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Effects of thiamine and pyridoxine on the lipid composition of Saccharomyces carlsbergensis 4228
483
Biochimica et Biophysiea Acta, 486 (1977) 483-489 @ Elsevier/North-Holland Biomedical Press
BBA 56943
EFFECTS OF THI~INE AND PYRIDOXINE ON THE LIPID COMPOSrT~ONOF SACCliA~~MYCESCA~LS~E~~E~SIS4228
YOSHIKI NISHIKAWA, ICHIRO NAKAMURA, SABURO FUKUI *
TEIJIRO KAMIHARA
~abo~to~ of Indu~~~l ~iochem~~~, ~e~artrn~~t ~n~nee~~ng, Kyoto Unive~ity~ Kyoto (Japan)
and
of ~~dus~~al Chemis~y, Faculty of
(Received August 24th, 1976)
Summary The lipid composition of Saccharomyces ca~ls~erge~sis 4228 cells grown aerobically in the presence of thiamine and absence of pyridoxine was markedly different from that of cells grown without addition of both of the growth factors. In addition to the previous observations showing a reduction in the levels of unsaturated fatty acids (Nishikawa, Y., Nakamura, I., Kamihara, T. and Fukui, S. (1974) Biochem. Biophys, Res. ~ommun. 59, 777-780) and lack of zymosterol and ergosterol (Nagai, J., Katsuki, H., Nishikawa, Y., Nakamura, I., Kamihara, T. and Fukui, S. (1974) Biochem, Biophys. Res. Commun. 60, 555-560), the thiamine-grown cells were found to contain low levels of total lipids, sterols (especially in the form of esters), triacylglycerols and total phospholipids. However, relative contents of triacylglycerols and phospholipids to total lipids were higher than those of control cells. Hydroc~bons and diacylglycerols accumulated to appreciable degrees. Phospholipid composition was also influenced by thiamine. The ratio of phosphatidylinositol to total phospholipids increased, whereas that of phosphatidylethanolamine decreased. The levels of phosphatidylcholine plus phosphatidylserine decreased in a similar ratio to that of total phospholipids. It was found that unsaturated fatty acid contents were low in all lipid esters tested. The effect of thiamine was particularly noteworthy in the case of sterol esters. Concomitant addition of pyridoxine with thiamine to the medium brought about a normal lipid composition in the yeast cells. Introduction As reported previously from our laboratory, Saccharomyces carlsbergensis 4228 (ATCC 9080) grown in the presence of thiamine and absence of pyri* To whom correspondence and reprint requests should be sent.
484
doxine exhibited a markedly low level of unsaturated fatty acids [l] and contained no zymosterol and ergosterol 121. We have also reported that the cells showed a markedly low respiration rate, low activities of respiratory enzymes and had no cytochromes [3,4). It was also found that the cells had a low level of vitamin B-6 [3], and that the addition of pyridoxine to the medium abolished the effects of thiamine [l-4]. These findings, therefore, suggest that thiamine causes some fundamental changes in the cellular lipid composition as a consequence of induced vitamin B-6 deficiency. In this paper, we describe in detail the effects of thiamine and pyridoxine on the lipid composition of yeast cells. Materials and Methods Grouts of yeast 5’. carzsberge~s~s 4228 (ATCC 9080) was maintained on 2% agar slants containing 3% malt extract. Stock cultures were transferred to fresh slants every week, incubated at 30°C for 48 h, then stored at 5°C. The yeast was cultivated aerobically in a modified Atkin’s medium [ 51 in 500-ml Erlenmeyer flasks on a rotary shaker at 30°C. The medium contained per 100 ml; 5 g glucose, 0.4 g vitamin-free casamino acids, 1 g potassium citrate, 0.2 g citric acid, 0.11 g KH,PO,, 85 mg KCl, 25 mg CaClz - 2Hz0, 0.5 mg MnS04, 25 mg MgS04 * 7Hz0, 0.5 mg FeC13, 1.6 ng biotin, 5 mg inositol, 0.25 mg calcium pantothenate and 0.5 mg nicotinic acid. Addition of thiamine . HCl to the medium at a final concentration of 1 ng per ml caused a delay of the beginning of growth, a decrease in the growth rate, and a lowering of the maximum level of growth [3]. The cells grown with and without added thiamine - HCI are designated as “thiamine cells” and control cells, respectively, Concomitant addition of pyridoxine * HCl (0.02 ng per ml) prevented the growth inhibition by thiamine [3]. The cells grown with both thiamine and pyridoxine are called “thiamine-pyridoxine cells”. The growth of the yeast was measured turbidimetrically at 610 nm and the values were converted to the dry cell weights (mg per ml culture). Extraction of lipids Yeast cells (l-2 g dry cells) harvested in the middle of logarithmic growth phase were washed three times with distilled water. Lipids were extracted twice with 200 ml chloroform/methanol (1 : 1, v/v) for 24 h at 5°C and then with 100 ml of the same solvent containing 1 mM HCl, The combined extracts were washed as described by Folch et al. [ 61. Lipid analyses (1) Determination of total lipids. An aliquot of the washed extracts was dried over anhydrous Na2S0, and the remaining traces of solvent were allowed to evaporate under reduced pressure in a desiccator. The total lipid was determined by weighing the residue. (2) Separation of neutral lipids. An aliquot of the lipid extracts described above was applied to a thin-layer chromatographic plate (Silica gel H, Merck Co. G.F.R.). The plate was developed with petroleum ether/diethyl ether/acetic acid (90 : 10 : 1, v/v), Phospholipids remained at the origin on the chromato-
485
gram. Spots of neutral lipids were located with iodine vapor or with 50% H2S04. Individual neutral lipids were tentatively identified by comparing their RF values with those of authentic samples of squalene, triacylglycerol, diacylglycerol, cholesterol stearate, lanosterol, ergosterol and phosphatidylcholine. The spots corresponding to hydrocarbons, triacylglycerols, diacylglycerols, sterol esters, free sterols and phospholipids were scraped off after brief exposure (10 s) of the plate to iodine vapor, and then the lipids were eluted from the silica gel successively with chloroform/methanol (1 : 1, v/v 2 X 5 ml) and petroleum ether (5 ml). (a) Sterol analyses: Free sterols eluted from the thin-layer plate were analyzed with a gas-liquid chromatograph equipped with a hydrogen flame ionization detector (Nihon Denshi Co. model JGC-BOKFP, Japan). A glass column (2 mm X 2 m) packed with 1.5% OV-17 supported on 80-100 mesh ChromosorbW (Shimadzu Seisakusho Co. Japan) was used at 240°C with helium as a carrier gas at a flow rate of 32.6 ml per min. An authentic sample of cholestanol was used as an internal standard. To determine the sterol content, a part of the recorder tracings of peak areas was excised and weighed. Sterol esters were determined as above after. saponification with 20% KOH in methanol in the presence of pyrogallol for 2 h at 80°C under nitrogen. (b) ~~ucyZglyce~o1 and d~cylg~ycero~ unaiyses: The eluates from the thinlayer plate were saponified as above. Nonsaponifiable lipids and fatty acids released from the glycerol esters were removed with petroleum ether and acidic diethyl ether, respectively. The quantitative estimation of glycerol in the remaining soluble fraction was done by the method of Hanahan and Olley [7]. (c) Phospholipid analyses: Phospholipid phosphorus was determined according to the method of Bartlett [S]. The phosphorus contents were multiplied by 25 to give the total phospholipid content in mg per g dry weight cells. (3) Separation of phospholipids. The lipid extracts from whole cells were also analyzed for phospholipids by thin-layer chromatography. After development with chloroform/methanol/acetic acid/water (85 : 15 : 10 : 4, v/v/v/v) [9], phospholipids were identified by their respective RF values, using commercially available phospholipids as reference standards, and by their staining behavior with Dittmer’s reagent [lo], ninhydrin, anthrone and Dragendorff reagent [ 111. The zones corresponding to diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine plus phosphatidylserine and phosphatidylinositol on the plate were scraped off and the phospholipids were eluted from the silica gel with chloroform/methanol (1 : 1, v/v; 3 X 5 ml). The phosphorus content of each sample was determined as described above and expressed in mg lipid phosphorus per g of dry cells. (4) Fatty acid composition of neutral lipids and phospholipids. An aliquot of the extracts of the individual lipid substances was evaporated to dryness and saponified with 20% KOH in methanol for 3 h at 80°C. After removal of nonsaponifiable lipids, fatty acids were extracted with diethyl ether in a pH range below 2. The solvent was evaporated and the resulting residue was dissolved in 2.5 ml methanol. Methylation was carried out with 0.5 ml boron trifluoride at 100” C for 2 min. After 2 ml water was added, the reaction mixture was extracted three times with petroleum ether (3 ml each). All these procedures were carried out in an atmosphere of nitrogen. The fatty acid methyl
486
esters were analyzed by gas-liquid chromatography. A stainless steel column (4 mm X 2 m) packed with 15% diethylene glycol succinate supported on 80100 mesh Neopack AS (Nihon Kogyo Co. Japan) was used at 175°C with nitrogen as a carrier gas at a flow rate of 41.7 ml per min. All samples were dissolved in diethyl ether and an aliquot of the solution was injected onto the column. Peak areas were determined by triangulation. Chemicals Phosphatidylinositol idylserine from porcine idylglycerol from bovine from Serdary Research were those of analytical mercially.
from yeast, phosphatidylcholine from egg, phosphatbrain, phosphatidylethanolamine from egg, diphosphatheart, triacylglycerol and diacylglycerol were obtained Laboratories Inc. (Canada). All other chemicals used reagent grade or of the highest purity available com-
Results Lipid composition Data in Table I (A) show that the contents of various lipids were low in “thiamine cells” as compared with those of the control cells; 10% in sterol
TABLE
I
EFFECTS OF THIAMINE AND PYRIDOXINE LIPID COMPOSITION OF SACCHAROMYCES Growth of the cells and extraction, separation Materials and Methods. (A) lipid composition: Lipids (A) Hydrocarbons Free sterols Sterol esters Diacylglycerols Triacylglycerols Phospholipids Total lipids
Control
cells
ON THE
Phosphatidylinositol Phosphatidylcholine plus phosphatidylserine Phosphatidylethanolamine Diphosphatidylglycerdol Total phospholipids
COMPOSITION
AND THE PHOSPHO-
4228
and determination of lipids were carried (B) phospholipid composition. Thiamine
cells
mg per g dry cells a _____~~~~ squalene b squalene b 1.31 (2.6) 2.79 (3.0) 7.68 (8.4) 0.799 (1.6) 7.67 (15.4) 4.53 (4.9) 7.61 (15.3) 11.6 (12.6) 18.7 (37.6) 25.8 (28.1) 49.7 91.9 mg lipid phosphorus
(B)
LIPID
CARLSBERGENSIS
Thiamine-pyridoxine
out as described
in
cells
NDC 4.68 (3.3) 4.37 (3.0) 10.6 (7.4) 12.3 (8.5) 39.3 (27.3) 144
per g dry cells a
0.13
(12.6)
0.19
(25.4)
0.10
(6.4)
0.49 0.36 0.01 1.03
(47.6) (34.9) (1.0)
0.36 0.20 0.01 0.75
(48.1) (26.7) (1.3)
0.72 0.70 0.02 1.57
(45.9) (44.6) (1.3)
a Data in parentheses show % of total lipids (A) and % of total phospholipids (B), respectively. b Accumulation of hydrocarbons in thiamine cells was observed on the chromatogram. The major part of hydrocarbons was identified as squalene, but not determined quantitatively. C ND, not detectable. d Values from which phospholipid contents shown in (A) were calculated as described in Materials and Methods. section 2~.
487
esters, 50% in free sterols, and 70% in either triacylglycerols or phospholipids. However, the amount of diacylglycerols was about 1.7 times higher than that of the control cells. The total lipid content was also markedly low in thiamine cells. Data in Table I (A) are also given as percentage of total lipids, in parentheses. The values for triacylglycerols and phospholipids were rather higher in thiamine cells than in the control cells. Thus, thiamine cells were considerably different from the control cells not only in the absolute contents but also in the composition of cellular lipids. These effects of thiamine were prevented by the addition of pyridoxine to the growth medium. Phospholipid composition At least five different phospholipid spots were detected in the yeast cells by thin-layer chromatography. Based on the specific color reactions and respective RF values of individual lipid components, the major phospholipids of this yeast were tentatively identified as follows; phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and diphosphatidylglycerol. Phosphatidylcholine and phosphatidylserine could not be separated by the developing solvent used, but they were identified with Dragendorff reagent and ninhydrin, respectively. It was found that the phospholipid composition of thiamine cells was qualitatively similar to that of the control cells and of “thiamine-pyridoxine cells”. However, the addition of thiamine to the medium caused significant changes in the level of individual phospholipid compounds as shown in Table I (B). The contents of phosphatidylcholine plus phosphatidylserine and phosphatidylethanolamine in thiamine cells were substantially lower than in the control cells. However, the content of diphosphatidylglycerol was the same as that of the control cells, and thiamine cells had a higher level of phosphatidylinositol. The increase of phosphatidylinositol is clearer when the data are given as percentages of total phospholipids (shown in parentheses). On the other hand, the phospholipid composition of thiamine-pyridoxine cells was similar to that of the control cells, although there was a significant difference in the amounts of individual phospholipids between these cells. Effects of thiamine and pyridoxine on the fatty acid composition of individual lipid compounds A marked difference in the proportion of unsaturated to total fatty acids was observed between thiamine cells and control cells, that is, 50% in the former and 80% in the latter, respectively. Relevant to this, the level of shortchain fatty acids increased with the decreased level of unsaturated fatty acids (data not shown). The fatty acid composition of thiamine-pyridoxine cells was similar to that of the control cells. Data in Table II show the composition of fatty acids present in major lipid classes; sterol esters, triacylglycerols, diacylglycerols, phosphatidylcholine plus phosphatidylserine, phosphatidylethanolamine and phosphatidylinositol. In every lipid class, especially in sterol esters, the content of unsaturated fatty acids was much lower in thiamine cells than in the control cells. The reduction in the amounts of unsaturated fatty acids in different lipid classes reflects the marked decrease in unsaturated fatty acid content in thiamine cells. The
488
TABLE RATIO LIPIDS Growth layer
II OF
UNSATURATED
TO
FROM
SACCHAROMYCBS
of
cells
the
chromatography
chromatography.
For
and
extraction and
fatty
experimental
TOTAL
of
lipids
acids details
Control
Lipids
FATTY
ACIDS
CARLSBRRGEhrSI.9 were
from
lipid
carried
cells
NEUTRAL
out as in Table
classes
see Materials
IN
and
Thiamine
were
cells
esters
86.2
34.6
81.1
IO.6
43.1
62.6
86.5
57.5
74.2
phosphatidylserine
were
PHOSPHO-
separated
quantitatively
Thiamine-pyridoxine
Diacylglycrrols Phosphadidylcholine
AND
by
by thingas-liquid
Methods.
(%)
Triacylglycerols
I. Lipids
determined
(W) stem1
LIPIDS
4228
cells
(%)
plus 84.0
52.9
72.7
Phosphatidylethanolamine
78.2
62.1
76.1
Phosphaditylinositol
59.6
38.8
59.1
unsaturated fatty acid content of each lipid class in thiamine-pyridoxine cells was similar to that in the control cells, as was found for the cellular level of unsaturated fatty acids. Discussion The results presented here clearly demonstrate that thiamine caused a fundamental alteration in the cellular lipid composition of S. carlsbergensis and that pyridoxine prevented the thiamine effect completely. It can be deduced therefore that the effect of thiamine on the lipid composition is exerted via a deficiency of vitamin B-6, as found for the previously reported phenomena, namely deficiency in the respiratory activity [3], decrease in the content of unsaturated fatty acids [ 11 and absence of zymosterol and ergosterol [ 21. It would be of interest to know the cause of the changes in the lipid composition in thiamine cells. It might be possible that these changes were results of the decrease in the levels of unsaturated fatty acids and the loss of ergosterol. This concept would be at least partially supported by our finding that unsaturated fatty acid contents of individual lipids decreased in a similar ratio (See Table II). On the other hand, the decreased levels of unsaturated fatty acids and ergosterol, as well as the respiratory deficiency, would be caused by a lowering of heme synthesis via a serious depression of 8-aminolevulinate synthase (EC 2.3.1.37) due to the vitamin B-6 deficiency (data to be published elsewhere). Then, it might be concluded that abnormal lipid composition would result from the same cause as in the case of the respiratory deficiency, namely, the lowering of heme synthesis. Acknowledgments The authors wish to express their gratitude to Dr. J. Nagai and Dr. H. Katsuki, Department of Chemistry, Faculty of Science, Kyoto University, for valuable advice.
489
References 1
Nishikawa,
Y.,
Nakamura,
I.,
Kamihara,
T.
and
Fukui,
S.
(1974)
Biochem.
Biophys.
Res.
Commun.
59.177-180 2
Nagai,
J.,
Katsuki,
Biophys. 3
Res.
Nakamura. 59,
H.,
Nishikawa,
Commun.
I.,
60.
Nishikawa,
Y..
Nakamura,
I.,
Kamihara.
T.
and
Fukui,
S.
(1974)
Biochem.
555-560 Y.,
Kamihara.
T.
and
Fukui,
S.
(1974)
Biochem.
Biophys.
Res.
Commun.
771-776
4
Nakamura.
5
Atkin,
L.,
6
Folch.
J., Lees,
7
Hanahan.
8
Bartlett,
9
Yano,
10
Dittmer.
11
Wagner,
I.. Nishikawa, Schultz, D.J. G.R.
M. and and
J.C. H.,
and
Kamihara,
Williams, J.N.
J. Biol. Y.
Lester,
Horhammer,
and
(1964) Wolff,
M.
Fukui,
Frey,
J. Biol. 234.
Kusunose,
L. and
and G.H.
(1958) Chem.
R.L.
T. and
W.L.
Sloane-Stanley,
Olley,
(1959)
I.. Furukawa,
Y.,
A.S.,
S. (1976)
C.N.
(1957)
(1943)
J. Biol.
Chem.
237,
FEBS Ind.
Chem.
L&t.
Chem.
236.
497-509
813-828
466-468 (1969)
J. Lipid P. (1961)
J. Bacterial.
Res.
98,
124-130
5, 126-127
Biochem.
Z.
62.
Eng.
334,175-184
354-358 Anal.
Ed 15.
141-144
Biochimica et Biophysiea Acta, 486 (1977) 483-489 @ Elsevier/North-Holland Biomedical Press
BBA 56943
EFFECTS OF THI~INE AND PYRIDOXINE ON THE LIPID COMPOSrT~ONOF SACCliA~~MYCESCA~LS~E~~E~SIS4228
YOSHIKI NISHIKAWA, ICHIRO NAKAMURA, SABURO FUKUI *
TEIJIRO KAMIHARA
~abo~to~ of Indu~~~l ~iochem~~~, ~e~artrn~~t ~n~nee~~ng, Kyoto Unive~ity~ Kyoto (Japan)
and
of ~~dus~~al Chemis~y, Faculty of
(Received August 24th, 1976)
Summary The lipid composition of Saccharomyces ca~ls~erge~sis 4228 cells grown aerobically in the presence of thiamine and absence of pyridoxine was markedly different from that of cells grown without addition of both of the growth factors. In addition to the previous observations showing a reduction in the levels of unsaturated fatty acids (Nishikawa, Y., Nakamura, I., Kamihara, T. and Fukui, S. (1974) Biochem. Biophys, Res. ~ommun. 59, 777-780) and lack of zymosterol and ergosterol (Nagai, J., Katsuki, H., Nishikawa, Y., Nakamura, I., Kamihara, T. and Fukui, S. (1974) Biochem, Biophys. Res. Commun. 60, 555-560), the thiamine-grown cells were found to contain low levels of total lipids, sterols (especially in the form of esters), triacylglycerols and total phospholipids. However, relative contents of triacylglycerols and phospholipids to total lipids were higher than those of control cells. Hydroc~bons and diacylglycerols accumulated to appreciable degrees. Phospholipid composition was also influenced by thiamine. The ratio of phosphatidylinositol to total phospholipids increased, whereas that of phosphatidylethanolamine decreased. The levels of phosphatidylcholine plus phosphatidylserine decreased in a similar ratio to that of total phospholipids. It was found that unsaturated fatty acid contents were low in all lipid esters tested. The effect of thiamine was particularly noteworthy in the case of sterol esters. Concomitant addition of pyridoxine with thiamine to the medium brought about a normal lipid composition in the yeast cells. Introduction As reported previously from our laboratory, Saccharomyces carlsbergensis 4228 (ATCC 9080) grown in the presence of thiamine and absence of pyri* To whom correspondence and reprint requests should be sent.
484
doxine exhibited a markedly low level of unsaturated fatty acids [l] and contained no zymosterol and ergosterol 121. We have also reported that the cells showed a markedly low respiration rate, low activities of respiratory enzymes and had no cytochromes [3,4). It was also found that the cells had a low level of vitamin B-6 [3], and that the addition of pyridoxine to the medium abolished the effects of thiamine [l-4]. These findings, therefore, suggest that thiamine causes some fundamental changes in the cellular lipid composition as a consequence of induced vitamin B-6 deficiency. In this paper, we describe in detail the effects of thiamine and pyridoxine on the lipid composition of yeast cells. Materials and Methods Grouts of yeast 5’. carzsberge~s~s 4228 (ATCC 9080) was maintained on 2% agar slants containing 3% malt extract. Stock cultures were transferred to fresh slants every week, incubated at 30°C for 48 h, then stored at 5°C. The yeast was cultivated aerobically in a modified Atkin’s medium [ 51 in 500-ml Erlenmeyer flasks on a rotary shaker at 30°C. The medium contained per 100 ml; 5 g glucose, 0.4 g vitamin-free casamino acids, 1 g potassium citrate, 0.2 g citric acid, 0.11 g KH,PO,, 85 mg KCl, 25 mg CaClz - 2Hz0, 0.5 mg MnS04, 25 mg MgS04 * 7Hz0, 0.5 mg FeC13, 1.6 ng biotin, 5 mg inositol, 0.25 mg calcium pantothenate and 0.5 mg nicotinic acid. Addition of thiamine . HCl to the medium at a final concentration of 1 ng per ml caused a delay of the beginning of growth, a decrease in the growth rate, and a lowering of the maximum level of growth [3]. The cells grown with and without added thiamine - HCI are designated as “thiamine cells” and control cells, respectively, Concomitant addition of pyridoxine * HCl (0.02 ng per ml) prevented the growth inhibition by thiamine [3]. The cells grown with both thiamine and pyridoxine are called “thiamine-pyridoxine cells”. The growth of the yeast was measured turbidimetrically at 610 nm and the values were converted to the dry cell weights (mg per ml culture). Extraction of lipids Yeast cells (l-2 g dry cells) harvested in the middle of logarithmic growth phase were washed three times with distilled water. Lipids were extracted twice with 200 ml chloroform/methanol (1 : 1, v/v) for 24 h at 5°C and then with 100 ml of the same solvent containing 1 mM HCl, The combined extracts were washed as described by Folch et al. [ 61. Lipid analyses (1) Determination of total lipids. An aliquot of the washed extracts was dried over anhydrous Na2S0, and the remaining traces of solvent were allowed to evaporate under reduced pressure in a desiccator. The total lipid was determined by weighing the residue. (2) Separation of neutral lipids. An aliquot of the lipid extracts described above was applied to a thin-layer chromatographic plate (Silica gel H, Merck Co. G.F.R.). The plate was developed with petroleum ether/diethyl ether/acetic acid (90 : 10 : 1, v/v), Phospholipids remained at the origin on the chromato-
485
gram. Spots of neutral lipids were located with iodine vapor or with 50% H2S04. Individual neutral lipids were tentatively identified by comparing their RF values with those of authentic samples of squalene, triacylglycerol, diacylglycerol, cholesterol stearate, lanosterol, ergosterol and phosphatidylcholine. The spots corresponding to hydrocarbons, triacylglycerols, diacylglycerols, sterol esters, free sterols and phospholipids were scraped off after brief exposure (10 s) of the plate to iodine vapor, and then the lipids were eluted from the silica gel successively with chloroform/methanol (1 : 1, v/v 2 X 5 ml) and petroleum ether (5 ml). (a) Sterol analyses: Free sterols eluted from the thin-layer plate were analyzed with a gas-liquid chromatograph equipped with a hydrogen flame ionization detector (Nihon Denshi Co. model JGC-BOKFP, Japan). A glass column (2 mm X 2 m) packed with 1.5% OV-17 supported on 80-100 mesh ChromosorbW (Shimadzu Seisakusho Co. Japan) was used at 240°C with helium as a carrier gas at a flow rate of 32.6 ml per min. An authentic sample of cholestanol was used as an internal standard. To determine the sterol content, a part of the recorder tracings of peak areas was excised and weighed. Sterol esters were determined as above after. saponification with 20% KOH in methanol in the presence of pyrogallol for 2 h at 80°C under nitrogen. (b) ~~ucyZglyce~o1 and d~cylg~ycero~ unaiyses: The eluates from the thinlayer plate were saponified as above. Nonsaponifiable lipids and fatty acids released from the glycerol esters were removed with petroleum ether and acidic diethyl ether, respectively. The quantitative estimation of glycerol in the remaining soluble fraction was done by the method of Hanahan and Olley [7]. (c) Phospholipid analyses: Phospholipid phosphorus was determined according to the method of Bartlett [S]. The phosphorus contents were multiplied by 25 to give the total phospholipid content in mg per g dry weight cells. (3) Separation of phospholipids. The lipid extracts from whole cells were also analyzed for phospholipids by thin-layer chromatography. After development with chloroform/methanol/acetic acid/water (85 : 15 : 10 : 4, v/v/v/v) [9], phospholipids were identified by their respective RF values, using commercially available phospholipids as reference standards, and by their staining behavior with Dittmer’s reagent [lo], ninhydrin, anthrone and Dragendorff reagent [ 111. The zones corresponding to diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine plus phosphatidylserine and phosphatidylinositol on the plate were scraped off and the phospholipids were eluted from the silica gel with chloroform/methanol (1 : 1, v/v; 3 X 5 ml). The phosphorus content of each sample was determined as described above and expressed in mg lipid phosphorus per g of dry cells. (4) Fatty acid composition of neutral lipids and phospholipids. An aliquot of the extracts of the individual lipid substances was evaporated to dryness and saponified with 20% KOH in methanol for 3 h at 80°C. After removal of nonsaponifiable lipids, fatty acids were extracted with diethyl ether in a pH range below 2. The solvent was evaporated and the resulting residue was dissolved in 2.5 ml methanol. Methylation was carried out with 0.5 ml boron trifluoride at 100” C for 2 min. After 2 ml water was added, the reaction mixture was extracted three times with petroleum ether (3 ml each). All these procedures were carried out in an atmosphere of nitrogen. The fatty acid methyl
486
esters were analyzed by gas-liquid chromatography. A stainless steel column (4 mm X 2 m) packed with 15% diethylene glycol succinate supported on 80100 mesh Neopack AS (Nihon Kogyo Co. Japan) was used at 175°C with nitrogen as a carrier gas at a flow rate of 41.7 ml per min. All samples were dissolved in diethyl ether and an aliquot of the solution was injected onto the column. Peak areas were determined by triangulation. Chemicals Phosphatidylinositol idylserine from porcine idylglycerol from bovine from Serdary Research were those of analytical mercially.
from yeast, phosphatidylcholine from egg, phosphatbrain, phosphatidylethanolamine from egg, diphosphatheart, triacylglycerol and diacylglycerol were obtained Laboratories Inc. (Canada). All other chemicals used reagent grade or of the highest purity available com-
Results Lipid composition Data in Table I (A) show that the contents of various lipids were low in “thiamine cells” as compared with those of the control cells; 10% in sterol
TABLE
I
EFFECTS OF THIAMINE AND PYRIDOXINE LIPID COMPOSITION OF SACCHAROMYCES Growth of the cells and extraction, separation Materials and Methods. (A) lipid composition: Lipids (A) Hydrocarbons Free sterols Sterol esters Diacylglycerols Triacylglycerols Phospholipids Total lipids
Control
cells
ON THE
Phosphatidylinositol Phosphatidylcholine plus phosphatidylserine Phosphatidylethanolamine Diphosphatidylglycerdol Total phospholipids
COMPOSITION
AND THE PHOSPHO-
4228
and determination of lipids were carried (B) phospholipid composition. Thiamine
cells
mg per g dry cells a _____~~~~ squalene b squalene b 1.31 (2.6) 2.79 (3.0) 7.68 (8.4) 0.799 (1.6) 7.67 (15.4) 4.53 (4.9) 7.61 (15.3) 11.6 (12.6) 18.7 (37.6) 25.8 (28.1) 49.7 91.9 mg lipid phosphorus
(B)
LIPID
CARLSBERGENSIS
Thiamine-pyridoxine
out as described
in
cells
NDC 4.68 (3.3) 4.37 (3.0) 10.6 (7.4) 12.3 (8.5) 39.3 (27.3) 144
per g dry cells a
0.13
(12.6)
0.19
(25.4)
0.10
(6.4)
0.49 0.36 0.01 1.03
(47.6) (34.9) (1.0)
0.36 0.20 0.01 0.75
(48.1) (26.7) (1.3)
0.72 0.70 0.02 1.57
(45.9) (44.6) (1.3)
a Data in parentheses show % of total lipids (A) and % of total phospholipids (B), respectively. b Accumulation of hydrocarbons in thiamine cells was observed on the chromatogram. The major part of hydrocarbons was identified as squalene, but not determined quantitatively. C ND, not detectable. d Values from which phospholipid contents shown in (A) were calculated as described in Materials and Methods. section 2~.
487
esters, 50% in free sterols, and 70% in either triacylglycerols or phospholipids. However, the amount of diacylglycerols was about 1.7 times higher than that of the control cells. The total lipid content was also markedly low in thiamine cells. Data in Table I (A) are also given as percentage of total lipids, in parentheses. The values for triacylglycerols and phospholipids were rather higher in thiamine cells than in the control cells. Thus, thiamine cells were considerably different from the control cells not only in the absolute contents but also in the composition of cellular lipids. These effects of thiamine were prevented by the addition of pyridoxine to the growth medium. Phospholipid composition At least five different phospholipid spots were detected in the yeast cells by thin-layer chromatography. Based on the specific color reactions and respective RF values of individual lipid components, the major phospholipids of this yeast were tentatively identified as follows; phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and diphosphatidylglycerol. Phosphatidylcholine and phosphatidylserine could not be separated by the developing solvent used, but they were identified with Dragendorff reagent and ninhydrin, respectively. It was found that the phospholipid composition of thiamine cells was qualitatively similar to that of the control cells and of “thiamine-pyridoxine cells”. However, the addition of thiamine to the medium caused significant changes in the level of individual phospholipid compounds as shown in Table I (B). The contents of phosphatidylcholine plus phosphatidylserine and phosphatidylethanolamine in thiamine cells were substantially lower than in the control cells. However, the content of diphosphatidylglycerol was the same as that of the control cells, and thiamine cells had a higher level of phosphatidylinositol. The increase of phosphatidylinositol is clearer when the data are given as percentages of total phospholipids (shown in parentheses). On the other hand, the phospholipid composition of thiamine-pyridoxine cells was similar to that of the control cells, although there was a significant difference in the amounts of individual phospholipids between these cells. Effects of thiamine and pyridoxine on the fatty acid composition of individual lipid compounds A marked difference in the proportion of unsaturated to total fatty acids was observed between thiamine cells and control cells, that is, 50% in the former and 80% in the latter, respectively. Relevant to this, the level of shortchain fatty acids increased with the decreased level of unsaturated fatty acids (data not shown). The fatty acid composition of thiamine-pyridoxine cells was similar to that of the control cells. Data in Table II show the composition of fatty acids present in major lipid classes; sterol esters, triacylglycerols, diacylglycerols, phosphatidylcholine plus phosphatidylserine, phosphatidylethanolamine and phosphatidylinositol. In every lipid class, especially in sterol esters, the content of unsaturated fatty acids was much lower in thiamine cells than in the control cells. The reduction in the amounts of unsaturated fatty acids in different lipid classes reflects the marked decrease in unsaturated fatty acid content in thiamine cells. The
488
TABLE RATIO LIPIDS Growth layer
II OF
UNSATURATED
TO
FROM
SACCHAROMYCBS
of
cells
the
chromatography
chromatography.
For
and
extraction and
fatty
experimental
TOTAL
of
lipids
acids details
Control
Lipids
FATTY
ACIDS
CARLSBRRGEhrSI.9 were
from
lipid
carried
cells
NEUTRAL
out as in Table
classes
see Materials
IN
and
Thiamine
were
cells
esters
86.2
34.6
81.1
IO.6
43.1
62.6
86.5
57.5
74.2
phosphatidylserine
were
PHOSPHO-
separated
quantitatively
Thiamine-pyridoxine
Diacylglycrrols Phosphadidylcholine
AND
by
by thingas-liquid
Methods.
(%)
Triacylglycerols
I. Lipids
determined
(W) stem1
LIPIDS
4228
cells
(%)
plus 84.0
52.9
72.7
Phosphatidylethanolamine
78.2
62.1
76.1
Phosphaditylinositol
59.6
38.8
59.1
unsaturated fatty acid content of each lipid class in thiamine-pyridoxine cells was similar to that in the control cells, as was found for the cellular level of unsaturated fatty acids. Discussion The results presented here clearly demonstrate that thiamine caused a fundamental alteration in the cellular lipid composition of S. carlsbergensis and that pyridoxine prevented the thiamine effect completely. It can be deduced therefore that the effect of thiamine on the lipid composition is exerted via a deficiency of vitamin B-6, as found for the previously reported phenomena, namely deficiency in the respiratory activity [3], decrease in the content of unsaturated fatty acids [ 11 and absence of zymosterol and ergosterol [ 21. It would be of interest to know the cause of the changes in the lipid composition in thiamine cells. It might be possible that these changes were results of the decrease in the levels of unsaturated fatty acids and the loss of ergosterol. This concept would be at least partially supported by our finding that unsaturated fatty acid contents of individual lipids decreased in a similar ratio (See Table II). On the other hand, the decreased levels of unsaturated fatty acids and ergosterol, as well as the respiratory deficiency, would be caused by a lowering of heme synthesis via a serious depression of 8-aminolevulinate synthase (EC 2.3.1.37) due to the vitamin B-6 deficiency (data to be published elsewhere). Then, it might be concluded that abnormal lipid composition would result from the same cause as in the case of the respiratory deficiency, namely, the lowering of heme synthesis. Acknowledgments The authors wish to express their gratitude to Dr. J. Nagai and Dr. H. Katsuki, Department of Chemistry, Faculty of Science, Kyoto University, for valuable advice.
489
References 1
Nishikawa,
Y.,
Nakamura,
I.,
Kamihara,
T.
and
Fukui,
S.
(1974)
Biochem.
Biophys.
Res.
Commun.
59.177-180 2
Nagai,
J.,
Katsuki,
Biophys. 3
Res.
Nakamura. 59,
H.,
Nishikawa,
Commun.
I.,
60.
Nishikawa,
Y..
Nakamura,
I.,
Kamihara.
T.
and
Fukui,
S.
(1974)
Biochem.
555-560 Y.,
Kamihara.
T.
and
Fukui,
S.
(1974)
Biochem.
Biophys.
Res.
Commun.
771-776
4
Nakamura.
5
Atkin,
L.,
6
Folch.
J., Lees,
7
Hanahan.
8
Bartlett,
9
Yano,
10
Dittmer.
11
Wagner,
I.. Nishikawa, Schultz, D.J. G.R.
M. and and
J.C. H.,
and
Kamihara,
Williams, J.N.
J. Biol. Y.
Lester,
Horhammer,
and
(1964) Wolff,
M.
Fukui,
Frey,
J. Biol. 234.
Kusunose,
L. and
and G.H.
(1958) Chem.
R.L.
T. and
W.L.
Sloane-Stanley,
Olley,
(1959)
I.. Furukawa,
Y.,
A.S.,
S. (1976)
C.N.
(1957)
(1943)
J. Biol.
Chem.
237,
FEBS Ind.
Chem.
L&t.
Chem.
236.
497-509
813-828
466-468 (1969)
J. Lipid P. (1961)
J. Bacterial.
Res.
98,
124-130
5, 126-127
Biochem.
Z.
62.
Eng.
334,175-184
354-358 Anal.
Ed 15.
141-144