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The relationship between chain elongation of palmitoyl-CoA and phospholipid content in rat liver microsomes
173
Bioehimica et Biophysics Aetu, 441 (1976) 173-180 @ Elsevier Scientific Publishing Company, Amsterdam
- Printed
in The Netherlands
BBA 56841
THE RELATIONSHIP BETWEEN CHAIN ELONGATION OF PALMITOYLCoA AND PHOSPHOLIPID CONTENT IN RAT LIVER MICROSOMES
YOICHI KAWASHIMA, UCHIYAMA * * Pharmaceutical (Received
MITSUO
Institute, Tokoku
January
15th,
NAKAGAWA
*, YASUO
University, Aobayama,
SUZUKI
and MITSURA
Sendai (Japan)
1976)
Summary The relationship between the chain elongation of palmitoyl-CoA and phospholipid content in rat liver microsomes was studied. When liver mierosomes were incubated with phospholip~e C, mi~rosom~ phospholipids were linearly hydrolyzed during 10 min of incubation under the present experimental conditions. Coincident with the decrease in microsomal phospholipid content by phospholipase C treatment, the chain elongation activity also decreased linearly. The decreased chain elongation activity in phospholipase C-treated microsomes was completely or partially recovered by the addition of a sonicated dispersion of phosphatidylcholine, microsomal phospholipids or phosphatidylcholine/phosphatidylethanolamine mixtures. The extent of recovery of decreased activity by a sonicated dispersion of phosphatidyIcholine/phosphatidyleth~olam~e mixture was gradually reduced by increasing amounts of phosphatidyleth~ol~ine in the dispersion. In addition, the chain elongation activity in native nicrosomes was more stimulated by the addition of a sonicated dispersion of phosphatidylcholine alone than by that of phosphatidylcholine/phosphatidylethanolam~e mixtures. The chain elongation activity of palmitoyl-CoA was inhibited by the addition of stearoyl-CoA which is the end-product of this reaction. The inhibitory effect of stearoyl-CoA was partially eliminated by the addition of a sonicated dispersion of phosphatidylcholine. The increase of the chain elongation activity in native and phospholipase C-treated microsomes by the addition of a sonicated dispersion of phosphatidylcholine was not related to the activity of fatty acyl-CoA hydrolase.
----
* Present ** Present Japan.
address: address:
Faculty National
of Pharmaceutical Sciences. Kumamoto University, Institute of Hygienic Sciences, Kamiyoga-1-chome,
Kumamoto. Setagaya,
Japan. Tokyo,
174
Introduction It is well known that the enzymes that catalyze the desaturation and chain elongation of palmitoyl-CoA are located in microsomal fraction of rat liver [ 11. We have previously reported that the chain elongation of palmitoyl-CoA in liver microsomes of normal and refed rats is stimulated by the addition of a sonicated dispersion of phosphatidylcholine, and that chain elongation activity in liver microsomes treated with phospholipase C and acetone decreases remarkably [2] . In addition, we have also reported that when palmitoyl-CoA is incubated with liver microsomes in the presence of malonyl-CoA and reduced pyridine nucleotides, palmitoyl-CoA is preferentially introduced into the chain elongation pathway, rather than into the desaturation pathway by the addition of a sonicated dispersion of phosphatidylcholine [ 31. The present paper deals with the relationship between the chain elongation activity of palmitoyl-CoA and phospholipid content in rat liver microsomes. Materials and Methods Materials
Palmityol-1-[ “C]CoA (58.2 Ci/mol) was obtained from New England Nuclear Corp. (Boston, Mass., U.S.A.). Palmitoyl-CoA was purchased from Nutritional Biochemical Co. (Cleaveland, Ohio, U.S.A.); reduced nicotinamide adenine dinucleotide (NADH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) from Kyowa Hakko Co. (Tokyo, Japan): malonyl-CoA, stearoylCoA, bovine serum albumin and phospholipase C (from Clostridium welchii) from Sigma Chemical Co. (St. Louis, MO., U.S.A.). Animals
Female albino rats of the Wistar strain, weighing 80-130 g, were used. The animals were starved for 48 h and then fed ad libitum on the commercial diet obtained from Oriental Yeast Co. (Tokyo, Japan) for 24 h. Preparation
of the enzyme
All procedures were carried out at O-4” C. Rat livers were homogenized in 3 ~01s. of cold 0.25 M sucrose solution. The homogenate was then fractionated by differential centrifugation, as described previously [2] . The microsomal pellet obtained was suspended in 0.1 M Tris - HCl or sodium phosphate buffer (pH 7.4) containing 0.25 M sucrose. The protein content determinations were performed by the procedure described by Lowry et al. [4] with crystalline bovine serum albumin as standard. Preparation
of phospholipids
Phosphatidylcholine and phosphatidylethanolamine were prepared from egg yolk and purified by silicic acid-column chromatography. The purified phosphatidylcholine and phosphatidylethanolamine gave a single spot on silica gel G plates with chloroform/methanol/water (65 : 25 : 4, v/v) as developing solvent. Microsomal phospholipids were isolated from microsomal total lipids extracted by chloroform/methanol (2 : 1, v/v) containing 0.1 mg of butylated hydroxy-
175
per 100 ml as antioxidant by using silicicacid column chromatog. raphy. We have confirmed by thin layer chromatography that the micmom~
toluene
phospholipid
fraction
did not contain
microsomal
neutral
lipids.
Preparation af phospholipid
dispersions The sonicated dispersion of phospholipids was prepared in the same manner as described previously except for the use of water instead of 0.025% ethylenediamine tetraacetic acid (EDTA) as solvent [ 51. Lipid phosphorous content in the dispersion was determined by the method of Rouser et al. [6].
Assay of chain elongation of palmitoyl-CoA The standard incubation mixture contained 45 nmol palmitoyl-CoA, 3.4 nmol palmitoyl-l-[14C] CoA, 200 nmol of malonyl-CoA, 3 pmol NADH, 3 pmol NADPH, 25 pmol sucrose, 1 E.cmol KCN and 1 mg microsomal protein. The final volume was adjusted to 1 ml with Tris - HCl buffer (90 pmol, pH 7.4). Incubation was carried out at 37°C for 6 min under Nz with constant shaking. After the incubation, the reaction was stopped by the addition of 1 ml of 10% KOH/methanolic solution and then heated at 70°C for 30 min. After acidification with 6 M HCl, the extraction of lipids and isolation of fatty acid was completed by the same procedure as described previously [7]. Fatty acids extracted from the incubation medium were methylated and then analyzed by means of radio-gas liquid chromatography as described previously [ 21. Assay of fatty acyl-CoA hyclrolase activity The fatty acyl-CoA hydrolase activity in liver microsomes was determined by a partially modified procedure of Jones et al. [8]. The incubation mixture contained the same components as those used for the chain elongation reaction except for the absence of NADH, NADPH and malonyl-CoA. After the reaction was stopped by the addition of 0.7 ml of 4 N H2S04, the incubation medium was extracted three times with 5 ml hexane. The hexane solutions were combined and evaporated to dryness at 45°C in vacua. The lipids obtained were dissolved in a constant volume of hexane. An aliquot of the hexane solution was used for the determination of the recovery of radioactivity. After another aliquot of the hexane solution was evaporated, the hexane extract was separated to each lipid fraction on silica gel G plates by using hexane/ether/acetic acid (70 : 30 : 1, v/v) as developing solvent. After developing, the area of silica gel G plate corresponding to palmitic acid used as standard was scraped out into a counting vial and the radioactivity measured by a Packard Tri-Carb liquid scintillation spectrometer. Each vial contained 15 ml toluene with 0.4% 2,5diphenyloxazole, 0.01% 1,4-bis-(5-phenyloxazolyl)-benzene and 3% thixotropic gel powder (Cab-o-sil). Phospholipase C treatment Phospholipase C from Cl. welchii (3.6 units/mg) was used without further purification. 30 mg of liver microsomal protein were incubated with phospholipase C, 30 pmol /3-mercaptoethanol, 21 prnol CaClz and 375 pmol sucrose in 6.9 ml of 0.1 M Tris - HCl buffer (pH 7.4) at 24” C. The reaction was stopped by the addition of EDTA. For the determination of the extent of the micro-
176
somal phospholipid hydrolysis, total lipids were extracted from the incubation medium as described previously [ 71. Lipid phosphorus content in the extracted lipids was determined by the method of Rouser et al. [6]. Results and Discussion We have previously reported that the chain elongation activity of palmitoylCoA in rat liver microsomes treated with phospholipase C decreases remarkably
[a* In this paper, we study in further detail the relationship between the chain elongation activity of palmitoyl-CoA and the decrease of phospholipid content in rat liver microsomes treated with phospholipase C. As shown in Fig. 1, when 1 mg liver microsome protein was incubated with phospholipase C (0.2 unit/mg) at 24°C microsomal phospholipids were hydrolyzed linearly during 10 min of incubation (curve l), under the present experimental conditions. Coincident with the decrease of microsomal phospholipid content caused by phospholipase C treatment, the chain elongation activity decreased linearly (curve 2). Thus an incubation time of 6 min was used for the degradation of microsomal phospholipids by phospholipase C treatment. On the other hand, as shown in Table I (experiment I), the effect of a sonicated dispersion of phosphatidylcholine, microsomal phospholipids or phosphatidylcholine/phosphatidylethanolamine mixtures on the chain elongation of
0 10
Incubation
20 time,
min
Fig. 1. Effect of phospholipase C treatment on the chain elongation of palmitoyl-CoA in liver microsomes. Liver microsomes were pretreated with phospholipase C under the following experimental conditions. 30 mg of liver microsomal protein were incubated with phospholipase C (6 units), 30 wmol pmercaptoethanol, 21 qnol C&12 and 375 /.unol wcrose in 6.9 ml of 0.1 M Tris . HCl buffer (pH 7.4) at 24’C. After each preincubation 0.23 ml of the above incubation mixture containing 1 mg of microsomal protein were added to the incubation medium containing 10 ~.rmol EDTA for the assay of the pahnitoylCoA chain elongation. The incubation conditions for the assay of the chain elongation reaction and the determination of phospholipid content were described in the text.
177
3
10
20
30
Palmitoyl-CoA,
40
50
PM 1
50
40
30
20
Stearoyl-CoA.
10
0
PM
Fig. 2. Effect of stearoyl-CoA on the chain elongation activity of palmitoyl-CoA in liver microsomes. Incubation conditions were described in the text, except for the addition of various amounts of palmitoylCoA as substrate and of stearoyl-CoA as inhibitor. The sum of the amounts of palmitoyl-CoA and stearoylPalmitoyl-CoA + stearoylCoA (curves 2 and 3) was constant. r-3, Pahnitoyl-CoA alone: A-----A, CoA + phosphatidylcholine dispersion (602 gg); a---A, Palmitoyl-CoA + stearoyl-CoA.
palmitoyl-CoA in native and phospholipase C-treated microsomes was examined. The decreased chain elongation activity of palmitoyl-CoA in rat liver microsomes treated with phospholipase C was completely or partially recovered by the addition of a sonicated dispersion of phosphatidylcholine (58.2 pg as lipid phosphorus) to the same degree as previously reported [2] , though not to the level obtained with phosphatidylcholine alone by the addition of microsomal phospholipid dispersions (58.2 pg as lipid phosphorus) or phosphatidylcholine/phosphatidylethanolamine dispersions (58.2 pg as lipid phosphorus). The reactivation of the decreased chain elongation activity by the phosphatidylcholine dispersion depended on the concentration of phosphatidylcholine added to the incubation medium. A plateau was reached at a concentration of approx. 1.2 mg phosphatidylcholine (47.2 pug lipid phosphorus per incubation medium. However, microsomal phospholipids and phosphatidylcholine/phosphatidylethanolamine mixtures added to the incubation medium as the dispersion contained respectively 41% and 64% phospholipids other than phosphatidylcholine. Therefore, the effect of increasing amounts (87.0 (ug as lipid phosphorus) of microsomal phospholipid dispersions or phosphatidylcholine/phosphatidylethanolamine dispersion on the chain elongation activity in liver microsomes treated with phospholipase C was also examined. The chain elongation activity could not be further elevated by the addition of increasing amounts of these dispersions. In addition, the stimulator-y effect of phosphatidylcholine dispersion on the chain elongation activity may be depressed by the presence of
178 TABLE
I
EFFECT TION The
OF
VARIOUS
ACTIVITY
OF
pretreatment
various
sonicated
phosphorus
(LP)
of
SONICATED
DISPERSIONS
PALMITOYL-C!oA liver
microsomes
dispersions for the
added
experiment
OF
IN LIVER by to
the
I and
phospholipase incubation 80.0
C
pg as lipid
min
was
medium
Chain
Additions
PHOSPHOLIPIDS
ON
THE
CHAIN
as in
Fig.
1. The
pg and
87.0
ELONGA-
MICROSOMES the
were
phosphorus
elongation
same 58.2 for
activity
pg
the experiment
(stearic
acid
amounts
in the
form
of of
II.
formed,
nmol/
per mg protein)
Native
microsomes
Phospholipase
C-treated
microsomes
Experiment
I
NOIX
1.56
0.59
1.93
1.81
Phosphatidylcholine 58.2
/~g as LP
87.0
pg as LP
Microsomal
1.86
phospholipids
58.2
pg as LP
87.0
ug as LP
1.19 1.05
Phosphatidylethanolamine
(64%)-
phosphatidykcholine
(36%)
mixtures
58.2
gg as LP
1.56
87.0
pg as LP
1.58
Experiment
II
NOW? Phosphatidylcholine
(80
jug LP)
2.44
1.28
3.94
2.48
Phosphatidylethanolaminephosphatidylcholine
(80
fig LP)
1:9
2.81
1.83
2:8
2.71
1.90
4:6
2.52
1.80
6:4
2.10
1.65
phospholipids other than phosphatidylcholine. In fact, as shown in Table I (experiment II), when a sonicated dispersion of various ratios of phosphatidylethanolamine to phosphatidylcholine were added to the incubation medium containing native and phospholipase C-treated microsomes, the elevation of the chain elongation activity was progressively decreased by increasing amounts of phosphatidylethanolamine in the dispersion. In general, fatty acid metabolism in rat liver microsomes is significantly affected by the co-existing long chain fatty acyl-CoA esters [9,10] . Whether the chain elongation of palmitoyl-CoA to stearoyl-CoA is also affected in the presence of stearoyl-CoA, which is the end-product of this reaction, was investigated. As shown in Fig. 2, the sum of the amounts of palmitoyl- and stearoylCoA was constant in this experiment. The chain elongation of palmitoyl-CoA in the presence of various amounts of stearoyl-CoA (curve 3) was depressed as compared to the results obtained by palmitoyl-CoA alone (curve 1). The inhibitory effect of stearoyl-CoA may not be due to its detergent action. In addition, the inhibition of the chain elongation activity of palmitoyl-CoA in the presence of stearoyl-CoA (curve 3) was partially eliminated by the addition of a soni-
179
TABLEII EFFECT ACTIVITY
OF
PHOSPHATIDYLCHOLINE
IN LIVER
DISPERSION
ON
FATTY
ACYL-CoA
HYDROLASE
MICROSOMES
The pretreatment of liver microsomes with phosphlipase C was the same as in Fig. 1. 1 mg of native and phospholipase C-treated microsomes was used for the assay of fatty acyl-CoA hydrolase activity. The other incubation conditions were described in the text. n.d.. not determined. Additions
Phospholipase C-treated microsomes None 1.2 mg Phosphatidylcholine 1.48 mg Phosphatidylcholine Native microsomes Native microsomes None 0.38 mg Phosphatidylcholine 0.55 mg Phosphatidylcholine
Palmitoyl-CoA
hydrolyzed
I
II
0.58 2.53 n.d. 3.22
0.15 n.d. 2.65 3.41
4.19 n.d. 4.36
3.76 3.44 n.d.
(nmol/min
per mg protein)
cated dispersion of phosphatidylcholine (curve 2). Both palmitoyl-CoA that is used as the substrate and stearoyl-CoA that is the end-product of the chain elongation reaction, can be introduced into various lipids. Therefore, the recovery effect of phosphatidylcholine dispersion on the chain elongation activity inhibited by stearoyl-CoA may be partially due to the esterification of stearoyl-CoA with some acceptors such as lysophosphatidylcholine, monoacylglycerol, diacylglycerol and cholesterol, and/or the penetration of the stearoylCoA produced from palmitoyl-CoA into phosphatidylcholine dispersion. However, we have found that a sonicated dispersion of phosphatidylcholine did not contain the lysophosphatidylcholine which by the degradation of phosphatidylcholine produces during the sonication, and that the incorporation of stearoylCoA produced from palmitoyl-CoA into neutral lipid fraction is little affected by the addition of phosphatidylcholine dispersion. It has been reported that fatty acyl-CoA esters are degraded to fatty acid and coenzyme-A by fatty acyl-CoA hydrolase in rat liver microsomes [ 111. Whether the increase of the chain elongation activity of palmitoyl-CoA in native and phospholipase C-treated microsomes by the addition of a sonicated dispersion of phosphatidylcholine is related to fatty acyl-CoA hydrolase activity was also investigated. As shown in Table II, palmitoyl-CoA hydrolase activity in phospholipase C-treated microsomes was reduced to approx. 20% of that in native microsomes. The decreased fatty acyl-CoA hydrolase activity was recovered to approx. 80% of the activity in native microsomes by the addition of phosphatidylcholine dispersion. However, ‘palmitoyl-CoA hydrolase activity in native microsomes was not stimulated by the addition of the dispersion. Therefore, the chain elongation activity of palmitoyl-CoA that was decreased by phospholipase C treatment, may not bedue to a decrease of the availability of palmitoylCoA as substrate by the increase of fatty acyl-CoA hydrolase activity. Experiments are further in progress to study the mechanism of chain elonga-
180
tion stimulation phatidylcholine
of palmitoyl-CoA dispersion.
in liver rnicrosomes
by the addition
of phos-
References 1 2 3 4 5 6 7 8 9 10
Seubert. W. and Podack. E.R. (1973) Mol. Cell, Biochem. 1. 29-40 Nakagawa, M., Kawashima, Y. and Uchiyama, M. (1976) Chem. Pharm. Bull. 24, 46-51 Nakagawa, M., Kawashima, Y. and Uchifama, M. (1976) Lipids, 11. 241-243 Lowry. O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, Nakagawa, M. and Nishida. T. (1973) Riochim. Biophys. Acta, 296. 577-585 Rouser, G., Siakotos. A.N. and Fleisher. S. (1966) Lipids 1, 85-86 Uchiyama. M., Nakagawa, M. and Okui. S. (1967) J. Biochem. 62, l-6 Jones, P.D.. Holloway, P.W., Peluffo, R.O. and Wakil. S.J. (1969) J. Biol. Chem. 244, Nugteren, U.H. (1965) Biochim. Biopbys. Acta. 106, 280--290 Brenner. R.R. and Peluffo. R.O. (1966) J. Biol. Chem. 241. 5213-5219
265-275
744-754
Bioehimica et Biophysics Aetu, 441 (1976) 173-180 @ Elsevier Scientific Publishing Company, Amsterdam
- Printed
in The Netherlands
BBA 56841
THE RELATIONSHIP BETWEEN CHAIN ELONGATION OF PALMITOYLCoA AND PHOSPHOLIPID CONTENT IN RAT LIVER MICROSOMES
YOICHI KAWASHIMA, UCHIYAMA * * Pharmaceutical (Received
MITSUO
Institute, Tokoku
January
15th,
NAKAGAWA
*, YASUO
University, Aobayama,
SUZUKI
and MITSURA
Sendai (Japan)
1976)
Summary The relationship between the chain elongation of palmitoyl-CoA and phospholipid content in rat liver microsomes was studied. When liver mierosomes were incubated with phospholip~e C, mi~rosom~ phospholipids were linearly hydrolyzed during 10 min of incubation under the present experimental conditions. Coincident with the decrease in microsomal phospholipid content by phospholipase C treatment, the chain elongation activity also decreased linearly. The decreased chain elongation activity in phospholipase C-treated microsomes was completely or partially recovered by the addition of a sonicated dispersion of phosphatidylcholine, microsomal phospholipids or phosphatidylcholine/phosphatidylethanolamine mixtures. The extent of recovery of decreased activity by a sonicated dispersion of phosphatidyIcholine/phosphatidyleth~olam~e mixture was gradually reduced by increasing amounts of phosphatidyleth~ol~ine in the dispersion. In addition, the chain elongation activity in native nicrosomes was more stimulated by the addition of a sonicated dispersion of phosphatidylcholine alone than by that of phosphatidylcholine/phosphatidylethanolam~e mixtures. The chain elongation activity of palmitoyl-CoA was inhibited by the addition of stearoyl-CoA which is the end-product of this reaction. The inhibitory effect of stearoyl-CoA was partially eliminated by the addition of a sonicated dispersion of phosphatidylcholine. The increase of the chain elongation activity in native and phospholipase C-treated microsomes by the addition of a sonicated dispersion of phosphatidylcholine was not related to the activity of fatty acyl-CoA hydrolase.
----
* Present ** Present Japan.
address: address:
Faculty National
of Pharmaceutical Sciences. Kumamoto University, Institute of Hygienic Sciences, Kamiyoga-1-chome,
Kumamoto. Setagaya,
Japan. Tokyo,
174
Introduction It is well known that the enzymes that catalyze the desaturation and chain elongation of palmitoyl-CoA are located in microsomal fraction of rat liver [ 11. We have previously reported that the chain elongation of palmitoyl-CoA in liver microsomes of normal and refed rats is stimulated by the addition of a sonicated dispersion of phosphatidylcholine, and that chain elongation activity in liver microsomes treated with phospholipase C and acetone decreases remarkably [2] . In addition, we have also reported that when palmitoyl-CoA is incubated with liver microsomes in the presence of malonyl-CoA and reduced pyridine nucleotides, palmitoyl-CoA is preferentially introduced into the chain elongation pathway, rather than into the desaturation pathway by the addition of a sonicated dispersion of phosphatidylcholine [ 31. The present paper deals with the relationship between the chain elongation activity of palmitoyl-CoA and phospholipid content in rat liver microsomes. Materials and Methods Materials
Palmityol-1-[ “C]CoA (58.2 Ci/mol) was obtained from New England Nuclear Corp. (Boston, Mass., U.S.A.). Palmitoyl-CoA was purchased from Nutritional Biochemical Co. (Cleaveland, Ohio, U.S.A.); reduced nicotinamide adenine dinucleotide (NADH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) from Kyowa Hakko Co. (Tokyo, Japan): malonyl-CoA, stearoylCoA, bovine serum albumin and phospholipase C (from Clostridium welchii) from Sigma Chemical Co. (St. Louis, MO., U.S.A.). Animals
Female albino rats of the Wistar strain, weighing 80-130 g, were used. The animals were starved for 48 h and then fed ad libitum on the commercial diet obtained from Oriental Yeast Co. (Tokyo, Japan) for 24 h. Preparation
of the enzyme
All procedures were carried out at O-4” C. Rat livers were homogenized in 3 ~01s. of cold 0.25 M sucrose solution. The homogenate was then fractionated by differential centrifugation, as described previously [2] . The microsomal pellet obtained was suspended in 0.1 M Tris - HCl or sodium phosphate buffer (pH 7.4) containing 0.25 M sucrose. The protein content determinations were performed by the procedure described by Lowry et al. [4] with crystalline bovine serum albumin as standard. Preparation
of phospholipids
Phosphatidylcholine and phosphatidylethanolamine were prepared from egg yolk and purified by silicic acid-column chromatography. The purified phosphatidylcholine and phosphatidylethanolamine gave a single spot on silica gel G plates with chloroform/methanol/water (65 : 25 : 4, v/v) as developing solvent. Microsomal phospholipids were isolated from microsomal total lipids extracted by chloroform/methanol (2 : 1, v/v) containing 0.1 mg of butylated hydroxy-
175
per 100 ml as antioxidant by using silicicacid column chromatog. raphy. We have confirmed by thin layer chromatography that the micmom~
toluene
phospholipid
fraction
did not contain
microsomal
neutral
lipids.
Preparation af phospholipid
dispersions The sonicated dispersion of phospholipids was prepared in the same manner as described previously except for the use of water instead of 0.025% ethylenediamine tetraacetic acid (EDTA) as solvent [ 51. Lipid phosphorous content in the dispersion was determined by the method of Rouser et al. [6].
Assay of chain elongation of palmitoyl-CoA The standard incubation mixture contained 45 nmol palmitoyl-CoA, 3.4 nmol palmitoyl-l-[14C] CoA, 200 nmol of malonyl-CoA, 3 pmol NADH, 3 pmol NADPH, 25 pmol sucrose, 1 E.cmol KCN and 1 mg microsomal protein. The final volume was adjusted to 1 ml with Tris - HCl buffer (90 pmol, pH 7.4). Incubation was carried out at 37°C for 6 min under Nz with constant shaking. After the incubation, the reaction was stopped by the addition of 1 ml of 10% KOH/methanolic solution and then heated at 70°C for 30 min. After acidification with 6 M HCl, the extraction of lipids and isolation of fatty acid was completed by the same procedure as described previously [7]. Fatty acids extracted from the incubation medium were methylated and then analyzed by means of radio-gas liquid chromatography as described previously [ 21. Assay of fatty acyl-CoA hyclrolase activity The fatty acyl-CoA hydrolase activity in liver microsomes was determined by a partially modified procedure of Jones et al. [8]. The incubation mixture contained the same components as those used for the chain elongation reaction except for the absence of NADH, NADPH and malonyl-CoA. After the reaction was stopped by the addition of 0.7 ml of 4 N H2S04, the incubation medium was extracted three times with 5 ml hexane. The hexane solutions were combined and evaporated to dryness at 45°C in vacua. The lipids obtained were dissolved in a constant volume of hexane. An aliquot of the hexane solution was used for the determination of the recovery of radioactivity. After another aliquot of the hexane solution was evaporated, the hexane extract was separated to each lipid fraction on silica gel G plates by using hexane/ether/acetic acid (70 : 30 : 1, v/v) as developing solvent. After developing, the area of silica gel G plate corresponding to palmitic acid used as standard was scraped out into a counting vial and the radioactivity measured by a Packard Tri-Carb liquid scintillation spectrometer. Each vial contained 15 ml toluene with 0.4% 2,5diphenyloxazole, 0.01% 1,4-bis-(5-phenyloxazolyl)-benzene and 3% thixotropic gel powder (Cab-o-sil). Phospholipase C treatment Phospholipase C from Cl. welchii (3.6 units/mg) was used without further purification. 30 mg of liver microsomal protein were incubated with phospholipase C, 30 pmol /3-mercaptoethanol, 21 prnol CaClz and 375 pmol sucrose in 6.9 ml of 0.1 M Tris - HCl buffer (pH 7.4) at 24” C. The reaction was stopped by the addition of EDTA. For the determination of the extent of the micro-
176
somal phospholipid hydrolysis, total lipids were extracted from the incubation medium as described previously [ 71. Lipid phosphorus content in the extracted lipids was determined by the method of Rouser et al. [6]. Results and Discussion We have previously reported that the chain elongation activity of palmitoylCoA in rat liver microsomes treated with phospholipase C decreases remarkably
[a* In this paper, we study in further detail the relationship between the chain elongation activity of palmitoyl-CoA and the decrease of phospholipid content in rat liver microsomes treated with phospholipase C. As shown in Fig. 1, when 1 mg liver microsome protein was incubated with phospholipase C (0.2 unit/mg) at 24°C microsomal phospholipids were hydrolyzed linearly during 10 min of incubation (curve l), under the present experimental conditions. Coincident with the decrease of microsomal phospholipid content caused by phospholipase C treatment, the chain elongation activity decreased linearly (curve 2). Thus an incubation time of 6 min was used for the degradation of microsomal phospholipids by phospholipase C treatment. On the other hand, as shown in Table I (experiment I), the effect of a sonicated dispersion of phosphatidylcholine, microsomal phospholipids or phosphatidylcholine/phosphatidylethanolamine mixtures on the chain elongation of
0 10
Incubation
20 time,
min
Fig. 1. Effect of phospholipase C treatment on the chain elongation of palmitoyl-CoA in liver microsomes. Liver microsomes were pretreated with phospholipase C under the following experimental conditions. 30 mg of liver microsomal protein were incubated with phospholipase C (6 units), 30 wmol pmercaptoethanol, 21 qnol C&12 and 375 /.unol wcrose in 6.9 ml of 0.1 M Tris . HCl buffer (pH 7.4) at 24’C. After each preincubation 0.23 ml of the above incubation mixture containing 1 mg of microsomal protein were added to the incubation medium containing 10 ~.rmol EDTA for the assay of the pahnitoylCoA chain elongation. The incubation conditions for the assay of the chain elongation reaction and the determination of phospholipid content were described in the text.
177
3
10
20
30
Palmitoyl-CoA,
40
50
PM 1
50
40
30
20
Stearoyl-CoA.
10
0
PM
Fig. 2. Effect of stearoyl-CoA on the chain elongation activity of palmitoyl-CoA in liver microsomes. Incubation conditions were described in the text, except for the addition of various amounts of palmitoylCoA as substrate and of stearoyl-CoA as inhibitor. The sum of the amounts of palmitoyl-CoA and stearoylPalmitoyl-CoA + stearoylCoA (curves 2 and 3) was constant. r-3, Pahnitoyl-CoA alone: A-----A, CoA + phosphatidylcholine dispersion (602 gg); a---A, Palmitoyl-CoA + stearoyl-CoA.
palmitoyl-CoA in native and phospholipase C-treated microsomes was examined. The decreased chain elongation activity of palmitoyl-CoA in rat liver microsomes treated with phospholipase C was completely or partially recovered by the addition of a sonicated dispersion of phosphatidylcholine (58.2 pg as lipid phosphorus) to the same degree as previously reported [2] , though not to the level obtained with phosphatidylcholine alone by the addition of microsomal phospholipid dispersions (58.2 pg as lipid phosphorus) or phosphatidylcholine/phosphatidylethanolamine dispersions (58.2 pg as lipid phosphorus). The reactivation of the decreased chain elongation activity by the phosphatidylcholine dispersion depended on the concentration of phosphatidylcholine added to the incubation medium. A plateau was reached at a concentration of approx. 1.2 mg phosphatidylcholine (47.2 pug lipid phosphorus per incubation medium. However, microsomal phospholipids and phosphatidylcholine/phosphatidylethanolamine mixtures added to the incubation medium as the dispersion contained respectively 41% and 64% phospholipids other than phosphatidylcholine. Therefore, the effect of increasing amounts (87.0 (ug as lipid phosphorus) of microsomal phospholipid dispersions or phosphatidylcholine/phosphatidylethanolamine dispersion on the chain elongation activity in liver microsomes treated with phospholipase C was also examined. The chain elongation activity could not be further elevated by the addition of increasing amounts of these dispersions. In addition, the stimulator-y effect of phosphatidylcholine dispersion on the chain elongation activity may be depressed by the presence of
178 TABLE
I
EFFECT TION The
OF
VARIOUS
ACTIVITY
OF
pretreatment
various
sonicated
phosphorus
(LP)
of
SONICATED
DISPERSIONS
PALMITOYL-C!oA liver
microsomes
dispersions for the
added
experiment
OF
IN LIVER by to
the
I and
phospholipase incubation 80.0
C
pg as lipid
min
was
medium
Chain
Additions
PHOSPHOLIPIDS
ON
THE
CHAIN
as in
Fig.
1. The
pg and
87.0
ELONGA-
MICROSOMES the
were
phosphorus
elongation
same 58.2 for
activity
pg
the experiment
(stearic
acid
amounts
in the
form
of of
II.
formed,
nmol/
per mg protein)
Native
microsomes
Phospholipase
C-treated
microsomes
Experiment
I
NOIX
1.56
0.59
1.93
1.81
Phosphatidylcholine 58.2
/~g as LP
87.0
pg as LP
Microsomal
1.86
phospholipids
58.2
pg as LP
87.0
ug as LP
1.19 1.05
Phosphatidylethanolamine
(64%)-
phosphatidykcholine
(36%)
mixtures
58.2
gg as LP
1.56
87.0
pg as LP
1.58
Experiment
II
NOW? Phosphatidylcholine
(80
jug LP)
2.44
1.28
3.94
2.48
Phosphatidylethanolaminephosphatidylcholine
(80
fig LP)
1:9
2.81
1.83
2:8
2.71
1.90
4:6
2.52
1.80
6:4
2.10
1.65
phospholipids other than phosphatidylcholine. In fact, as shown in Table I (experiment II), when a sonicated dispersion of various ratios of phosphatidylethanolamine to phosphatidylcholine were added to the incubation medium containing native and phospholipase C-treated microsomes, the elevation of the chain elongation activity was progressively decreased by increasing amounts of phosphatidylethanolamine in the dispersion. In general, fatty acid metabolism in rat liver microsomes is significantly affected by the co-existing long chain fatty acyl-CoA esters [9,10] . Whether the chain elongation of palmitoyl-CoA to stearoyl-CoA is also affected in the presence of stearoyl-CoA, which is the end-product of this reaction, was investigated. As shown in Fig. 2, the sum of the amounts of palmitoyl- and stearoylCoA was constant in this experiment. The chain elongation of palmitoyl-CoA in the presence of various amounts of stearoyl-CoA (curve 3) was depressed as compared to the results obtained by palmitoyl-CoA alone (curve 1). The inhibitory effect of stearoyl-CoA may not be due to its detergent action. In addition, the inhibition of the chain elongation activity of palmitoyl-CoA in the presence of stearoyl-CoA (curve 3) was partially eliminated by the addition of a soni-
179
TABLEII EFFECT ACTIVITY
OF
PHOSPHATIDYLCHOLINE
IN LIVER
DISPERSION
ON
FATTY
ACYL-CoA
HYDROLASE
MICROSOMES
The pretreatment of liver microsomes with phosphlipase C was the same as in Fig. 1. 1 mg of native and phospholipase C-treated microsomes was used for the assay of fatty acyl-CoA hydrolase activity. The other incubation conditions were described in the text. n.d.. not determined. Additions
Phospholipase C-treated microsomes None 1.2 mg Phosphatidylcholine 1.48 mg Phosphatidylcholine Native microsomes Native microsomes None 0.38 mg Phosphatidylcholine 0.55 mg Phosphatidylcholine
Palmitoyl-CoA
hydrolyzed
I
II
0.58 2.53 n.d. 3.22
0.15 n.d. 2.65 3.41
4.19 n.d. 4.36
3.76 3.44 n.d.
(nmol/min
per mg protein)
cated dispersion of phosphatidylcholine (curve 2). Both palmitoyl-CoA that is used as the substrate and stearoyl-CoA that is the end-product of the chain elongation reaction, can be introduced into various lipids. Therefore, the recovery effect of phosphatidylcholine dispersion on the chain elongation activity inhibited by stearoyl-CoA may be partially due to the esterification of stearoyl-CoA with some acceptors such as lysophosphatidylcholine, monoacylglycerol, diacylglycerol and cholesterol, and/or the penetration of the stearoylCoA produced from palmitoyl-CoA into phosphatidylcholine dispersion. However, we have found that a sonicated dispersion of phosphatidylcholine did not contain the lysophosphatidylcholine which by the degradation of phosphatidylcholine produces during the sonication, and that the incorporation of stearoylCoA produced from palmitoyl-CoA into neutral lipid fraction is little affected by the addition of phosphatidylcholine dispersion. It has been reported that fatty acyl-CoA esters are degraded to fatty acid and coenzyme-A by fatty acyl-CoA hydrolase in rat liver microsomes [ 111. Whether the increase of the chain elongation activity of palmitoyl-CoA in native and phospholipase C-treated microsomes by the addition of a sonicated dispersion of phosphatidylcholine is related to fatty acyl-CoA hydrolase activity was also investigated. As shown in Table II, palmitoyl-CoA hydrolase activity in phospholipase C-treated microsomes was reduced to approx. 20% of that in native microsomes. The decreased fatty acyl-CoA hydrolase activity was recovered to approx. 80% of the activity in native microsomes by the addition of phosphatidylcholine dispersion. However, ‘palmitoyl-CoA hydrolase activity in native microsomes was not stimulated by the addition of the dispersion. Therefore, the chain elongation activity of palmitoyl-CoA that was decreased by phospholipase C treatment, may not bedue to a decrease of the availability of palmitoylCoA as substrate by the increase of fatty acyl-CoA hydrolase activity. Experiments are further in progress to study the mechanism of chain elonga-
180
tion stimulation phatidylcholine
of palmitoyl-CoA dispersion.
in liver rnicrosomes
by the addition
of phos-
References 1 2 3 4 5 6 7 8 9 10
Seubert. W. and Podack. E.R. (1973) Mol. Cell, Biochem. 1. 29-40 Nakagawa, M., Kawashima, Y. and Uchiyama, M. (1976) Chem. Pharm. Bull. 24, 46-51 Nakagawa, M., Kawashima, Y. and Uchifama, M. (1976) Lipids, 11. 241-243 Lowry. O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, Nakagawa, M. and Nishida. T. (1973) Riochim. Biophys. Acta, 296. 577-585 Rouser, G., Siakotos. A.N. and Fleisher. S. (1966) Lipids 1, 85-86 Uchiyama. M., Nakagawa, M. and Okui. S. (1967) J. Biochem. 62, l-6 Jones, P.D.. Holloway, P.W., Peluffo, R.O. and Wakil. S.J. (1969) J. Biol. Chem. 244, Nugteren, U.H. (1965) Biochim. Biopbys. Acta. 106, 280--290 Brenner. R.R. and Peluffo. R.O. (1966) J. Biol. Chem. 241. 5213-5219
265-275
744-754