Pharmacology 14: 49 9 -5 1 0 (1976)
Effects of Hypolipidemic Agents on Lipid Synthesis in Subcellular Fractions from Tetraliymena pyriformis H. Y.M. Pan, S .C . Chou and K .A . Conklin Department o f Pharmacology, School o f Medicine. University o f Hawaii, Honolulu, Hawaii
Key Words. Hypolipidemic agents • Lipid synthesis • Microsomes • Mitochondria • Soluble cell fraction • Tetraliymena pyriformis Abstract. Lipid synthesizing systems have been prepared from subcellular fractions o f Tetraliymena pyriformis, G L . These fractions, the mitochondrial fraction, the microsomal fraction and the soluble cell fraction, have been characterized as to cofactors and cations required for optimal lipid synthesis. The effects o f hypolipidemic agents on lipid synthesis by all fractions are presented.
Introduction
Received: March 11, 1976.
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The biosynthetic pathways o f lipids in Tetraliymena pyriformis have previ ously been examined both in vivo (10, 29) and in vitro (16, 26). These studies were performed using 14C-labeled acetate or other suitable precursors for lipid synthesis, and have led to the identification of the lipids synthesized by this organism. Other studies have led to the identification o f the various classes o f lipids present in the membrane fragments o f organelles (22 25, 32, 33). These in vivo and in vitro systems have also been utilized to study the effects o f drugs, primarily hypocholesterolemic agents, on lipid synthesis (15, 21, 26, 27, 30). Cholesterol and other steroids have been shown to overcome the inhibitory effects by some o f these hypocholesterolemic agents (14). In this paper, we present the characterization o f lipid synthesis in subcellu lar fractions of T. pyriformis, strain G L , and the effects o f hypolipidemic agents on these processes.
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Materials and Methods Organism and Growth Condition T. pyriformis, strain G L , was grown and harvested as described previously (8). For experimental purposes, 2-day-old cultures were used to inoculate 200 ml (for mitochondrial preparation) or 1,000 ml (for microsomal and soluble cell fraction preparations) o f the proteose-peptone liver extract medium. Cultures were incubated 12 18 h at 29 °C . The cells were harvested when the population reached approximately 200.000/ml.
Preparation o f Subcellular Fractions Mitochondria were prepared by a modification o f the procedure o f Schwab-Stey et al. (28). The harvested ceils were washed and suspended in 10 ml o f buffer containing 0.1 M phosphate buffer (pH 7.2), 0.3 Af sucrose, and 10 mAf mercaptoethanol (solution L). The cells were then disrupted by homogenization in a glass homogenizer, and centrifuged at 500 g for 10 min at 4 °C . The pellet was resuspended and centrifuged at 500 g for 10 min. Tile supernatants were combined and again centrifuged at l,0 0 0 g for 10 min. The super natant was centrifuged at 7.000g for 10 min. The pellet was resuspended in 10 ml o f a Ficoll solution containing 10 g o f Ficoll in 100 ml o f solution L and centrifuged at 1.000g for 10 min. The supernatant containing mitochondria was then washed twice in the Ficoll solution by centrifugation at 7,000 g for 30 min. The final pellet containing the mitochon drial preparation was suspended in 10 ml o f solution L. This preparation yielded a mito chondrial preparation with a protein content o f 1—1.5 mg,'ml. The microsomal fraction was prepared from the pooled supernatants from the 1,000 g centrifugation for the preparation o f the mitochondrial fraction. This supernatant was cen trifuged at 10,000g for 30 min, the pellet was discarded and the microsomal fraction was sedimented by centrifugation at 105,000g for 60 min. The final microsomal pellet was suspended in 4 ml o f solution L. This preparation yielded a microsomal preparation with a protein content o f 3—4 mg/ml. The supernatant after the final centrifugation at 105,000g was used as the soluble cell fraction. This preparation contained a protein concentration o f 7 -1 0 mg/ml.
Assay for mitochondrial lipid synthesis was done using a 0.5-ml reaction mixture containing 200-400 jug o f protein as determined by the procedure o f Lowry et al. (17), 0.1 Af phosphate buffer (pH 7.2), 0.3 M sucrose, 10 mAf mercaptoethanol, 0.1 mM E D T A , 2.5 mM M gC L, 10 mAf ATP. 0.4 mM N A D H , 0.4 mM NADPH, 0.1 mM C o A , and 0.1 nCi o f 2-‘,'C-acetate (25 juCi/mmol, Calatomic). The reaction mixtures were incubated at 29 °C , the optimal growth temperature for T. pyriformis, and samples (0.1 ml) were taken in duplicate at 0 and 30 min. The samples were placed on filter paper discs and assayed by the filter paper disc procedure to determine the incorporation o f acetate into lipids (5). Assay for microsomal lipid synthesis was done using a 0.5-ml reaction mixture contain ing 0.1 M phosphate buffer (pH 7.2), 0.3 M sucrose, 10 mAf mercaptoethanol, 0.8 mAf N A D H , 1.0 mAf C o A , 10 mAf A TP , 5 mAf MgCl2 and 0.1 yCi o f 2-l4C-acetate (25 juCi/mmol, Calatomic). The reaction tube was incubated at 29 °C and samples (0.1 ml) were taken in duplicate at 0 and 30 min and assayed as described above for the mitochondrial fraction. Assay for lipid synthesis in the soluble cell fraction was performed in a reaction mixture containing 0.1 Af phosphate buffer (pH 7.2), 0.3 Af sucrose, 10 mAf mercapto ethanol, 1 mAf E D T A . 2 mAf N A D H , 2 mAf N A D PH , 0.4 mAf C o A , 4 mAf A TP , 4 mAf M n C l,, 2 mAf M gCl, and 0.1 juCi o f 2-14C-acetate in a total volume o f 0.5 ml. The amount o f protein in each reaction mixture was 1- 2 mg. The reaction mixture was incubated at
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Assay for Lipid Synthesis in the Subcellular Fractions
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29 °C and samples were removed in duplicate at 0 and 90 min. The samples were then assayed by the filter paper disc procedure.
Isolation and Identification o f Lipids Synthesized Lipids were isolated from the reaction mixtures by extraction with chloroform:methanol (2:1 v/v) according to the method o f Bligh and Dyer (4). Extracted lipids were washed overnight by the method o f Folcli et at. (11). General classes o f lipids were separated and identified in Silica gel G thin-layer plates by developing with a solvent system containing petroleum ctheridiethyl cthcr:acctic acid (75:25:1 v/v/v). Phospholipids were further sepa rated by developing in a solvent system containing chloroform:acctic acid:methanol:water (75:25:5:2.2 v/v/v/v). After developing the plates, the spots were located by exposure to iodine vapors, the stained areas carefully circled with a lead pencil, and following sublima tion o f the iodine the spots were scraped directly into scintillation vials. Concentrated sulfuric acid was used to identify tetrahymenol on the plates. The radioactivity present on each spot was then determined by measurement in a Beckman liquid scintillation counter, model LS 150, using toluene-PPO (5 g/1) scintillation fluid.
Results
Requirements fo r Acetate Incorporation into Lipids by the Microsomal Fraction Table II shows the requirements for the incorporation of acetate into lipids by the microsomal fraction. The complete reaction mixture incorporated 990 cpm/100/rg protein. Omission o f NADH and Mg++ reduced the activity to 22 and 39%, respectively. Omission o f ATP and CoA abolished the incorpora-
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Requirements fo r Acetate Incorporation into Lipids by the Mitochondrial Fraction Table 1 shows the requirements for lipid synthesis by the mitochondrial fraction. The complete reaction mixture incorporated 6,350 cpm/100 Mg pro tein. Omission of ATP or CoA almost abolished the activity (3 % remaining for ATP, 4 % for CoA). Both NADH and NADPH were required for the system. Omission of NADH reduced the activity to 35 % and absence o f NADPH re duced activity to 89 %. Magnesium ions were also required (9 % remaining with out Mg++), but Mn++ was inhibitory to the system at 2.5 and 5.0 mM . ADP did not support lipid synthesis in the absence of ATP. The reaction mixture minus EDTA showed 94 % of the activity o f the complete system. Figure 1a shows the time course o f lipid synthesis by the mitochondrial preparation. Linear incorporation was observed for the first 30 min and maxi mum incorporation was attained after 60 min of incubation. Figure 2a shows the optimal protein concentration for incorporation using the optimal condi tions. Linear incorporation was observed with increasing protein concentration up to 500 Mg per reaction mixture.
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Fig. 1. Time course for lipid synthesis in the mitochondrial fraction (a), the micro somal fraction (b) and the soluble cell fraction (c). The reaction mixture for each is as described in Materials and Methods, with samples being assayed at the indicated times by the filter paper disc procedure for lipid synthesis. Each value is the average o f two determi nations from a representative set o f experiments.
Table I. Requirements for acetate incorporation into lipids in the mitochondrial frac tion Incubation mixture
Incorporation cpm/100 Mg protein
Relative incorporation %
Complété Minus ATP Minus ATP, plus 10 mM ADP Minus CoA Minus NADH Minus NADPH Minus Mg” Minus ED TA Minus E D T A , plus Mn** 2.5 mM Minus E D T A , plus Mn** 5.0 mM
6,350 180 200 230 2,210 5,080 560 5,960 5,410 4,250
100 3 3 4 35 89 9 94 85 67
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The complete incubation mixture is as described in Materials and Methods. Samples were incubated at 29 °C and 0.1-ml aliquots in duplicate were removed at 0 and 30 min, and acetate incorporation into lipids was determined by the filter paper disc procedure. Results presented are from one representative set o f experiments.
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Lipid Synthesis in Tetrahymena pyriformis
P ro te in , m g / re a c tio n sam p le
Fig. 2. Protein concentration for lipid synthesis in the mitochondrial fraction (a), the microsomal fraction (b) and the soluble cell fraction (c). The reaction mixture for each is as described in Materials and Methods. Samples were taken at 0 and 30 min (a); 0 and 30 min (b); and 0 and 90 min (c), and were assayed by the filter paper disc procedure for lipid synthesis. Each value is the average o f two determinations from a representative set o f experiments.
Table II. Requirements for the incorporation o f acetate into lipids in the microsomal fraction Reaction mixture
Incorporation cpm/100 ug protein
Relative incorporation
% Complete Minus ATP Minus CoA Minus N A D U Minus Mg'* Plus N A D PH , 0.8 mAf Plus Mn**, 5 mM Plus E D T A , 0.1 mM
990 8 8 220 390 960 740 960
100 1 1 22 39 97 75 97
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The complete incubation mixture is as described in Materials and Methods. Samples were incubated at 29 °C and 0.1-ml aliquots in duplicates were removed at 0 and 30 min, and acetate incorporation into lipids was determined by the filter paper disc procedure. Each value is the average o f two determinations. Results presented are from one representative set o f experiments.
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Table III. Requirements for the incorporation o f acelate into lipids in the soluble cell fraction Reaction mixture
Incorporation cpm/mg protein
Relative incorporation
% Complete Minus ATP Minus CoA Minus NADH Minus NADPH Minus Mg** Minus Mn** Minus ED TA
2,610 30 70 120 120 2,480 1,530 1,780
100 1 3 5 5 95 59 68
The complete incubation mixture is as described in Materials and Methods. Samples were incubated at 29 °C and 0.1-ml aliquots in duplicate were removed at 0 and 90 min, and acetate incorporation into lipids was determined by the filter paper disc procedure. Each value is the average o f two determinations. Results presented are from one representative set o f experiments.
Requirements fo r Acetate Incorporation into Lipids by the Soluble Cell Fraction Table III shows the requirements for the incorporation o f acetate into lipids by the soluble cell fraction. This fraction, like the mitochondrial fraction, required both NADH and NADPH for optimal activity. Omission o f either o f these cofactors reduced the incorporation to 5 %. Omission o f ATP reduced the activity to 1 % and omission o f CoA reduced activity to 3 %. Both Mn++ and Mg++ were required by the system for maximal synthesis of lipids. EDTA was also required by this fraction. Figure l c shows the time course for lipid synthesis by the soluble cell fraction. Linear incorporation was observed in the first 90 min and maximal incorporation was attained at approximately 120 min. Figure 2c shows linear incorporation for protein concentration up to 2 mg per reaction mixture.
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tion almost completely. NADPH, Mn++ and EDTA were not required by this fraction for the synthesis o f lipids. Figure 1b shows the time course for lipid synthesis by the microsomal fraction. The rate o f incorporation was linear up to 30 min and reached maximal incorporation at 60 min. Figure 2 b gives the relationship of protein concentra tion to the incorporation o f acetate into lipids. Linear incorporation was ob served with protein concentration up to 1 mg per reaction mixture.
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Table IV . Acetate incorporation into individual classes o f lipids in the subcellular frac tions Lipid
Triglycerides Free fatty acids Tetrahymenol Total neutral lipids Phospholipids Total lipids
Incorporation, cpm/100 jug protein mitochondria
microsomes
soluble cell fraction
24 (0.5) 156(3) 1,260 (24) 1,440 3,839 (72.5) 5.279
38 (5) 435 (58) 52(7) 525 225 (30) 750
109 (5) 620(28) 745 (33.50) 1,474 746 (33.5) 2,220
The separation and identification o f the various classes o f lipids are as described in Materials and Methods. Each value is the average o f two determinations. Total neutral lipids is the total o f triglycerides, free fatty acids and tetrahymenol. The percentage o f total lipids synthesized for each lipid class is in parentheses following each value.
Table V. Acetate incorporation into individual classes o f phospholipids in the subcellu lar fractions Lipid class
Cardiolipin 2-Aminoethylphosphonolipids Ethanolamine phosphatides Choline phosphatides Total phospholipids
Incorporation, cpm/100 Mg protein mitochondria
microsomes
soluble cell fraction
480(11) 524(12) 3,152 (74) 132(3) 4,288
8 (4) 30(15) 165 (80) 2(1) 205
0 40 (8) 321 (60) 172 (32) 533
Characterization o f the Products Synthesized in the Subcellular Fractions Table IV and V shows the percentages o f the various classes o f lipids synthe sized by these subcellular fractions. The mitochondria produced 0.5 % triglycer ides, 3 % free fatty acids, 24 % tetrahymenol and 72.5 % phospholipids. O f the phospholipids synthesized, ethanolamine phosphatides comprised the largest percentage (74%). Lipids synthesized in the microsomal fraction were primarily
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The separation and identification o f the various classes o f lipids are as described in Materials and Methods. Each value is the average of two determinations. The percent age o f total phospholipids for each phospholipid class is in parentheses following each value.
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free fatty acids (58 % of total lipids), and phospholipids (30 % o f total lipids). As in the mitochondrial fraction, ethanolamine phosphatides were the primary phospholipids synthesized (80 %). O f the lipids synthesized in the soluble cell fraction, 5 % were triglycerides, 28% free fatty acids, and 33.5% were tetrahymenol. The remaining lipids were phospholipids (33.5 %), and o f these 60 % were ethanolamine phosphatides, 32 % choline phosphatides, and 8 % 2-aminoethylphosphonates. Effects o f Hypolipidemic Agents on Lipid Synthesis Table VI shows the degree of inhibition o f lipid synthesis by hypolipidemic agents in each o f the subcellular fractions.
Subcellular fractions from T. pyriformis have been shown to incorporate l4C-acetate into several classes o f lipids. The mitochondrial fraction synthesized more phospholipids (both relatively and absolutely) than either the microsomal or the soluble cell fraction. Free fatty acid synthesis was more active in the soluble cell fraction. It was not determined, however, whether incorporation of acetate into free fatty acids was by de novo synthesis or by chain elongation. Tetrahymenol, a triterpenoid synthesized exclusively in T. pyriformis in place o f cholesterol (18), was synthesized primarily in the mitochondrial and the soluble cell fractions. The cofactor requirements for optimal lipid synthesis differed between the subcellular fractions. The microsomal fraction did not require NADPH, whereas the soluble cell fraction showed a 95 % decrease in activity when this cofactor was omitted. The mitochondrial fraction also showed less activity in the absence of NADPH. ATP was essential for activity of all fractions. It is generally believed that ATP does not traverse the mitochondrial membrane (7), but is formed by oxidative phosphorylation from ADP which is transported across the mitochon drial membrane. In the characterization o f lipid synthesis in the mitochondrial fraction, however, ADP in place o f ATP did not support lipid synthesis. This may indicate that, under the conditions o f the reaction, ADP did not cross the mitochondrial membrane or that oxidative phosphorylation was not functioning. Clofibrate, a hypolipidemic agent which inhibits hepatic synthesis o f choles terol and triglycerides (1, 2), produced a marked inhibition o f lipid synthesis by mitochondria. The degree of inhibition o f lipid synthesis by the microsomal and the soluble cell fraction was considerably less. S-8527, a hypolipidemic agent more potent than clofibrate (34), produced marked inhibition in all fractions. a-Phenylbutyrate, which inhibits the activation of acetate to acetyl CoA (20), showed inhibition o f lipid synthesis in all fractions. TPIA, known to inhibit the
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Discussion
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Table VI. Effects o f hypolipidemic agents on l4C-acetate incorporation into lipids Drug
Concentration mol/1
Mitochondria
Inhibition, % microsomes
soluble cell fraction
10’ 4 10’ 3
38 63
6 19
0 1
S-8527
1 X 1 0 -3 2 X 1 0 -3
51 78
58 75
50 91
a-Phenylbutyrate
3 X 1 0 -3 6 X 1 0 -3
28 49
4 17
14 33
TP1A
8 8
X
1 0 -5 10-4
40 83
12 38
12 59
4 4
X
1 0 -5 10-4
52 82
14 31
4 12
1.4 1.4
X
10-4 1 0 '3
52 94
68 97
8 19
6 6
X
10-* 10-4
54 88
52 78
18 31
2 2
X
10-4 1 0 -3
19 69
0 2
12 26
1 0 -4 1 0 -3
28 43
0 5
0 0
1 0 -3 1 0 -J
2 17
0 4
0 2
1 0 -3 1 0 -3
8 30
0 5
0 0
5 X 10-4 1 X 1 0 -3
0 0
0 0
0 0
Clofibrate
Triparanol
S K F 3301A
S K F 525A
S K F 2314
S K F 16467 A
Nicotinic acid
A 4-Cholestenone
Probucol
4 2
X X
X
X
X
X
X
1.4 1.4
X
8 1.6
X
4 8
X
X
X X
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The complete reaction mixture is as described in Materials and Methods, with the hypolipidemic agents added at the indicated concentrations. Incubation time was as follows: mitochondria, 30 min; microsomes, 30 min; soluble cell fraction, 90 min. Acetate incorporation was determined by the filter paper disc procedure. Each value is the average o f two determinations.
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activity o f acetyl CoA carboxylase (19), and triparanol which has numerous sites o f action (13), also showed consistent inhibition. The SKF experimental drugs, which have been shown to be hypocholes térolémie agents (12, 13), showed inhibitory effects on the incorporation o f acetate into lipids o f most fractions. However, as compared to SKF 3301 A and 525 A , SK F 16467A and 2314 produced considerably less inhibition o f lipid synthesis in the microsomal fraction. Since tetrahymenol synthesis is very low in this fraction, these results may indicate a primary' effect on steroid synthesis by the latter compounds, while the former compounds have in addition a promi nent effect on synthesis o f other neutral lipids or phospholipids. Nicotinic acid, A4-cholestenone, and probucol did not show potent inhibi tory effects in any fraction. However, for nicotinic acid and probucol these results may be consistent with proposed sites of action for these agents as the former inhibits release o f fatty acids from adipose tissue (6) and the latter inhibits transport o f cholesterol from the gut (3). Neither o f these actions would be expected to inhibit lipid synthesis in cell-free preparations. A4-cholestenone has been proposed to inhibit the conversion of hydroxymethylglutaryl CoA to mevalonic acid (31), a reaction common to cholesterol and tetrahymenol bio synthesis (9). Therefore, this agent should inhibit lipid synthesis by mitochon dria and the soluble fraction as these fractions synthesize tetrahymenol. The explanation for the minimal inhibition observed with mitochondria, and the lack o f inhibition with the soluble fraction is unclear.
A cknowledgement This is contribution No. 1354 to the Army Research Program on Malaria, and was supported by contract D A D A 17-71-C-l 116 from the United States Army Medical Research and Development Command.
References
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Adams, L .L .; Webb. W. W., and P'allon, H .J.: Inhibition o f hepatic triglyceride synthesis by clofibrate. Clin. Res. 18: 50 (1970). Avoy, D .R .; Swyryd, E .A ., and Gould, R .G .: Effects o f CPIB with and without androsterone on cholesterol biosynthesis in rat liver. J . Lipid Res. 6: 369 -376 (1965). Barnhart, J.W . and Johnson, J .D .: The effects o f probucol (DH-581) on cholesterol metabolism. Abstr. IVth lnt. Sym p. on Drugs Affecting Lipid Metabolism, p. 25 (1971). Bligh, E.G. and Dyer, W.J.: A rapid method o f total lipid extraction and purification. Can. J . Biochem. Physiol. 37: 9 11-917 (1959). Byfield, J.E .: Henze, J ., and Scherbaum, O .H .: Micro-assay for in vivo lipid synthesis in cell suspensions. Life Sci. 6: 1099-1105 (1967).
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The effect o f nicotinic acid on the plasma free fatty acid. Demonstration o f a metabolic type o f sympathiocolysis. Acta med. Scand. 172: 641 645 (1962). Chappell, J.B .: Systems for the transport o f substances into mitochondria. Br. med. Bull. 24: 150 157 (1968). Chou, S.C. and Ramanathan, S .: Quinacrine site of inhibition o f synchronized cell division in Tetrahymena. Life Sci. 7: 1053 1062 (1968). Connor, R .L .; Landrey, J .R .: Borns, C.H ., and Mallory, F .B .: Cholesterol inhibition o f pentacyclic triterpenoid biosynthesis in Tetrahymena pyriformis. J . Protozool. 15: 600-605 (1968). Erwin, J. and Block, K .: Lipid metabolism o f ciliated protozoa. J . biol. Chem. 238: 1618 1624 (1963). Folch, J .: Lees, M., and Sloane-Stanley, G .H .: A simple method for the isolation and purification o f total lipids from animal tissues. J . biol. Chem. 226: 4 9 6 -5 0 9 (1957). Holmes, W.L. and Bentz, J .D .: Inhibition o f cholesterol biosynthesis in vitro by or dicthylaminocthyldiphenyl-propylacetate hydrochloride (SK F 525A). J . biol. Chem. 235: 3118 3122(1960). Holmes, W.L. and Detullio, N.W.: Inhibitors o f cholesterol biosynthesis which act at or beyond the mevalonic acid stage. Am . J . clin. Nutr. 10: 310 322 (1962). Holmlund, G.W.: Growth inhibition o f Tetrahymena pyriformis by hypocholesteremic compound and the mechanism o f its reversal by various lipids. Biochim. biophys. Acta 296: 221-233 (1973). Holz, G .G .: Erwin, J .: Rosebaum, N., and Aaronson, S .: Triparanol inhibition o f Tetra hymena and its prevention by lipids. Archs Biochem. Biophys. 98: 31 2 -3 2 2 (1962). Kapoulas, V.M. and Thompson, G .A ., jr.: The formation o f glycerol ethers by cell free Tetrahymena extracts. Biochim. biophys. Acta 187: 594 597 (1969). Lowry, O.H .; Rosebrough, N .J.; Farr, A .L ., and Randall, R .J .: Protein measurement with the Folin phenol reagent. J . biol. Chem. 193: 265-275 (1951). Mallory, F.B .: Gordon, J.H ., and Connor, R .C .: The isolation o f the pentacyclic triterpenoid alcohol from a protozoan. J . A m . chem. Soc. 85: 1362-1363 (1963). Maragoudakis, M .E .: Inhibition o f hepatic acetyl coenzyme A carboxylase by hypo lipidemic agents. J . biol. Chem. 244: 5005 5013 (1969). Masters, R . and Steinberg, D .: Studies on the mechanism o f action o f a-phenylbutyrate. Biochim. biophys. Acta 27: 592- 597 (1958). Nozawa, Y .: Inhibition o f lipid biosynthesis by p-chlorophenyoxy isobutyrate (CP1B) in Tetrahymena pyriformis. J . Biochem. 74: 1157 1163 (19 7 3). Nozawa, Y.; Fukushima, H ., and lida, H .: Isolation and lipid composition o f nuclear membranes from macronuclei o f Tetrahymena pyriformis. Biochim. biophys. Acta 318: 335-344 (1973). Nozawa, Y. and Thompson, G .A .,jr .: Studies o f membrane formation in Tetrahymena pyriformis. II. Isolation and lipid analysis o f cell fractions. J . Cell Biol. 49: 712-721 (1971). Nozawa, Y. and Thompson, G .A ., jr.: Studies o f membrane formation in Tetra hymena pyriformis. III. Lipid incorporation into various cellular membranes o f log phase cultures. J . Cell Biol. 49: 722-730 (1971). Nozawa, Y. and Thompson, G .A .,jr .: Studies o f membrane formation in Tetrahymena pyriformis. V . Lipid incorporation into various cellular membranes o f stationary phase cells, starving cells and cells treated with metabolic inhibitors. Biochim. biophys. Acta 282: 93-1 0 4 (1972). Pan, H .Y .M .; Chou, S .C , and Conklin, K .A .: Effects o f antimalarial drugs and clofi-
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brate on in vitro lipid synthesis in Tetrahymena pyriformis, G L . Pharmacology 12: 4 8 -5 6 (1974). Pollard, W. V.; Shorb, M .S.; Lund, P .G ., and Vasaitis, V.: The effect o f triparanol on synthesis o f lipids by Tetrahymena pyriformis. J. Protozool. 10: Suppl., p. 7 (1963). Schwab-Stey, H.; Schwab, D ., and Krebs, W.: Electron microscopic examination o f isolated mitochondria o f Tetrahymena pyriformis. J . Uttrastruct. Res. 37: 8 2 -9 3 (1971). Seaman, G .R .: Utilization o f acetate by Tetrahymena geleii (s). J . biol. Chem. 186: 97-1 0 4 (1950). Shorb, M .S.: Dunlop, B.E ., and Pollard, W.O.: Effects by triparanol on synthesis o f squalene and tetrahymenol by Tetrahymena pyriformis. Proc. Soc. exp. Biol. Med. 118: 1140-1145 (1963). Steinberg, D .: Frederickson, D .S., and Avigan, J .: Effect o f A 4-cholestenone in animals and man. Proc. Soc. exp. Biol. Med. 97: 784-790 (1958). Thompson, G .A ., jr.: Studies on membrane formation in Tetrahymena pyriformis. 1. Rates o f phospholipid biosynthesis. Biochemistry 6: 2015-2022 (1967). Thompson, G .A ., jr.; Bambery, R .J ., and Nozawa, Y.: Further studies o f lipid composi tion and biochemical properties o f Tetrahymena pyriformis membrane systems. Bio chemistry 10: 4441 4447 (1971). Toki, K.; Nakamura, Y.; Agatsuma, K .; Nakatani, H., and Aono, S .: Hypolipidemic action o f a new aryloxy compound (S-8527) in rats. Atherosclerosis 18: 101-108 (1973).
Dr. K .A . Conklin, Department o f Anesthesiology, School o f Medicine, University o f Cali fornia, Los Angeles. CA 90024 (USA)
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Effects of Hypolipidemic Agents on Lipid Synthesis in Subcellular Fractions from Tetraliymena pyriformis H. Y.M. Pan, S .C . Chou and K .A . Conklin Department o f Pharmacology, School o f Medicine. University o f Hawaii, Honolulu, Hawaii
Key Words. Hypolipidemic agents • Lipid synthesis • Microsomes • Mitochondria • Soluble cell fraction • Tetraliymena pyriformis Abstract. Lipid synthesizing systems have been prepared from subcellular fractions o f Tetraliymena pyriformis, G L . These fractions, the mitochondrial fraction, the microsomal fraction and the soluble cell fraction, have been characterized as to cofactors and cations required for optimal lipid synthesis. The effects o f hypolipidemic agents on lipid synthesis by all fractions are presented.
Introduction
Received: March 11, 1976.
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The biosynthetic pathways o f lipids in Tetraliymena pyriformis have previ ously been examined both in vivo (10, 29) and in vitro (16, 26). These studies were performed using 14C-labeled acetate or other suitable precursors for lipid synthesis, and have led to the identification of the lipids synthesized by this organism. Other studies have led to the identification o f the various classes o f lipids present in the membrane fragments o f organelles (22 25, 32, 33). These in vivo and in vitro systems have also been utilized to study the effects o f drugs, primarily hypocholesterolemic agents, on lipid synthesis (15, 21, 26, 27, 30). Cholesterol and other steroids have been shown to overcome the inhibitory effects by some o f these hypocholesterolemic agents (14). In this paper, we present the characterization o f lipid synthesis in subcellu lar fractions of T. pyriformis, strain G L , and the effects o f hypolipidemic agents on these processes.
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Materials and Methods Organism and Growth Condition T. pyriformis, strain G L , was grown and harvested as described previously (8). For experimental purposes, 2-day-old cultures were used to inoculate 200 ml (for mitochondrial preparation) or 1,000 ml (for microsomal and soluble cell fraction preparations) o f the proteose-peptone liver extract medium. Cultures were incubated 12 18 h at 29 °C . The cells were harvested when the population reached approximately 200.000/ml.
Preparation o f Subcellular Fractions Mitochondria were prepared by a modification o f the procedure o f Schwab-Stey et al. (28). The harvested ceils were washed and suspended in 10 ml o f buffer containing 0.1 M phosphate buffer (pH 7.2), 0.3 Af sucrose, and 10 mAf mercaptoethanol (solution L). The cells were then disrupted by homogenization in a glass homogenizer, and centrifuged at 500 g for 10 min at 4 °C . The pellet was resuspended and centrifuged at 500 g for 10 min. Tile supernatants were combined and again centrifuged at l,0 0 0 g for 10 min. The super natant was centrifuged at 7.000g for 10 min. The pellet was resuspended in 10 ml o f a Ficoll solution containing 10 g o f Ficoll in 100 ml o f solution L and centrifuged at 1.000g for 10 min. The supernatant containing mitochondria was then washed twice in the Ficoll solution by centrifugation at 7,000 g for 30 min. The final pellet containing the mitochon drial preparation was suspended in 10 ml o f solution L. This preparation yielded a mito chondrial preparation with a protein content o f 1—1.5 mg,'ml. The microsomal fraction was prepared from the pooled supernatants from the 1,000 g centrifugation for the preparation o f the mitochondrial fraction. This supernatant was cen trifuged at 10,000g for 30 min, the pellet was discarded and the microsomal fraction was sedimented by centrifugation at 105,000g for 60 min. The final microsomal pellet was suspended in 4 ml o f solution L. This preparation yielded a microsomal preparation with a protein content o f 3—4 mg/ml. The supernatant after the final centrifugation at 105,000g was used as the soluble cell fraction. This preparation contained a protein concentration o f 7 -1 0 mg/ml.
Assay for mitochondrial lipid synthesis was done using a 0.5-ml reaction mixture containing 200-400 jug o f protein as determined by the procedure o f Lowry et al. (17), 0.1 Af phosphate buffer (pH 7.2), 0.3 M sucrose, 10 mAf mercaptoethanol, 0.1 mM E D T A , 2.5 mM M gC L, 10 mAf ATP. 0.4 mM N A D H , 0.4 mM NADPH, 0.1 mM C o A , and 0.1 nCi o f 2-‘,'C-acetate (25 juCi/mmol, Calatomic). The reaction mixtures were incubated at 29 °C , the optimal growth temperature for T. pyriformis, and samples (0.1 ml) were taken in duplicate at 0 and 30 min. The samples were placed on filter paper discs and assayed by the filter paper disc procedure to determine the incorporation o f acetate into lipids (5). Assay for microsomal lipid synthesis was done using a 0.5-ml reaction mixture contain ing 0.1 M phosphate buffer (pH 7.2), 0.3 M sucrose, 10 mAf mercaptoethanol, 0.8 mAf N A D H , 1.0 mAf C o A , 10 mAf A TP , 5 mAf MgCl2 and 0.1 yCi o f 2-l4C-acetate (25 juCi/mmol, Calatomic). The reaction tube was incubated at 29 °C and samples (0.1 ml) were taken in duplicate at 0 and 30 min and assayed as described above for the mitochondrial fraction. Assay for lipid synthesis in the soluble cell fraction was performed in a reaction mixture containing 0.1 Af phosphate buffer (pH 7.2), 0.3 Af sucrose, 10 mAf mercapto ethanol, 1 mAf E D T A . 2 mAf N A D H , 2 mAf N A D PH , 0.4 mAf C o A , 4 mAf A TP , 4 mAf M n C l,, 2 mAf M gCl, and 0.1 juCi o f 2-14C-acetate in a total volume o f 0.5 ml. The amount o f protein in each reaction mixture was 1- 2 mg. The reaction mixture was incubated at
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Assay for Lipid Synthesis in the Subcellular Fractions
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29 °C and samples were removed in duplicate at 0 and 90 min. The samples were then assayed by the filter paper disc procedure.
Isolation and Identification o f Lipids Synthesized Lipids were isolated from the reaction mixtures by extraction with chloroform:methanol (2:1 v/v) according to the method o f Bligh and Dyer (4). Extracted lipids were washed overnight by the method o f Folcli et at. (11). General classes o f lipids were separated and identified in Silica gel G thin-layer plates by developing with a solvent system containing petroleum ctheridiethyl cthcr:acctic acid (75:25:1 v/v/v). Phospholipids were further sepa rated by developing in a solvent system containing chloroform:acctic acid:methanol:water (75:25:5:2.2 v/v/v/v). After developing the plates, the spots were located by exposure to iodine vapors, the stained areas carefully circled with a lead pencil, and following sublima tion o f the iodine the spots were scraped directly into scintillation vials. Concentrated sulfuric acid was used to identify tetrahymenol on the plates. The radioactivity present on each spot was then determined by measurement in a Beckman liquid scintillation counter, model LS 150, using toluene-PPO (5 g/1) scintillation fluid.
Results
Requirements fo r Acetate Incorporation into Lipids by the Microsomal Fraction Table II shows the requirements for the incorporation of acetate into lipids by the microsomal fraction. The complete reaction mixture incorporated 990 cpm/100/rg protein. Omission o f NADH and Mg++ reduced the activity to 22 and 39%, respectively. Omission o f ATP and CoA abolished the incorpora-
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Requirements fo r Acetate Incorporation into Lipids by the Mitochondrial Fraction Table 1 shows the requirements for lipid synthesis by the mitochondrial fraction. The complete reaction mixture incorporated 6,350 cpm/100 Mg pro tein. Omission of ATP or CoA almost abolished the activity (3 % remaining for ATP, 4 % for CoA). Both NADH and NADPH were required for the system. Omission of NADH reduced the activity to 35 % and absence o f NADPH re duced activity to 89 %. Magnesium ions were also required (9 % remaining with out Mg++), but Mn++ was inhibitory to the system at 2.5 and 5.0 mM . ADP did not support lipid synthesis in the absence of ATP. The reaction mixture minus EDTA showed 94 % of the activity o f the complete system. Figure 1a shows the time course o f lipid synthesis by the mitochondrial preparation. Linear incorporation was observed for the first 30 min and maxi mum incorporation was attained after 60 min of incubation. Figure 2a shows the optimal protein concentration for incorporation using the optimal condi tions. Linear incorporation was observed with increasing protein concentration up to 500 Mg per reaction mixture.
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Fig. 1. Time course for lipid synthesis in the mitochondrial fraction (a), the micro somal fraction (b) and the soluble cell fraction (c). The reaction mixture for each is as described in Materials and Methods, with samples being assayed at the indicated times by the filter paper disc procedure for lipid synthesis. Each value is the average o f two determi nations from a representative set o f experiments.
Table I. Requirements for acetate incorporation into lipids in the mitochondrial frac tion Incubation mixture
Incorporation cpm/100 Mg protein
Relative incorporation %
Complété Minus ATP Minus ATP, plus 10 mM ADP Minus CoA Minus NADH Minus NADPH Minus Mg” Minus ED TA Minus E D T A , plus Mn** 2.5 mM Minus E D T A , plus Mn** 5.0 mM
6,350 180 200 230 2,210 5,080 560 5,960 5,410 4,250
100 3 3 4 35 89 9 94 85 67
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The complete incubation mixture is as described in Materials and Methods. Samples were incubated at 29 °C and 0.1-ml aliquots in duplicate were removed at 0 and 30 min, and acetate incorporation into lipids was determined by the filter paper disc procedure. Results presented are from one representative set o f experiments.
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P ro te in , m g / re a c tio n sam p le
Fig. 2. Protein concentration for lipid synthesis in the mitochondrial fraction (a), the microsomal fraction (b) and the soluble cell fraction (c). The reaction mixture for each is as described in Materials and Methods. Samples were taken at 0 and 30 min (a); 0 and 30 min (b); and 0 and 90 min (c), and were assayed by the filter paper disc procedure for lipid synthesis. Each value is the average o f two determinations from a representative set o f experiments.
Table II. Requirements for the incorporation o f acetate into lipids in the microsomal fraction Reaction mixture
Incorporation cpm/100 ug protein
Relative incorporation
% Complete Minus ATP Minus CoA Minus N A D U Minus Mg'* Plus N A D PH , 0.8 mAf Plus Mn**, 5 mM Plus E D T A , 0.1 mM
990 8 8 220 390 960 740 960
100 1 1 22 39 97 75 97
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The complete incubation mixture is as described in Materials and Methods. Samples were incubated at 29 °C and 0.1-ml aliquots in duplicates were removed at 0 and 30 min, and acetate incorporation into lipids was determined by the filter paper disc procedure. Each value is the average o f two determinations. Results presented are from one representative set o f experiments.
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Table III. Requirements for the incorporation o f acelate into lipids in the soluble cell fraction Reaction mixture
Incorporation cpm/mg protein
Relative incorporation
% Complete Minus ATP Minus CoA Minus NADH Minus NADPH Minus Mg** Minus Mn** Minus ED TA
2,610 30 70 120 120 2,480 1,530 1,780
100 1 3 5 5 95 59 68
The complete incubation mixture is as described in Materials and Methods. Samples were incubated at 29 °C and 0.1-ml aliquots in duplicate were removed at 0 and 90 min, and acetate incorporation into lipids was determined by the filter paper disc procedure. Each value is the average o f two determinations. Results presented are from one representative set o f experiments.
Requirements fo r Acetate Incorporation into Lipids by the Soluble Cell Fraction Table III shows the requirements for the incorporation o f acetate into lipids by the soluble cell fraction. This fraction, like the mitochondrial fraction, required both NADH and NADPH for optimal activity. Omission o f either o f these cofactors reduced the incorporation to 5 %. Omission o f ATP reduced the activity to 1 % and omission o f CoA reduced activity to 3 %. Both Mn++ and Mg++ were required by the system for maximal synthesis of lipids. EDTA was also required by this fraction. Figure l c shows the time course for lipid synthesis by the soluble cell fraction. Linear incorporation was observed in the first 90 min and maximal incorporation was attained at approximately 120 min. Figure 2c shows linear incorporation for protein concentration up to 2 mg per reaction mixture.
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tion almost completely. NADPH, Mn++ and EDTA were not required by this fraction for the synthesis o f lipids. Figure 1b shows the time course for lipid synthesis by the microsomal fraction. The rate o f incorporation was linear up to 30 min and reached maximal incorporation at 60 min. Figure 2 b gives the relationship of protein concentra tion to the incorporation o f acetate into lipids. Linear incorporation was ob served with protein concentration up to 1 mg per reaction mixture.
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Table IV . Acetate incorporation into individual classes o f lipids in the subcellular frac tions Lipid
Triglycerides Free fatty acids Tetrahymenol Total neutral lipids Phospholipids Total lipids
Incorporation, cpm/100 jug protein mitochondria
microsomes
soluble cell fraction
24 (0.5) 156(3) 1,260 (24) 1,440 3,839 (72.5) 5.279
38 (5) 435 (58) 52(7) 525 225 (30) 750
109 (5) 620(28) 745 (33.50) 1,474 746 (33.5) 2,220
The separation and identification o f the various classes o f lipids are as described in Materials and Methods. Each value is the average o f two determinations. Total neutral lipids is the total o f triglycerides, free fatty acids and tetrahymenol. The percentage o f total lipids synthesized for each lipid class is in parentheses following each value.
Table V. Acetate incorporation into individual classes o f phospholipids in the subcellu lar fractions Lipid class
Cardiolipin 2-Aminoethylphosphonolipids Ethanolamine phosphatides Choline phosphatides Total phospholipids
Incorporation, cpm/100 Mg protein mitochondria
microsomes
soluble cell fraction
480(11) 524(12) 3,152 (74) 132(3) 4,288
8 (4) 30(15) 165 (80) 2(1) 205
0 40 (8) 321 (60) 172 (32) 533
Characterization o f the Products Synthesized in the Subcellular Fractions Table IV and V shows the percentages o f the various classes o f lipids synthe sized by these subcellular fractions. The mitochondria produced 0.5 % triglycer ides, 3 % free fatty acids, 24 % tetrahymenol and 72.5 % phospholipids. O f the phospholipids synthesized, ethanolamine phosphatides comprised the largest percentage (74%). Lipids synthesized in the microsomal fraction were primarily
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The separation and identification o f the various classes o f lipids are as described in Materials and Methods. Each value is the average of two determinations. The percent age o f total phospholipids for each phospholipid class is in parentheses following each value.
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free fatty acids (58 % of total lipids), and phospholipids (30 % o f total lipids). As in the mitochondrial fraction, ethanolamine phosphatides were the primary phospholipids synthesized (80 %). O f the lipids synthesized in the soluble cell fraction, 5 % were triglycerides, 28% free fatty acids, and 33.5% were tetrahymenol. The remaining lipids were phospholipids (33.5 %), and o f these 60 % were ethanolamine phosphatides, 32 % choline phosphatides, and 8 % 2-aminoethylphosphonates. Effects o f Hypolipidemic Agents on Lipid Synthesis Table VI shows the degree of inhibition o f lipid synthesis by hypolipidemic agents in each o f the subcellular fractions.
Subcellular fractions from T. pyriformis have been shown to incorporate l4C-acetate into several classes o f lipids. The mitochondrial fraction synthesized more phospholipids (both relatively and absolutely) than either the microsomal or the soluble cell fraction. Free fatty acid synthesis was more active in the soluble cell fraction. It was not determined, however, whether incorporation of acetate into free fatty acids was by de novo synthesis or by chain elongation. Tetrahymenol, a triterpenoid synthesized exclusively in T. pyriformis in place o f cholesterol (18), was synthesized primarily in the mitochondrial and the soluble cell fractions. The cofactor requirements for optimal lipid synthesis differed between the subcellular fractions. The microsomal fraction did not require NADPH, whereas the soluble cell fraction showed a 95 % decrease in activity when this cofactor was omitted. The mitochondrial fraction also showed less activity in the absence of NADPH. ATP was essential for activity of all fractions. It is generally believed that ATP does not traverse the mitochondrial membrane (7), but is formed by oxidative phosphorylation from ADP which is transported across the mitochon drial membrane. In the characterization o f lipid synthesis in the mitochondrial fraction, however, ADP in place o f ATP did not support lipid synthesis. This may indicate that, under the conditions o f the reaction, ADP did not cross the mitochondrial membrane or that oxidative phosphorylation was not functioning. Clofibrate, a hypolipidemic agent which inhibits hepatic synthesis o f choles terol and triglycerides (1, 2), produced a marked inhibition o f lipid synthesis by mitochondria. The degree of inhibition o f lipid synthesis by the microsomal and the soluble cell fraction was considerably less. S-8527, a hypolipidemic agent more potent than clofibrate (34), produced marked inhibition in all fractions. a-Phenylbutyrate, which inhibits the activation of acetate to acetyl CoA (20), showed inhibition o f lipid synthesis in all fractions. TPIA, known to inhibit the
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Discussion
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Table VI. Effects o f hypolipidemic agents on l4C-acetate incorporation into lipids Drug
Concentration mol/1
Mitochondria
Inhibition, % microsomes
soluble cell fraction
10’ 4 10’ 3
38 63
6 19
0 1
S-8527
1 X 1 0 -3 2 X 1 0 -3
51 78
58 75
50 91
a-Phenylbutyrate
3 X 1 0 -3 6 X 1 0 -3
28 49
4 17
14 33
TP1A
8 8
X
1 0 -5 10-4
40 83
12 38
12 59
4 4
X
1 0 -5 10-4
52 82
14 31
4 12
1.4 1.4
X
10-4 1 0 '3
52 94
68 97
8 19
6 6
X
10-* 10-4
54 88
52 78
18 31
2 2
X
10-4 1 0 -3
19 69
0 2
12 26
1 0 -4 1 0 -3
28 43
0 5
0 0
1 0 -3 1 0 -J
2 17
0 4
0 2
1 0 -3 1 0 -3
8 30
0 5
0 0
5 X 10-4 1 X 1 0 -3
0 0
0 0
0 0
Clofibrate
Triparanol
S K F 3301A
S K F 525A
S K F 2314
S K F 16467 A
Nicotinic acid
A 4-Cholestenone
Probucol
4 2
X X
X
X
X
X
X
1.4 1.4
X
8 1.6
X
4 8
X
X
X X
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The complete reaction mixture is as described in Materials and Methods, with the hypolipidemic agents added at the indicated concentrations. Incubation time was as follows: mitochondria, 30 min; microsomes, 30 min; soluble cell fraction, 90 min. Acetate incorporation was determined by the filter paper disc procedure. Each value is the average o f two determinations.
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activity o f acetyl CoA carboxylase (19), and triparanol which has numerous sites o f action (13), also showed consistent inhibition. The SKF experimental drugs, which have been shown to be hypocholes térolémie agents (12, 13), showed inhibitory effects on the incorporation o f acetate into lipids o f most fractions. However, as compared to SKF 3301 A and 525 A , SK F 16467A and 2314 produced considerably less inhibition o f lipid synthesis in the microsomal fraction. Since tetrahymenol synthesis is very low in this fraction, these results may indicate a primary' effect on steroid synthesis by the latter compounds, while the former compounds have in addition a promi nent effect on synthesis o f other neutral lipids or phospholipids. Nicotinic acid, A4-cholestenone, and probucol did not show potent inhibi tory effects in any fraction. However, for nicotinic acid and probucol these results may be consistent with proposed sites of action for these agents as the former inhibits release o f fatty acids from adipose tissue (6) and the latter inhibits transport o f cholesterol from the gut (3). Neither o f these actions would be expected to inhibit lipid synthesis in cell-free preparations. A4-cholestenone has been proposed to inhibit the conversion of hydroxymethylglutaryl CoA to mevalonic acid (31), a reaction common to cholesterol and tetrahymenol bio synthesis (9). Therefore, this agent should inhibit lipid synthesis by mitochon dria and the soluble fraction as these fractions synthesize tetrahymenol. The explanation for the minimal inhibition observed with mitochondria, and the lack o f inhibition with the soluble fraction is unclear.
A cknowledgement This is contribution No. 1354 to the Army Research Program on Malaria, and was supported by contract D A D A 17-71-C-l 116 from the United States Army Medical Research and Development Command.
References
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The effect o f nicotinic acid on the plasma free fatty acid. Demonstration o f a metabolic type o f sympathiocolysis. Acta med. Scand. 172: 641 645 (1962). Chappell, J.B .: Systems for the transport o f substances into mitochondria. Br. med. Bull. 24: 150 157 (1968). Chou, S.C. and Ramanathan, S .: Quinacrine site of inhibition o f synchronized cell division in Tetrahymena. Life Sci. 7: 1053 1062 (1968). Connor, R .L .; Landrey, J .R .: Borns, C.H ., and Mallory, F .B .: Cholesterol inhibition o f pentacyclic triterpenoid biosynthesis in Tetrahymena pyriformis. J . Protozool. 15: 600-605 (1968). Erwin, J. and Block, K .: Lipid metabolism o f ciliated protozoa. J . biol. Chem. 238: 1618 1624 (1963). Folch, J .: Lees, M., and Sloane-Stanley, G .H .: A simple method for the isolation and purification o f total lipids from animal tissues. J . biol. Chem. 226: 4 9 6 -5 0 9 (1957). Holmes, W.L. and Bentz, J .D .: Inhibition o f cholesterol biosynthesis in vitro by or dicthylaminocthyldiphenyl-propylacetate hydrochloride (SK F 525A). J . biol. Chem. 235: 3118 3122(1960). Holmes, W.L. and Detullio, N.W.: Inhibitors o f cholesterol biosynthesis which act at or beyond the mevalonic acid stage. Am . J . clin. Nutr. 10: 310 322 (1962). Holmlund, G.W.: Growth inhibition o f Tetrahymena pyriformis by hypocholesteremic compound and the mechanism o f its reversal by various lipids. Biochim. biophys. Acta 296: 221-233 (1973). Holz, G .G .: Erwin, J .: Rosebaum, N., and Aaronson, S .: Triparanol inhibition o f Tetra hymena and its prevention by lipids. Archs Biochem. Biophys. 98: 31 2 -3 2 2 (1962). Kapoulas, V.M. and Thompson, G .A ., jr.: The formation o f glycerol ethers by cell free Tetrahymena extracts. Biochim. biophys. Acta 187: 594 597 (1969). Lowry, O.H .; Rosebrough, N .J.; Farr, A .L ., and Randall, R .J .: Protein measurement with the Folin phenol reagent. J . biol. Chem. 193: 265-275 (1951). Mallory, F.B .: Gordon, J.H ., and Connor, R .C .: The isolation o f the pentacyclic triterpenoid alcohol from a protozoan. J . A m . chem. Soc. 85: 1362-1363 (1963). Maragoudakis, M .E .: Inhibition o f hepatic acetyl coenzyme A carboxylase by hypo lipidemic agents. J . biol. Chem. 244: 5005 5013 (1969). Masters, R . and Steinberg, D .: Studies on the mechanism o f action o f a-phenylbutyrate. Biochim. biophys. Acta 27: 592- 597 (1958). Nozawa, Y .: Inhibition o f lipid biosynthesis by p-chlorophenyoxy isobutyrate (CP1B) in Tetrahymena pyriformis. J . Biochem. 74: 1157 1163 (19 7 3). Nozawa, Y.; Fukushima, H ., and lida, H .: Isolation and lipid composition o f nuclear membranes from macronuclei o f Tetrahymena pyriformis. Biochim. biophys. Acta 318: 335-344 (1973). Nozawa, Y. and Thompson, G .A .,jr .: Studies o f membrane formation in Tetrahymena pyriformis. II. Isolation and lipid analysis o f cell fractions. J . Cell Biol. 49: 712-721 (1971). Nozawa, Y. and Thompson, G .A ., jr.: Studies o f membrane formation in Tetra hymena pyriformis. III. Lipid incorporation into various cellular membranes o f log phase cultures. J . Cell Biol. 49: 722-730 (1971). Nozawa, Y. and Thompson, G .A .,jr .: Studies o f membrane formation in Tetrahymena pyriformis. V . Lipid incorporation into various cellular membranes o f stationary phase cells, starving cells and cells treated with metabolic inhibitors. Biochim. biophys. Acta 282: 93-1 0 4 (1972). Pan, H .Y .M .; Chou, S .C , and Conklin, K .A .: Effects o f antimalarial drugs and clofi-
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Dr. K .A . Conklin, Department o f Anesthesiology, School o f Medicine, University o f Cali fornia, Los Angeles. CA 90024 (USA)
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