyl alcohol (2-phenylethanol, C6HsCH2CH:OH, Nakarai Kagaku Chemicals Co., K y o t o ) was added dropwise into the culture medium to a final concentration of 5.5 mM and then incubation was further continued. Isolation of various membrane fractions from control and phenethyl alcohol-treated cells was performed according to the procedure of Nozawa and Thompson [27]. Lipid extraction and analysis. Lipids were extracted from whole cells and various membrane fractions b y the m e t h o d of Bligh and Dyer [28], and individual phospholipids were separated on silica gel H plate. After charring the developed plate with 50% H2SO4, the bands corresponding to individual phospholipids were scraped o f f the plate and their phosphorus contents were determined by the Rouser's m e t h o d [29]. The phospholipid fraction was separated from neutral lipids by a silicic acid-Hyflo Super Cel column chromatography as previously described [ 26 ]. Total phosphorus in extracted lipids was determined by the method of Bartlett [30], as modified by Marinetti [31]. Quantitative analysis of fatty acids was carried o u t by gas-liquid chromatography as previously described [26]. Radioisotope labeling. For determination of lipid synthesis, 20-ml aliquots of the growth culture were withdrawn at several times during incubation with phenethyl alcohol and labeled with 5 pCi of [14C]acetate (NEN, 58.3 Ci/mol) for 30 min before extraction. The incorporation of [~4C]acetate into the individual lipid fractions was measured by counting the radioactivities in areas corresponding to lipid fractions separated on silica gel H plate using a Beckman LS-8000 scintillation counter. The desaturation rate of palmitate to palmitoleate was measrued according to the procedure described previously [32]. Samples (20 ml) were taken at desired intervals from the control and phenethyl alcohol-treated cultures, and pulse-labeled with 0.25 ~Ci of [~4C]palmitate (NEN, 50.2 Ci/mol) for 20 min. Furthermore, the rate of conversion of phosphatidylethanolamine into phosphatidylcholine was estimated b y incorporation of [~4C]ethanolamine into phosphatidylcholine fraction. The 200-ml culture was labeled with 5 ~Ci of [~4C]ethanolamine (The Radiochemical Centre, Amersham, 55 Ci/mol) for 165 min, and labeled cells were harvested by centrifugation at 164 × g for 5 min and the resuspended in the fresh medium. The culture was divided into two portions and to one of them phenethyl alcohol was added to a concentration of 5.5 mM. Lipids were extracted from both cultures at different intervals during labeling. The radioactivities in individual phospholipids separated on the thin-layer plate were measured as above. Freeze-fracture electron microscopy. Freeze-fracture electron microscopy has been proved to be an useful technique for the quantitative analysis of physical states of membrane lipids. Detailed procedures have been described in the previous paper [33]. The particle density index was used, which can be calculated from the equation (X --A/B - - A ) × 100, where A, B and X is the particle n u m b e r of 39.5°C -, 0.5°C-fixed membranes and membrane to be examined at a given temperature, respectively. Cells grown at 39.50C in the presence and absence of phenethyl alcohol were cooled to the desired temperatures over 4 min. After 5 min incubation at each temperature samples were fixed by glutaraldehyde at a final concentration of 1%. The fixed samples were freeze-fractured in Hitachi device and the obtained replicas were examined
41
with a JEM 100U electron microscope. The protoplasmic fracture face of the outer alveolar membrane was used to monitor distribution of membraneintercalated particles for determination of the particle density index. Results
Effects o f phenethyl alcohol on growth and lipid synthesis in T. pyriformis NT-I Phenethyl alcohol at different concentration (0.6--9.8 mM) was added to the culture in the early logarithmic phase. There was a somewhat dose
