Journal
of Molecular
and Cellular
Cardiology
(1975)
7, 577-590
Some Properties and Subcellular Distribution of Palmitoyl-CoA Hydrolase and Phosphatidate Phosphohydrolase in Rabbit Hearts MAW-SHUNG Department
of Physiology,
Faculty
(Received
LIU of Medicine,
23 January
AND K. JOE University Canada
1974,
KAKO*t
of Ottawa,
and accepted 8 August
Ottawa,
Ontario,
KIN
6NN5,
1974)
M. S. Lru AND K. J. I(AKo. Some Properties and Subcellular Distribution of PalmitoylCoA Hydrolase and Phosphatidate Phosphohydrolase in Rabbit Hearts.Journal of Molecular andCellular Cardiology (1975), 7,577-590). This report deals with the basic characteristics of two enzymes which are related to myocardial glyceride biosynthesis and which have been studied relatively little. Appropriate assay conditions for palmitoyl-CoA hydrolase and phosphatidate phosphohydrolase were established with respect to the time courses of reactions, amounts of enzyme source and pH optima. (+)-Decanoylcarnitine (4 - 12mM) potentiated palmitoyl-CoA hydrolase activity of rabbit heart homogenates under our assay conditions, while 5mM of diisopropylfluorophosphate inhibited its activity by half. The highest activity of the above enzyme was found in the lysosomal and the microsomal fractions, followed by the mitochondrial and the soluble fraction. The Km of phosphatidate phosphohydrolase in the lysosomal-microsomal fraction was 0.33 mM. This enzyme also showed its highest specific activity in the lysosomal and the microsomal fractions. The activity of the enzyme in the soluble fraction was less than l/15 that of the microsomal enzyme when phosphatidate suspension was used as the substrate, whereas when membrane-bound phosphatidate was used as substrate, its measured activity was increased tenfold, suggesting the existence of a distinct soluble enzyme. KEY WOROS: Decanoylcarnitine; drial; Lysosomal; Soluble enzyme; Cysteine.
Diisopropylfluorophosphate; pH; Kinetics; Membrane-bound
Microsomal; Mitochonphosphatidate; DTT:
1. Introduction Studies on fatty acid (FA) and oxygen uptake by the heart in situ were initiated by Bing and his associates in the early 50s. They concluded that not all the FA taken up by the myocardium was completely oxidized, but some of it entered the tissue glyceride pool [4, 51. This conclusion was later confirmed by various investigators [I, 33, 351, who utilized more advanced methodology of lipid biochemistry. In fact, the acylation-deacylation process of some stored glycerolipids was found to be relatively rapid: thus, neutral lipids in the heart were readily mobilized in the isolated, perfused heart preparation [IZ, 341, and fasting of animals, alcohol administration or treatment with thyroid hormone induced the accumulation of * Reprint requests should be addressed to K. J.K. t Dedicated to Dr. Y. Kako on the occasion of his 77th birthday.
578
M.-S.
LIU
AND
K.
J.
KAKO
triglycerides in the heart [9, 22, 2.5, 3.51. Similarly, heart homogenates prepared from the alcohol- or thyroid-treated animals were found to esterify in vitro exogenous FAs to tissue glycerides to a greater extent than the control homogenates [9,23,25]. There are at least four enzymes directly involved in the esterification of FA into triglyceride [17, 311. Therefore, in order to elucidate the mechanism for the increased esterification under pathological conditions, it is essential to provide information concerning the kinetics of these enzymes and factors controlling the reaction rates. While there are numerous reports describing esterifying enzymes in the liver and intestine, [17, 311, those dealing with heart enzymes have been scarce. Accordingly, we have attempted to characterize these enzymes in the cardiac subcellular fractions. Our study dealing with acyl-CoA:sn-glycerol 3-phosphate acyltransferase (EC 2.3.1.15) has been reported elsewhere [24, 27, 281; this paper contains a study of palmitoyl-CoA hydrolase (EC 3.1.2.2.) and phosphatidate phosphohydrolase (EC 3.1.3.4).
2. Materials
and
Methods
Materials Palmitoyl-CoA, palmitic acid, bovine serum albumin, fraction V, ATP and CoA were purchased from Sigma Chemical Co. [1-i*C]palmitoyl-CoA and [l-t%] palmitic acid were obtained from New England Nuclear Corp. Their stated purity was more than 98.5%; they were stored at -20°C for not more than 3 months. The purity of [U-14C]-sn-glycerol-3-phosphate was checked by t.1.c. [27]. Diisopropylfluorophosphate was a product of K & K laboratories and phosphatidic acid, sodium salt, was obtained from Pierce Chemical Co. The material showed a single peak on the thin layer chromatographic plate, and its stated composition was 36% palmitic, 37% oleic, 15% stearic and 12% linoleic acids. (+)-Decanoylcarnitine was prepared according to the method of Brendel and Bressler [8], and crystallized twice by acidification from a hot aqueous solution. Its purity was checked by t.1.c. with a solvent system of chloroform:methanol:O. 1 M sodium acetate (4:4:1, v/v/v) [G]. All solvents were of the highest grade available and lipid solvents were redistilled before use.
Preparation
of subcellular fractions
Albino male rabbits were anesthetized by an intraperitoneal injection of 2.5 ml of 2% chloralose in 20% urethane per kg body weight. The hearts were excised, trimmed to remove fat, and were homogenized with 4 volumes of 0.25 M sucrose containing 0.02 M Tris-HCI, pH 7.4, with a Teflon pestle, as described previously
ACYL-COA
AND
PHOSPHATIDATE
HYDROLASE
579
[27]. The homogenized tissue mixture was centrifuged at 800 x g for 15 min to remove nuclei and cell debris, and the supernatant was used as a homogenate fraction. In the preparation of subcellular organelles, the homogenate was centrifuged at 10000 x g for 15 min. The resulting supernatant was then spun at 100000 x g for 60 min and the precipitate used as a microsomal fraction [15, 271. The precipitate from the 10000 x g centrifugation was resuspended and centrifuged at 8000 x g for 15 min, yielding a mitochondrial pellet [15, 271. In some experiments a “lysosomal” fraction was isolated by collecting a fraction precipitated between 10000 x g (15 min) and 25000 x g (15 min). The protein concentration of each fraction was determined by the method of Lowry et al. [29]. Assay of palrnitoyl-CoA
hydrolase
The enzyme preparation (homogenates or subcellular fractions) was incubated with 0.2 pmol [i%]palmitoyl-CoA with a radioactivity of approximately 35000 ct/min, 80 pmol Tris-phosphate buffer, pH 7.4, and 20 mg of bovine serum albumin in a final volume of 2.0 ml. In some experiments, the pH was adjusted by altering the amount of Tris-base and phosphoric acid. The incubation was carried out at 37°C in a metabolic shaker, either under Na gas or in the presence of 4 pmol (+)decanoylcarnitine in the reaction mixture, to prevent the possible oxidation of added substrate. The reaction was stopped by the addition of 10 ml of a mixture of isopropanol :heptane : 1~ HzS04 (20 :5 : 1, v/v/v) [ 14,161 after a period of incubation as stated in the individual experiment, followed by the addition of 6 ml heptane and 4 ml H20. The mixture was then transferred to a separator-y funnel, shaken vigorously, and was allowed to stand for 5 to 10 min. After the separation of two phases, the heptane phase was re-washed with heptane-saturated HzO, and the aqueous phase with HzO-saturated heptane [16]. An aliquot from the combined heptane phases was taken and dissolved in a naphthalene1.4-dioxane-based scintillating solution [7] for the determination of radioactivity. The combined heptane extracts recovered 93 to 96% of radioactivity when [i%]palmitic acid was tested alone. In contrast, a negligible amount of [1-14C]palmitoyl-CoA was extracted by the heptane, indicating a satisfactory isolation of the radioactive product from its precursor. A Nuclear-Chicago liquid scintillation counter, Mark I, was used for the determination of radioactivity. A counting efficiency of more than 70% was obtained as judged by the channels ratio method. Assay of phosphatidate phosphohydrolase This activity was determined by measuring the release of inorganic phosphate from phosphatidic acid (diacylglycerol 3-phosphate). The incubation mixture was prepared immediately prior to each experiment and contained 3.0 pmol sodium
580
M.-S.
LIU
AND
K. J. KAKO
phosphatidate, 160 pmol Tris-acetate buffer, pH 7.0, and an enzyme preparation in a final volume of 2.0 ml. The reaction was stopped by the addition of 2.0 ml 01 12% trichloroacetic acid. After removal of the precipitated protein by centrifugation, the inorganic phosphate in the reaction mixture was determined by the method of Berenblum and Chain [3]. The latter method was found to be more suitable than the methods of Fiske and Subbarow, and Lowry and Lopez, which tended to produce a higher blank value either at zero time incubation with substrate, or after incubation in the absence of enzyme preparation. Reproducibility of the Berenblum and Chain method was found to be excellent. The effect of pH on the enzyme activity was examined by using the buffer described above, except for pH 11, in which case the pH of the Tris-acetate was adjusted with 1~ NaOH. So-called particle-bound phosphatidic acid [,?I, 321 was prepared by incubating the cardiac or hepatic microsomal preparation in the presence of [U-l%]glycerol 3-phosphate for 5 min at 37°C [24, 271. The incubation mixture contained 50 mM Tris-phosphate buffer, pH 7.4, 1 mM potassium palmitate, 0.4 mM CoA, 6 mM ATP, 3 mM MgC12, 20 mg fatty acid-poor bovine serum albumin, 3 mM glycerol 3phosphate (approximately 1 @Ii) and microsomes (c. 2 mg protein) in a final volume of 2.0 ml. After incubation, the contents of ten tubes were pooled, layered on a 0.3 M sucrose solution and centrifuged at 100000 x g for 60 min. The microsomal pellet was resuspended and used as particle-bound phosphatidic acid. As reported earlier [27], 93 to 98% of the acylation products were monoacyland diacylglycerol 3-phosphate under these experimental conditions. The activity of phosphatidate phosphohydrolase was measured as above by incubating this form of phosphatidic acid (approximately 30 nmol in 5 mg microsomal protein) in the presence and in the absence of 1.5 ml (17.1 mg protein) of supernatant fraction in a final volume of 3.0 ml. Water-saturated butanol was added to the blanks (time zero samples) and assay tubes, and lipids were extracted by the procedure previously published [27]. The products were analyzed by t.l.c., with the use of the solvent systems, petroleum ether :ether :acetic acid (40 : 10 : 1) and chloroform :methanol : acetic acid :water (65 :25 :8 :4), and their radioactivity determined [27]. From these data, the effect of the supernatant fraction on the conversion of phosphatidate to glycerides was assessed.
3. Results Palmitoyl-CoA
hydrolase
The rate of hydrolysis of palmitoyl-CoA by rabbit heart homogenates was constant up to 120 min under the experimental conditions selected, as is shown in Figure 1 (a). The optimal pH for palmitoyl-CoA hydrolase in heart homogenates was approximately 8.5 [Figure 1 (b)].
ACYL-COA
ov 0
AND
I
I
30
60 Time
PHOSPHATIDATE
90
'
120
0' 5.5
I 6.5
(mud
FIGURE 1 (a). The time course of hydrolysis ‘The incubation mixture, in a final volume of
581
HYDROLASE
I 7.5
8.5
' 0-J 9.5
10.5
PH
of palmitoyl-CoA
by rabbit
heart
homogenates.
2.0 ml, contained 0.2 pmol [I-i%]palmitoyl-CoA (226000 d/min/pmol), 80 pmol Tris-phosphate buffer, pH 7.4, 20 mg bovine serum albumin, 4 pmol (+)-decanoylcarnitine and 0.4 mg of freshly prepared homogenates. The temperature of incubation was 37°C and gas phase, room air. (b) The effect of different pHs on the activity of palmitoyl-CoA hydrolase. The assay conditions are described in the legend to Figure l(a), except that the pH of the assay medium was varied as shown and (+)-decanoylcarnitine was omitted. The amount of heart homogenates was 0.3 mg, incubation time, 60 min, temperature, 37°C and gas
phase, nitrogen. Each value was corrected by the blank value, which was the value obtained in the absence of homogenates after the same period of incubation at the corresponding pH. The rate of hydrolysis is shown on the ordinate.
(+)-Decanoylcarnitine was reported to be a competitive inhibitor for carnitine palmitoyltransferase [38]. The effects of various concentrations of this compound are illustrated in Figure 2(a). The activity of palmitoyl-CoA hydrolase was increased as the concentration of decanoylcarnitine increased up to 7.5 mM and stayed constant as the concentration was further increased to 12 mM. It is unlikely that this activation is secondary to the inhibition of palmitoyl-CoA oxidation by decanoylcarnitine, since the activities measured in air and in a nitrogen atmosphere, both in the absence (0.45 vs. 0.51 nmol/mg.min) and in the presence of decanoylcarnitine (3.10 vs. 2.76 nmol/mg.min), were almost identical. On the other hand, the activity of palmitoyl-CoA hydrolase was inhibited by diisopropylflurophosphate, but only at rather high concentrations of the inhibitor, as compared to the liver enzyme [.?6] ; half maximal inhibition occurred at 5 mM [Figure 2(b)]. The rate of hydrolysis of palmitoyl-CoA was proportional to the amount of enzyme preparation up to 1.2 and 3.0 mg protein for microsomal and soluble fraction, respectively. [Figure 3 (a)]. The activity was similar when Na or room air was used as the atmosphere during incubation. The distribution pattern of the hydrolase in subcellular fractions of rabbit heart is shown in Figure 3(b). The mitochondrial, lysosomal, microsomal and soluble fractions all possess activity but the highest specific activity was noted in lysosomal and microsomal fractions. Since a large proportion of the total protein distributes
582
M.-S.
LIU
AND
K. J. KAKO 0.4
(b) \ A
-AlA
*
0.3
/ 0 A 0 A /
9“ 0
2
(+I
4
6
8
- Decanoylcarnitine
IO
12
0
0
(mM)
5
IO DFP
30
(rnt.4)
FIGURE 2(a). The effect of various concentrations of( +)-decanoylcarnitine on the activity of palmitoyl-CoA hydrolase in heart homogenates. The assay conditions are described in the legend to Figure l(a), except that the amount of (+)-decanoylcarnitine was varied as indicated (mM). The protein contents of homogenates were 0.39 and 0.41 mg protein in these experiments (a = 2). Incubation time was 60 min, the temperature, 37°C and gas phase, room air. The ordinate indicates the rate of hydrolysis. (b) The effect of preincubation of heart homogenates with diisopropylfluorophosphate (DFP) upon the activity of palmitoyl-CoA hydrolase. The homogenate containing approximately 4.9 mg protein in 2.0 ml was pre-incubated in the presence of various concentrations (mn) of DFP for 60 min at 0°C in this experiment (a = 2). Palmitoyl-CoA hydrolase activity was assayed subsequently by taking 0.1 ml aliquots from the pre-incubation mixture under the conditions similar to those described in the legend to Figure 1 (a). The incubation was performed under Ns for 60 min.
in the soluble and mitochondrial fractions, the total activities of these fractions are considerable. Dithiothreitol (5-30 mM) and cysteine (25 and 50 mM) accelerated spontaneous decomposition of palmitoyl-Cob, particularly at high pH values (> 7) [Figure 4(a)]. These sulfhydryl reagents are frequently used for the synthetic reactions involving acyl-CoA, and therefore, the present finding points to a possible error which may arise in determining an accurate concentration of acyl-CoA in the reaction mixture.
Phosphatidate phosphohydrolase The relationship between the rate of hydrolysis of phosphatidic acid and the amount of heart homogenate (O-2.55 mg protein) was linear under the experimental conditions selected. Furthermore, the reaction rate was constant for nearly 3 h with our assay system [Figure 4(b)]. The pH optimum of the heart phosphatidate
ACYL-COA
AND
PHOSPHATIDATE
(b)
016-
Ccl)
.
0 IF?/ 2 L 0.00 z E c
-
0.04
-
.
0
/
/
/
,
/
/
/
,
/
! /
I
I
Mt
I /
MC
Ly
’
/
583
HYDROLASE
1’
Y
/a/’ .,’ o/l
0
I 1.0 Protein
I 2.0
30
% of torol
protein
(mg)
FIGURE 3(a). The relationship between different amounts of microsomal or soluble fractions and rates of hydrolysis of palmitoyl-CoA. For the assay conditions see the legend to Figure 1 (a), except that a nitrogen atmosphere was introduced during a 40 min incubation and (+)-decanoylcarnitine was omitted. The ordinate indicates the rate of hydrolysis of palmitoyl-CoA, while the abscissa with a 100000 x g preindicates the amounts of subcellular preparations. (a) results obtained cipitate containing microsomes and lysosomes; (0) results with the soluble fraction. (b) The distribution of palmitoyl-CoA hydrolase in rabbit heart subcellular fractions. The ordinate indicates the activity in nmol palmitoyl-CoA hydrolyxed/mg protein/min and the abscissa shows yo distribution of protein in subcellular fractions. The symbols are: mitochondrial fraction = Mt, lysosomal fraction = Ly, microsomal fraction = MC, and soluble fraction = s. n = 6.
phosphohydroiase was 7.0 [Figure 5(a)]. The Michaelis-Menten constant, Km, of the enzyme in the microsomal-lysosomal fraction was 0.33 mM [Figure 5(b)], a value similar to that previously obtained with other organs [IO, 18, 361. The rate of hydrolysis of phosphatidic acid was proportional to the amount of subcellular fractions up to 1.0 and 1.3 mg protein per flask for microsomal and mitochondrial fractions, respectively, whereas the linear relationship with soluble (cytosomal) fraction was sustained up to 3.0 mg protein [Figure 6(a)]. The distribution of phosphatidate phosphohydrolase in cardiac subcellular fractions is shown in Figure 6(b). The highest specific activity of this enzyme was found in the microsomal and lysosomal fractions. The activity found in the cytosomal fraction was extremely low; thus it is difficult to state, from this analysis, whether or not the observed activity is due to microsomal contamination. The above question was further investigated by using a membrane-bound substrate, which was shown to be preferentially dephosphorylated by the enzyme
584
(a)
M.-S.
LIU
AND
K. J.
KAKO
200 ITT
1.2 -
160 c
0
/
/
.E 5 0.8E c 0.4 -
ODTT Cysteine
nil -
5 -
I 15 -
30 -
25
50
(mMj (mtd)
0
60
120 Time (min)
180
FIGURE 4(a). The effect of an addition of dithiothreitol (DTT) and cysteine on the spontaneous decomposition of palmitoyl-CoA. The reaction mixture contained 0.15 pmol [ l-14C]palmitoyl-CoA, 80 ymol Tris-phosphate buffer, pH 7.4,20 mg bovine serum albumin and different concentrations of DTT or cysteine in a final volume of 2.0 ml. The incubation was carried out for 30 min at 37°C in a nitrogen atmosphere. The ordinate indicates the amount of palmitoyl-CoA decomposed in nmoljmin. (0) the value obtained in the absence of DTT and cysteine; (%) the value obtained in the presence of DTT; ( ) the value obtained in the presence of cysteine. (b) The time course of the hydrolysis of phosphatidic acid by rabbit heart homogenates. The assay mixture contained, in a final volume of 2.0 ml, 3.0 pmol of phosphatidate, 160 pmol Tris-acetate buffer, pH 7.0 and 2.55 or 3.10 mg (protein) of homogenates (n = 2). Incubation was carried out in air at 37°C. The ordinate indicates the activity of phosphatidate phosphohydrolase in nmol inorganic phosphate released/mg protein, whereas the abscissa indicated the incubation time in min.
6
7
8 PH
9
5 Phosphotidote
(mM)
FIGURE 5(a). The pH-activity relationship of phosphatidate phosphohydrolase of rabbit heart homogenates. The assay conditions are identical to those described for Figure 4(b). The heart homogenate in this experiment was dialyzed against a medium consisting of 0.25 M sucrose, 0.02 M Tris-HCl and 0.001 M EDTA for 16 h at 4°C. The amount of homogenates was 1.9 mg and the incubation lasted for 45 min. Each value was corrected for a small blank value obtained at the same pH in the absence of phosphatidate. (b) The effect of various concentrations of phosphatidate on the activity of phosphatidate phosphohydrolase. The assay conditions are described in the legend to Figure 4(b). The incubation was continued for 60 min, and the lysosomal-microsomal fraction ( 1.56 mg protein) was used as the enzyme source in this experiment.
ACYL-COA
AND
PHOSPHATIDATE
583
HYDROLASE
(b) 3.0
2.5
6
LY -
-MC
.g 2.0 E” > E c
.-E4 \ zc
1.5
1.0
2 0.5
0 1.0 Protein
2.0 (mg)
Ml G
0
20
40
% of total
,
s,
60
80
100
protein
FIGURE 6(a). The relationship between amounts of different subcell&r fractions and phobphatidate phosphohydrolase activities. The assay mixture contained, in a final volume of 2.0 ml. 3.0 pmol of phosphatidate, 160 pmol of Tris-acetate buffer, pH 7.0, and various amounts of subcellular fractions. The assay was carried out for 45 min at 37°C in air. The activity is expressed as nmol of inorganic phosphate released per min. (0) activities of the lysosomal-microsomal preparations; (0) activities of the mitochondrial preparations; (0) activities of the soluble (cytosomalj fractions. (b) Subcellular distribution of phosphatidate phosphohydrolase in rabbit heart homogenates. See the legend to Figure 3(b) for the symbols used. n = 7.
existing in the soluble fraction of the liver or intestine [17, 21, 321. The microsomal fraction synthesized a very small amount of neutral lipid after the first incubation, namely, 6.5 -j= 0.7% of the total lipids, the remainder being mono- and diacylglycerol 3-phosphate, as reported in the previous paper [27]. A second, prolonged incubation of the microsomal fraction resulted in further glyceride formation. The rate of synthesis was 1.70 5 0.19 nmol/mg microsomal protein per min (n = 81, which is approximately half of the rate of the phosphatidate phosphohydrolase reaction assayed by using phosphatidate suspension as the substrate [Figure 6(b)]. The addition of the supernatant fraction to the second incubation mixture containing microsome-bound phosphatidate resulted in a considerable increase in neutral glyceride formation; the glyceride amounted to 47% of the total lipids in a 30-min incubation period, the rate of the reaction being 2.36 j, 0.28 nmol/mg soluble protein per min (n = 8). This reaction rate was ten-fold greater than the value recorded from the assay utilizing non-membrane-bound phosphatidate.
586
M.-S.
LIU
AND
K. J. KAKO
4. Discussion With the assay method adopted for palmitoyl-CoA hydrolase activity, hydrolysis was constant up to 2h of incubation, and the rate of reaction was proportional to the amount of enzyme. The pH-activity relationship of this enzyme determined with heart homogenates resembled that obtained with dog lung microsomes [13] and rat serum [19], but it differed from that found in adipose tissue homogenates [II]. Palmitoyl-CoA hydrolase can be rate-limiting for FA esterification in adipose tissue of fasted rats [II], but its activity in cardiac subcellular fractions was relatively insignificant (Results) and changed little in the hyperthyroid state (unpublished observation), so that it plays probably a relatively minor role in controlling the intracellular concentration of acyl-CoA. An apparent activation of palmitoyl-CoA hydrolase by a relatively high concentration of (+)-decanoylcarnitine has not been previously reported. It is unknown whether this enzyme is potentiated by the detergent property of decanoylcarnitine, or whether the latter compound inhibits palmitoyl-Cob oxidation, thus providing a relatively greater amount of substrate for the enzyme. However, a high concentration of palmitoyl-CoA used in our assay system (100 FM) and comparison of data obtained in the presence and absence of oxygen make the latter possibility less likely. It was reported earlier that the enzyme is active with the substrate in a micelle form [2], and thus it is possible that under the conditions of these experiments, the physico-chemical state of the substrate might have changed, causing the potentiation. An inhibition of enzyme activity by the detergent property of the palmitoyl-CoA is improbable in view of a study with hepatic microsomes, in which CoA esters of saturated FAs did not inhibit the hydrolase activity [ZO]. The study of cardiac subcellular fractions revealed uneven distribution of this enzyme activity; the enzyme activity of the mitochondria showed approximately 64% of that of the microsomal activity, suggesting that the observed activity cannot be ascribed to microsomal contamination but must be due to an independent mitochondrial enzyme. A relatively high specific activity in the lysosomal fraction has not been previously described, but the microsomal fraction has been known to possess a high hydrolase activity [13, 20, 261. This pattern of distribution of the enzyme [Figure 3(b)] implies that some acyl-CoA may be hydrolyzed and thus prevented from being utilized in oxidative energy production. On the other hand, a high microsomal hydrolase activity could be beneficial in limiting the availability of acyl-CoA for esterification, although this enzyme has a preference towards micelle form substrates, as mentioned above [2]. Our determinations of phosphatidate phosphohydrolase in heart muscle showed a pH optimum and subcellular distribution pattern similar to those provided by previous workers using organs other than the heart [IO, 18, 36, 371. A relatively high specific activity found in the mitochondrial fraction (approximately l/3 of the
ACYL-COA
AND
PHOSPHATIDATE
HYDROLASE
587
a possibility that the mitomicrosomal activity) [Figure 6(b)] makes unlikely chondrial activity is a result of microsomal contamination to the mitochondrial preparation. High specific activities of both phosphatidate phosphohydrolase and palmitoylCoA hydrolase in the lysosomal fraction suggest their role in degenerative heart disease or in myocardial fat infiltration. However, the activities in the hearts of hamsters suffering from hereditary cardiomyopathy were not increased (unpublished observation), and the functional significance of these lysosomal enzymes in the heart has not yet been elucidated. A report from another laboratory indicated that the phosphohydrolase showed some FA specificity, oleoyl-palmitoyl-glycerol S-phosphate being the most favourable substrate for rat liver enzyme preparations [32]. However, our observations with the crude enzyme preparation from the rat liver lysosomal fraction [37] in the presence of various synthesized monoacylglycerol3-phosphates suggest that the FA preference by this enzyme is not distinct [ZS]. Glyceride biosynthesis by subcellular fractions of rat liver or the small intestinal mucosa was reported to be greatly stimulated by the addition of the particle-free supernatant fraction [17, 21, 321. The effect by the latter was mostly attributed to phosphatidate phosphohydrolase existing in the soluble fraction [17]. These investigators demonstrated that the subcellular distribution of the enzyme activity varied depending upon the nature of the substrate; thus when aqueous dispersion of phosphatidic acid was used as the substrate, 90% of the total activity was in the particulate fractions, whereas, when membrane-bound phosphatidate was used as the substrate [17, 321, the particulate fractions possessed low activities and the soluble fraction was most active. It was also suggested that the enzyme in the soluble fraction is rate-governing in the biosynthesis of neutral glycerides [30]. This postulate was based on the finding that the activity of phosphohydrolase increased simultaneously with the increased triglyceride content in the liver following subtotal hepatectomy [30]. Th e p recise mechanism may be more complicated, however, since the same investigators observed, in addition, that treatment with actinomycin D suppressed the increased enzyme activity, whereas hepatic triglyceride accumulation was uninfluenced [3U]. Moreover, recent evidence is at variance with the older literature, in that the difference that was observed by using the membrane-bound and non-membrane-bound substrate can be attributed to the effect of Mgs+, at least, in adipose tissue [18]. Our results with cardiac subcellular fractions differ from the results of the above studies, in that, although the cardiac soluble fraction is more active than the microsomal fraction in hydrolyzing membrane-bound phosphatidate, this activity is lower than the microsomal enzyme activity assayed with the use of unbound phosphatidic acid. Our study thus demonstrated that the highest activity in heart homogenates is found in the lysosomal and microsomal fractions [see also, 32, 36, 371, and that the properties of the soluble enzyme are different from those of
M.-S. LIU AND K. J. KAKO
588
particle-bound enzymes. Furthermore, we have previously reported that acyl-CoA: glycerol 3-phosphate acyltransferase and acyl-CoA:monoacylglycerol 3-phosphate acyltransferase are under hormonal control [24]. Similarly, particulate enzyme activities were found to be increased under the influence of triiodothyronine treatment, but there was no change in the soluble phosphohydrolase (unpublished observation). Consequently, evidence obtained so far supports a view that each subcellular fraction contains individual, different phosphohydrolases.
Acknowledgement
This work was supported by grants from the Medical Research Council and Ontario Heart Foundation. M.S.L. is a recipient of Scholarship by the Alcoholism and Drug Addiction Research Foundation of Ontario, and K. J.K. is an MRC Research Associate. The valuable assistance of Miss Dale Patterson is gratefully acknowledged.
REFERENCES BALLARD, F.B.,DANFORTH, W. H., NAEGLE, S. & BING, R.J. Myocardialmetabolism of fatty acids. 3ourruzl of Clinical Investigation 39, 7 17-723 (1960). BARDEN, R. E. & CLELAND, W. W. L-Acylglycerol 3-phosphate acyltransferase rat liver. Journal of Biological Chemistry 244, 3677-3684 (1969). BERENBLUM, I. & CHAIN, E. An improved method for the calorimetric determination
of BiochemicalJournal 32, 295-298 (1938). BING, R. J., SIEGEL, A., UNGAR, I. & GILBERT, M. Metabolism of the human heart. II. Studies of fat, ketone and amino acid metabolism. Atnerican Journal of Medicine 16, 504-515 (1954). BING, R. J. The metabolism of the heart. In The Harvey Lectures, Series L, pp. 27-70, New York : Academic Press ( 1956). BBHMER, T., NORUM, K. R. & BREMER, J. The relative amounts of long-chain acylcarnitine, acetylcarnitine, and free carnitine in organs of rats in different nutritional states and with alloxan diabetes. Biochimica et biophysics acta 125, 24425 1 (1966). BRAY, G. A. A simple efficient liquid scintillator for counting aqueous solution in a liquid scintillation counter. Analytical Biochemistry 1, 279-285 (1960). BRENDEL, K. & BRESSLER, R. The resolution of (&)-carnitine and the synthesis of acylcarnitines. Biochimica et biofihysica acta 137, 98-106 (1967). BRESSLER, R. & W~I-~ELS, B. The effect of thyroxine on lipid and carbohydrate metabolism in the heart. Journal of Clinical Investigation 45, 1326-1333 (1966). COLEMAN, R. & H~~BSCHER, G. Metabolism of phospholipids. V. Studies of phosphatidic acid phosphatase. Biochimica et biofihysica acta 56, 479-490 (1962). DANIEL, A. M. & RUBINSTEIN, D. Fatty acid esterifying enzymes in rat adipose tissue homegenates. Canadian Journal of Biochemistry 46, 1039-1045 (1968). DENTON, R. M. & RANDLE, P. J. Hormonal control of lipid concentration in rat heart and gastrocnemius. Nature 208, 488 (1965). phosphate.
5. 6.
7. 8. 9. 10. 11. 12.
from
ACYL-COA 13. 1-l. 15.
16.
17. 18.
19.
20. 21.
22. 23.
24.
25. 26.
27.
28.
29. 30. 31. 32.
AND
PHOSPHATIDATE
HYDROLASE
589
FROSOLONO, M. F., SLIVKA, S. & CHARMS, B. L. Acyl transferase activities in dog lung microsomes. 3ournal of Lipid Research 12, 96-102 (1971). Goss, J. E. & LEIN, A. Microtitration offree fatty acids in plasma. Clinicul Chemistry 13, 36-39 (1967). HIJANG, C. C. & KAKO, K. J. Lipid composition of the subcellular elements and mitochondrial respiration of the heart and liver in hypercholesterolemic rabbits. Canadian 3oumal of Physiology and Pharmacology 46, 823-828 ( 1968). HUANG, C. C. & KAKO, K. J. Mechanism of triglyceridemia in hypercholesterolemic rabbits. Circulation Research 26, 771-782 (1970). H~~BSCHER, G. Glyceride metabolism. In Lipid Metabolism, S. J. Wakil, Ed. pp. 280-370. New York: Academic Press (1970). JAMDAR, S. C. & FALLON, H. J. Glycerolipid biosynthesis in rat adipose tissue. II. Properties and distribution of phosphatidate phosphatase. Journal of Lipid Research 14, 517-524 (1973). JANSEN, H. & H~~LSMANN, W. C. Long-chain acyl-CoA hydrolase activity in serum: Identity with clearing factor lipase. Biochimica et biophysics acta 296, 241-248 (1973). JEZYK, P. F. & HUGHES, H. N. Studies on the hydrolysis and utilization of long-chain acyl CoA thioesters by liver microsomes. Lipids 6, 107-I 12 (1971). JOHNSTON, J. M., RAO, G. A., LOWE, P. A. & SCHWARZ, B. E. The nature of the stimulatory role of the supernatant fraction on triglyceride synthesis by the a-glycerophosphate pathway. Ligids 2, 14-20 (1967). KAKO, K. & DUBUC, M. J. G. Changes in esterified fatty acids in the isolated perfused rabbit heart. Canadian 3ournal of Biochemistry 46, 1241-1246 (1968). KAKO, K. J., LIU, M. S. & THORNTON, M. J. Changes in fatty acid composition of myocardial triglyceride following a single administration of ethanol to rabbits. Journal of Molecular and Cellular Cardiology 5, 473489 (1973). KAKO, K. J. & LIU, M. S. Acylation of glycerol-3-phosphate by rabbit heart mitochondria and microsomes: Triiodothyronine-induced increase in its activity. FEBS Letters 39, 243-246 (1974). KIKUCHI, T. & KAKO, K. J. Metabolic effects of ethanol on the rabbit heart. Circulation Research 26, 625-634 (1970). LANDS, W. E. M. & HART, P. Metabolism of glycerolipids. VI. Specificities of acyl coenzyme A: phospholipid acyltransferases. 3oumal of Biological Chemistry 240, 19051911 (1965). Lru, M. S. & KAKO, K. J. Characteristics of mitochondrial and microsomal monoacyland diacylglycerol 3-phosphate biosynthesis in rabbit heart. Biochemical Journal 138, 11-21 (1974). LIU, M. S., BROOKS, P. J. & KAKO, K. J. Biosynthesis of monoacyl-sn-glycerol-3phosphate by rabbit heart mitochondria: Positional specificity differing from liver enzyme. Lipids 9, 391-396 (1974). LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, R. J. Protein measurement with the Folin phenol reagent. 3ournal of Biological Chemistry 193, 265-275 (195 1). MANGIAPANE, E. H., LLOYD-DAVIES, K. A. & BRINDLEY, D. N. A study of some enzymes of glycerolipid biosynthesis in rat liver after subtotal hepactomy. Biochemical 3oumal134, 103-l 12 (1973). MCMUIUZAY, W. C. & MAGEE, W. L. Phospholipid metabolism. Annual Review of Biochemistry 41, 129-160 (1972). MITCHELL, M. P., BRINDLEY, D. N. & H~~SCHER, G. Properties of phosphatidate phosphohydrolase. European 3oumd of Biochemistry 18, 2 14-220 (197 1).
590 33. 34. 35. 36. 37.
38.
M.-S.
LIU AND
K.J.
KAKO
MOST, A. S., BRACHFELD,N.,GORLIN,R. & WAHREN, J.Freefatty acid metabolism of the human heart at rest. Journal of Clinical Investigation 48, 1177-l 188 (1969). OLSON, R. E. & HOESCHEN, R. J. Utilization of endogenous lipid by the isolated perfused rat heart. BiochemicalJournal 103, 796801 (1967). OPJE, L. H. Metabolism of the heart in health and disease. American Heart Journal 76, 685-698 (1968), 77, 100-122, 383410 (1969). SEDGWICK, B. & H~~BSCHER, G. Metabolism of phospholipids. IX. Phosphatidate phosphohydrolase in rat liver. Biochimica et biophysics acta 106, 63-77 (1965). WILGRAM, G. F. & KENNEDY, E. P. Intracellular distribution of some enzymes catalyzing reactions in the biosynthesis of complex lipids. Journal of Biological Chemistry 238 2615-2619 (1963). WILLIAMSON, J. R., BROWNING, E. T., SCHOLZ, R., KREISBERG, R. A. & FRITZ, I. B. Inhibition of fatty acid stimulation of gluconeogenesis by (+)-decanoylcamitine in perfused rat liver. Diabetes 17, 194-208 (1968).
of Molecular
and Cellular
Cardiology
(1975)
7, 577-590
Some Properties and Subcellular Distribution of Palmitoyl-CoA Hydrolase and Phosphatidate Phosphohydrolase in Rabbit Hearts MAW-SHUNG Department
of Physiology,
Faculty
(Received
LIU of Medicine,
23 January
AND K. JOE University Canada
1974,
KAKO*t
of Ottawa,
and accepted 8 August
Ottawa,
Ontario,
KIN
6NN5,
1974)
M. S. Lru AND K. J. I(AKo. Some Properties and Subcellular Distribution of PalmitoylCoA Hydrolase and Phosphatidate Phosphohydrolase in Rabbit Hearts.Journal of Molecular andCellular Cardiology (1975), 7,577-590). This report deals with the basic characteristics of two enzymes which are related to myocardial glyceride biosynthesis and which have been studied relatively little. Appropriate assay conditions for palmitoyl-CoA hydrolase and phosphatidate phosphohydrolase were established with respect to the time courses of reactions, amounts of enzyme source and pH optima. (+)-Decanoylcarnitine (4 - 12mM) potentiated palmitoyl-CoA hydrolase activity of rabbit heart homogenates under our assay conditions, while 5mM of diisopropylfluorophosphate inhibited its activity by half. The highest activity of the above enzyme was found in the lysosomal and the microsomal fractions, followed by the mitochondrial and the soluble fraction. The Km of phosphatidate phosphohydrolase in the lysosomal-microsomal fraction was 0.33 mM. This enzyme also showed its highest specific activity in the lysosomal and the microsomal fractions. The activity of the enzyme in the soluble fraction was less than l/15 that of the microsomal enzyme when phosphatidate suspension was used as the substrate, whereas when membrane-bound phosphatidate was used as substrate, its measured activity was increased tenfold, suggesting the existence of a distinct soluble enzyme. KEY WOROS: Decanoylcarnitine; drial; Lysosomal; Soluble enzyme; Cysteine.
Diisopropylfluorophosphate; pH; Kinetics; Membrane-bound
Microsomal; Mitochonphosphatidate; DTT:
1. Introduction Studies on fatty acid (FA) and oxygen uptake by the heart in situ were initiated by Bing and his associates in the early 50s. They concluded that not all the FA taken up by the myocardium was completely oxidized, but some of it entered the tissue glyceride pool [4, 51. This conclusion was later confirmed by various investigators [I, 33, 351, who utilized more advanced methodology of lipid biochemistry. In fact, the acylation-deacylation process of some stored glycerolipids was found to be relatively rapid: thus, neutral lipids in the heart were readily mobilized in the isolated, perfused heart preparation [IZ, 341, and fasting of animals, alcohol administration or treatment with thyroid hormone induced the accumulation of * Reprint requests should be addressed to K. J.K. t Dedicated to Dr. Y. Kako on the occasion of his 77th birthday.
578
M.-S.
LIU
AND
K.
J.
KAKO
triglycerides in the heart [9, 22, 2.5, 3.51. Similarly, heart homogenates prepared from the alcohol- or thyroid-treated animals were found to esterify in vitro exogenous FAs to tissue glycerides to a greater extent than the control homogenates [9,23,25]. There are at least four enzymes directly involved in the esterification of FA into triglyceride [17, 311. Therefore, in order to elucidate the mechanism for the increased esterification under pathological conditions, it is essential to provide information concerning the kinetics of these enzymes and factors controlling the reaction rates. While there are numerous reports describing esterifying enzymes in the liver and intestine, [17, 311, those dealing with heart enzymes have been scarce. Accordingly, we have attempted to characterize these enzymes in the cardiac subcellular fractions. Our study dealing with acyl-CoA:sn-glycerol 3-phosphate acyltransferase (EC 2.3.1.15) has been reported elsewhere [24, 27, 281; this paper contains a study of palmitoyl-CoA hydrolase (EC 3.1.2.2.) and phosphatidate phosphohydrolase (EC 3.1.3.4).
2. Materials
and
Methods
Materials Palmitoyl-CoA, palmitic acid, bovine serum albumin, fraction V, ATP and CoA were purchased from Sigma Chemical Co. [1-i*C]palmitoyl-CoA and [l-t%] palmitic acid were obtained from New England Nuclear Corp. Their stated purity was more than 98.5%; they were stored at -20°C for not more than 3 months. The purity of [U-14C]-sn-glycerol-3-phosphate was checked by t.1.c. [27]. Diisopropylfluorophosphate was a product of K & K laboratories and phosphatidic acid, sodium salt, was obtained from Pierce Chemical Co. The material showed a single peak on the thin layer chromatographic plate, and its stated composition was 36% palmitic, 37% oleic, 15% stearic and 12% linoleic acids. (+)-Decanoylcarnitine was prepared according to the method of Brendel and Bressler [8], and crystallized twice by acidification from a hot aqueous solution. Its purity was checked by t.1.c. with a solvent system of chloroform:methanol:O. 1 M sodium acetate (4:4:1, v/v/v) [G]. All solvents were of the highest grade available and lipid solvents were redistilled before use.
Preparation
of subcellular fractions
Albino male rabbits were anesthetized by an intraperitoneal injection of 2.5 ml of 2% chloralose in 20% urethane per kg body weight. The hearts were excised, trimmed to remove fat, and were homogenized with 4 volumes of 0.25 M sucrose containing 0.02 M Tris-HCI, pH 7.4, with a Teflon pestle, as described previously
ACYL-COA
AND
PHOSPHATIDATE
HYDROLASE
579
[27]. The homogenized tissue mixture was centrifuged at 800 x g for 15 min to remove nuclei and cell debris, and the supernatant was used as a homogenate fraction. In the preparation of subcellular organelles, the homogenate was centrifuged at 10000 x g for 15 min. The resulting supernatant was then spun at 100000 x g for 60 min and the precipitate used as a microsomal fraction [15, 271. The precipitate from the 10000 x g centrifugation was resuspended and centrifuged at 8000 x g for 15 min, yielding a mitochondrial pellet [15, 271. In some experiments a “lysosomal” fraction was isolated by collecting a fraction precipitated between 10000 x g (15 min) and 25000 x g (15 min). The protein concentration of each fraction was determined by the method of Lowry et al. [29]. Assay of palrnitoyl-CoA
hydrolase
The enzyme preparation (homogenates or subcellular fractions) was incubated with 0.2 pmol [i%]palmitoyl-CoA with a radioactivity of approximately 35000 ct/min, 80 pmol Tris-phosphate buffer, pH 7.4, and 20 mg of bovine serum albumin in a final volume of 2.0 ml. In some experiments, the pH was adjusted by altering the amount of Tris-base and phosphoric acid. The incubation was carried out at 37°C in a metabolic shaker, either under Na gas or in the presence of 4 pmol (+)decanoylcarnitine in the reaction mixture, to prevent the possible oxidation of added substrate. The reaction was stopped by the addition of 10 ml of a mixture of isopropanol :heptane : 1~ HzS04 (20 :5 : 1, v/v/v) [ 14,161 after a period of incubation as stated in the individual experiment, followed by the addition of 6 ml heptane and 4 ml H20. The mixture was then transferred to a separator-y funnel, shaken vigorously, and was allowed to stand for 5 to 10 min. After the separation of two phases, the heptane phase was re-washed with heptane-saturated HzO, and the aqueous phase with HzO-saturated heptane [16]. An aliquot from the combined heptane phases was taken and dissolved in a naphthalene1.4-dioxane-based scintillating solution [7] for the determination of radioactivity. The combined heptane extracts recovered 93 to 96% of radioactivity when [i%]palmitic acid was tested alone. In contrast, a negligible amount of [1-14C]palmitoyl-CoA was extracted by the heptane, indicating a satisfactory isolation of the radioactive product from its precursor. A Nuclear-Chicago liquid scintillation counter, Mark I, was used for the determination of radioactivity. A counting efficiency of more than 70% was obtained as judged by the channels ratio method. Assay of phosphatidate phosphohydrolase This activity was determined by measuring the release of inorganic phosphate from phosphatidic acid (diacylglycerol 3-phosphate). The incubation mixture was prepared immediately prior to each experiment and contained 3.0 pmol sodium
580
M.-S.
LIU
AND
K. J. KAKO
phosphatidate, 160 pmol Tris-acetate buffer, pH 7.0, and an enzyme preparation in a final volume of 2.0 ml. The reaction was stopped by the addition of 2.0 ml 01 12% trichloroacetic acid. After removal of the precipitated protein by centrifugation, the inorganic phosphate in the reaction mixture was determined by the method of Berenblum and Chain [3]. The latter method was found to be more suitable than the methods of Fiske and Subbarow, and Lowry and Lopez, which tended to produce a higher blank value either at zero time incubation with substrate, or after incubation in the absence of enzyme preparation. Reproducibility of the Berenblum and Chain method was found to be excellent. The effect of pH on the enzyme activity was examined by using the buffer described above, except for pH 11, in which case the pH of the Tris-acetate was adjusted with 1~ NaOH. So-called particle-bound phosphatidic acid [,?I, 321 was prepared by incubating the cardiac or hepatic microsomal preparation in the presence of [U-l%]glycerol 3-phosphate for 5 min at 37°C [24, 271. The incubation mixture contained 50 mM Tris-phosphate buffer, pH 7.4, 1 mM potassium palmitate, 0.4 mM CoA, 6 mM ATP, 3 mM MgC12, 20 mg fatty acid-poor bovine serum albumin, 3 mM glycerol 3phosphate (approximately 1 @Ii) and microsomes (c. 2 mg protein) in a final volume of 2.0 ml. After incubation, the contents of ten tubes were pooled, layered on a 0.3 M sucrose solution and centrifuged at 100000 x g for 60 min. The microsomal pellet was resuspended and used as particle-bound phosphatidic acid. As reported earlier [27], 93 to 98% of the acylation products were monoacyland diacylglycerol 3-phosphate under these experimental conditions. The activity of phosphatidate phosphohydrolase was measured as above by incubating this form of phosphatidic acid (approximately 30 nmol in 5 mg microsomal protein) in the presence and in the absence of 1.5 ml (17.1 mg protein) of supernatant fraction in a final volume of 3.0 ml. Water-saturated butanol was added to the blanks (time zero samples) and assay tubes, and lipids were extracted by the procedure previously published [27]. The products were analyzed by t.l.c., with the use of the solvent systems, petroleum ether :ether :acetic acid (40 : 10 : 1) and chloroform :methanol : acetic acid :water (65 :25 :8 :4), and their radioactivity determined [27]. From these data, the effect of the supernatant fraction on the conversion of phosphatidate to glycerides was assessed.
3. Results Palmitoyl-CoA
hydrolase
The rate of hydrolysis of palmitoyl-CoA by rabbit heart homogenates was constant up to 120 min under the experimental conditions selected, as is shown in Figure 1 (a). The optimal pH for palmitoyl-CoA hydrolase in heart homogenates was approximately 8.5 [Figure 1 (b)].
ACYL-COA
ov 0
AND
I
I
30
60 Time
PHOSPHATIDATE
90
'
120
0' 5.5
I 6.5
(mud
FIGURE 1 (a). The time course of hydrolysis ‘The incubation mixture, in a final volume of
581
HYDROLASE
I 7.5
8.5
' 0-J 9.5
10.5
PH
of palmitoyl-CoA
by rabbit
heart
homogenates.
2.0 ml, contained 0.2 pmol [I-i%]palmitoyl-CoA (226000 d/min/pmol), 80 pmol Tris-phosphate buffer, pH 7.4, 20 mg bovine serum albumin, 4 pmol (+)-decanoylcarnitine and 0.4 mg of freshly prepared homogenates. The temperature of incubation was 37°C and gas phase, room air. (b) The effect of different pHs on the activity of palmitoyl-CoA hydrolase. The assay conditions are described in the legend to Figure l(a), except that the pH of the assay medium was varied as shown and (+)-decanoylcarnitine was omitted. The amount of heart homogenates was 0.3 mg, incubation time, 60 min, temperature, 37°C and gas
phase, nitrogen. Each value was corrected by the blank value, which was the value obtained in the absence of homogenates after the same period of incubation at the corresponding pH. The rate of hydrolysis is shown on the ordinate.
(+)-Decanoylcarnitine was reported to be a competitive inhibitor for carnitine palmitoyltransferase [38]. The effects of various concentrations of this compound are illustrated in Figure 2(a). The activity of palmitoyl-CoA hydrolase was increased as the concentration of decanoylcarnitine increased up to 7.5 mM and stayed constant as the concentration was further increased to 12 mM. It is unlikely that this activation is secondary to the inhibition of palmitoyl-CoA oxidation by decanoylcarnitine, since the activities measured in air and in a nitrogen atmosphere, both in the absence (0.45 vs. 0.51 nmol/mg.min) and in the presence of decanoylcarnitine (3.10 vs. 2.76 nmol/mg.min), were almost identical. On the other hand, the activity of palmitoyl-CoA hydrolase was inhibited by diisopropylflurophosphate, but only at rather high concentrations of the inhibitor, as compared to the liver enzyme [.?6] ; half maximal inhibition occurred at 5 mM [Figure 2(b)]. The rate of hydrolysis of palmitoyl-CoA was proportional to the amount of enzyme preparation up to 1.2 and 3.0 mg protein for microsomal and soluble fraction, respectively. [Figure 3 (a)]. The activity was similar when Na or room air was used as the atmosphere during incubation. The distribution pattern of the hydrolase in subcellular fractions of rabbit heart is shown in Figure 3(b). The mitochondrial, lysosomal, microsomal and soluble fractions all possess activity but the highest specific activity was noted in lysosomal and microsomal fractions. Since a large proportion of the total protein distributes
582
M.-S.
LIU
AND
K. J. KAKO 0.4
(b) \ A
-AlA
*
0.3
/ 0 A 0 A /
9“ 0
2
(+I
4
6
8
- Decanoylcarnitine
IO
12
0
0
(mM)
5
IO DFP
30
(rnt.4)
FIGURE 2(a). The effect of various concentrations of( +)-decanoylcarnitine on the activity of palmitoyl-CoA hydrolase in heart homogenates. The assay conditions are described in the legend to Figure l(a), except that the amount of (+)-decanoylcarnitine was varied as indicated (mM). The protein contents of homogenates were 0.39 and 0.41 mg protein in these experiments (a = 2). Incubation time was 60 min, the temperature, 37°C and gas phase, room air. The ordinate indicates the rate of hydrolysis. (b) The effect of preincubation of heart homogenates with diisopropylfluorophosphate (DFP) upon the activity of palmitoyl-CoA hydrolase. The homogenate containing approximately 4.9 mg protein in 2.0 ml was pre-incubated in the presence of various concentrations (mn) of DFP for 60 min at 0°C in this experiment (a = 2). Palmitoyl-CoA hydrolase activity was assayed subsequently by taking 0.1 ml aliquots from the pre-incubation mixture under the conditions similar to those described in the legend to Figure 1 (a). The incubation was performed under Ns for 60 min.
in the soluble and mitochondrial fractions, the total activities of these fractions are considerable. Dithiothreitol (5-30 mM) and cysteine (25 and 50 mM) accelerated spontaneous decomposition of palmitoyl-Cob, particularly at high pH values (> 7) [Figure 4(a)]. These sulfhydryl reagents are frequently used for the synthetic reactions involving acyl-CoA, and therefore, the present finding points to a possible error which may arise in determining an accurate concentration of acyl-CoA in the reaction mixture.
Phosphatidate phosphohydrolase The relationship between the rate of hydrolysis of phosphatidic acid and the amount of heart homogenate (O-2.55 mg protein) was linear under the experimental conditions selected. Furthermore, the reaction rate was constant for nearly 3 h with our assay system [Figure 4(b)]. The pH optimum of the heart phosphatidate
ACYL-COA
AND
PHOSPHATIDATE
(b)
016-
Ccl)
.
0 IF?/ 2 L 0.00 z E c
-
0.04
-
.
0
/
/
/
,
/
/
/
,
/
! /
I
I
Mt
I /
MC
Ly
’
/
583
HYDROLASE
1’
Y
/a/’ .,’ o/l
0
I 1.0 Protein
I 2.0
30
% of torol
protein
(mg)
FIGURE 3(a). The relationship between different amounts of microsomal or soluble fractions and rates of hydrolysis of palmitoyl-CoA. For the assay conditions see the legend to Figure 1 (a), except that a nitrogen atmosphere was introduced during a 40 min incubation and (+)-decanoylcarnitine was omitted. The ordinate indicates the rate of hydrolysis of palmitoyl-CoA, while the abscissa with a 100000 x g preindicates the amounts of subcellular preparations. (a) results obtained cipitate containing microsomes and lysosomes; (0) results with the soluble fraction. (b) The distribution of palmitoyl-CoA hydrolase in rabbit heart subcellular fractions. The ordinate indicates the activity in nmol palmitoyl-CoA hydrolyxed/mg protein/min and the abscissa shows yo distribution of protein in subcellular fractions. The symbols are: mitochondrial fraction = Mt, lysosomal fraction = Ly, microsomal fraction = MC, and soluble fraction = s. n = 6.
phosphohydroiase was 7.0 [Figure 5(a)]. The Michaelis-Menten constant, Km, of the enzyme in the microsomal-lysosomal fraction was 0.33 mM [Figure 5(b)], a value similar to that previously obtained with other organs [IO, 18, 361. The rate of hydrolysis of phosphatidic acid was proportional to the amount of subcellular fractions up to 1.0 and 1.3 mg protein per flask for microsomal and mitochondrial fractions, respectively, whereas the linear relationship with soluble (cytosomal) fraction was sustained up to 3.0 mg protein [Figure 6(a)]. The distribution of phosphatidate phosphohydrolase in cardiac subcellular fractions is shown in Figure 6(b). The highest specific activity of this enzyme was found in the microsomal and lysosomal fractions. The activity found in the cytosomal fraction was extremely low; thus it is difficult to state, from this analysis, whether or not the observed activity is due to microsomal contamination. The above question was further investigated by using a membrane-bound substrate, which was shown to be preferentially dephosphorylated by the enzyme
584
(a)
M.-S.
LIU
AND
K. J.
KAKO
200 ITT
1.2 -
160 c
0
/
/
.E 5 0.8E c 0.4 -
ODTT Cysteine
nil -
5 -
I 15 -
30 -
25
50
(mMj (mtd)
0
60
120 Time (min)
180
FIGURE 4(a). The effect of an addition of dithiothreitol (DTT) and cysteine on the spontaneous decomposition of palmitoyl-CoA. The reaction mixture contained 0.15 pmol [ l-14C]palmitoyl-CoA, 80 ymol Tris-phosphate buffer, pH 7.4,20 mg bovine serum albumin and different concentrations of DTT or cysteine in a final volume of 2.0 ml. The incubation was carried out for 30 min at 37°C in a nitrogen atmosphere. The ordinate indicates the amount of palmitoyl-CoA decomposed in nmoljmin. (0) the value obtained in the absence of DTT and cysteine; (%) the value obtained in the presence of DTT; ( ) the value obtained in the presence of cysteine. (b) The time course of the hydrolysis of phosphatidic acid by rabbit heart homogenates. The assay mixture contained, in a final volume of 2.0 ml, 3.0 pmol of phosphatidate, 160 pmol Tris-acetate buffer, pH 7.0 and 2.55 or 3.10 mg (protein) of homogenates (n = 2). Incubation was carried out in air at 37°C. The ordinate indicates the activity of phosphatidate phosphohydrolase in nmol inorganic phosphate released/mg protein, whereas the abscissa indicated the incubation time in min.
6
7
8 PH
9
5 Phosphotidote
(mM)
FIGURE 5(a). The pH-activity relationship of phosphatidate phosphohydrolase of rabbit heart homogenates. The assay conditions are identical to those described for Figure 4(b). The heart homogenate in this experiment was dialyzed against a medium consisting of 0.25 M sucrose, 0.02 M Tris-HCl and 0.001 M EDTA for 16 h at 4°C. The amount of homogenates was 1.9 mg and the incubation lasted for 45 min. Each value was corrected for a small blank value obtained at the same pH in the absence of phosphatidate. (b) The effect of various concentrations of phosphatidate on the activity of phosphatidate phosphohydrolase. The assay conditions are described in the legend to Figure 4(b). The incubation was continued for 60 min, and the lysosomal-microsomal fraction ( 1.56 mg protein) was used as the enzyme source in this experiment.
ACYL-COA
AND
PHOSPHATIDATE
583
HYDROLASE
(b) 3.0
2.5
6
LY -
-MC
.g 2.0 E” > E c
.-E4 \ zc
1.5
1.0
2 0.5
0 1.0 Protein
2.0 (mg)
Ml G
0
20
40
% of total
,
s,
60
80
100
protein
FIGURE 6(a). The relationship between amounts of different subcell&r fractions and phobphatidate phosphohydrolase activities. The assay mixture contained, in a final volume of 2.0 ml. 3.0 pmol of phosphatidate, 160 pmol of Tris-acetate buffer, pH 7.0, and various amounts of subcellular fractions. The assay was carried out for 45 min at 37°C in air. The activity is expressed as nmol of inorganic phosphate released per min. (0) activities of the lysosomal-microsomal preparations; (0) activities of the mitochondrial preparations; (0) activities of the soluble (cytosomalj fractions. (b) Subcellular distribution of phosphatidate phosphohydrolase in rabbit heart homogenates. See the legend to Figure 3(b) for the symbols used. n = 7.
existing in the soluble fraction of the liver or intestine [17, 21, 321. The microsomal fraction synthesized a very small amount of neutral lipid after the first incubation, namely, 6.5 -j= 0.7% of the total lipids, the remainder being mono- and diacylglycerol 3-phosphate, as reported in the previous paper [27]. A second, prolonged incubation of the microsomal fraction resulted in further glyceride formation. The rate of synthesis was 1.70 5 0.19 nmol/mg microsomal protein per min (n = 81, which is approximately half of the rate of the phosphatidate phosphohydrolase reaction assayed by using phosphatidate suspension as the substrate [Figure 6(b)]. The addition of the supernatant fraction to the second incubation mixture containing microsome-bound phosphatidate resulted in a considerable increase in neutral glyceride formation; the glyceride amounted to 47% of the total lipids in a 30-min incubation period, the rate of the reaction being 2.36 j, 0.28 nmol/mg soluble protein per min (n = 8). This reaction rate was ten-fold greater than the value recorded from the assay utilizing non-membrane-bound phosphatidate.
586
M.-S.
LIU
AND
K. J. KAKO
4. Discussion With the assay method adopted for palmitoyl-CoA hydrolase activity, hydrolysis was constant up to 2h of incubation, and the rate of reaction was proportional to the amount of enzyme. The pH-activity relationship of this enzyme determined with heart homogenates resembled that obtained with dog lung microsomes [13] and rat serum [19], but it differed from that found in adipose tissue homogenates [II]. Palmitoyl-CoA hydrolase can be rate-limiting for FA esterification in adipose tissue of fasted rats [II], but its activity in cardiac subcellular fractions was relatively insignificant (Results) and changed little in the hyperthyroid state (unpublished observation), so that it plays probably a relatively minor role in controlling the intracellular concentration of acyl-CoA. An apparent activation of palmitoyl-CoA hydrolase by a relatively high concentration of (+)-decanoylcarnitine has not been previously reported. It is unknown whether this enzyme is potentiated by the detergent property of decanoylcarnitine, or whether the latter compound inhibits palmitoyl-Cob oxidation, thus providing a relatively greater amount of substrate for the enzyme. However, a high concentration of palmitoyl-CoA used in our assay system (100 FM) and comparison of data obtained in the presence and absence of oxygen make the latter possibility less likely. It was reported earlier that the enzyme is active with the substrate in a micelle form [2], and thus it is possible that under the conditions of these experiments, the physico-chemical state of the substrate might have changed, causing the potentiation. An inhibition of enzyme activity by the detergent property of the palmitoyl-CoA is improbable in view of a study with hepatic microsomes, in which CoA esters of saturated FAs did not inhibit the hydrolase activity [ZO]. The study of cardiac subcellular fractions revealed uneven distribution of this enzyme activity; the enzyme activity of the mitochondria showed approximately 64% of that of the microsomal activity, suggesting that the observed activity cannot be ascribed to microsomal contamination but must be due to an independent mitochondrial enzyme. A relatively high specific activity in the lysosomal fraction has not been previously described, but the microsomal fraction has been known to possess a high hydrolase activity [13, 20, 261. This pattern of distribution of the enzyme [Figure 3(b)] implies that some acyl-CoA may be hydrolyzed and thus prevented from being utilized in oxidative energy production. On the other hand, a high microsomal hydrolase activity could be beneficial in limiting the availability of acyl-CoA for esterification, although this enzyme has a preference towards micelle form substrates, as mentioned above [2]. Our determinations of phosphatidate phosphohydrolase in heart muscle showed a pH optimum and subcellular distribution pattern similar to those provided by previous workers using organs other than the heart [IO, 18, 36, 371. A relatively high specific activity found in the mitochondrial fraction (approximately l/3 of the
ACYL-COA
AND
PHOSPHATIDATE
HYDROLASE
587
a possibility that the mitomicrosomal activity) [Figure 6(b)] makes unlikely chondrial activity is a result of microsomal contamination to the mitochondrial preparation. High specific activities of both phosphatidate phosphohydrolase and palmitoylCoA hydrolase in the lysosomal fraction suggest their role in degenerative heart disease or in myocardial fat infiltration. However, the activities in the hearts of hamsters suffering from hereditary cardiomyopathy were not increased (unpublished observation), and the functional significance of these lysosomal enzymes in the heart has not yet been elucidated. A report from another laboratory indicated that the phosphohydrolase showed some FA specificity, oleoyl-palmitoyl-glycerol S-phosphate being the most favourable substrate for rat liver enzyme preparations [32]. However, our observations with the crude enzyme preparation from the rat liver lysosomal fraction [37] in the presence of various synthesized monoacylglycerol3-phosphates suggest that the FA preference by this enzyme is not distinct [ZS]. Glyceride biosynthesis by subcellular fractions of rat liver or the small intestinal mucosa was reported to be greatly stimulated by the addition of the particle-free supernatant fraction [17, 21, 321. The effect by the latter was mostly attributed to phosphatidate phosphohydrolase existing in the soluble fraction [17]. These investigators demonstrated that the subcellular distribution of the enzyme activity varied depending upon the nature of the substrate; thus when aqueous dispersion of phosphatidic acid was used as the substrate, 90% of the total activity was in the particulate fractions, whereas, when membrane-bound phosphatidate was used as the substrate [17, 321, the particulate fractions possessed low activities and the soluble fraction was most active. It was also suggested that the enzyme in the soluble fraction is rate-governing in the biosynthesis of neutral glycerides [30]. This postulate was based on the finding that the activity of phosphohydrolase increased simultaneously with the increased triglyceride content in the liver following subtotal hepatectomy [30]. Th e p recise mechanism may be more complicated, however, since the same investigators observed, in addition, that treatment with actinomycin D suppressed the increased enzyme activity, whereas hepatic triglyceride accumulation was uninfluenced [3U]. Moreover, recent evidence is at variance with the older literature, in that the difference that was observed by using the membrane-bound and non-membrane-bound substrate can be attributed to the effect of Mgs+, at least, in adipose tissue [18]. Our results with cardiac subcellular fractions differ from the results of the above studies, in that, although the cardiac soluble fraction is more active than the microsomal fraction in hydrolyzing membrane-bound phosphatidate, this activity is lower than the microsomal enzyme activity assayed with the use of unbound phosphatidic acid. Our study thus demonstrated that the highest activity in heart homogenates is found in the lysosomal and microsomal fractions [see also, 32, 36, 371, and that the properties of the soluble enzyme are different from those of
M.-S. LIU AND K. J. KAKO
588
particle-bound enzymes. Furthermore, we have previously reported that acyl-CoA: glycerol 3-phosphate acyltransferase and acyl-CoA:monoacylglycerol 3-phosphate acyltransferase are under hormonal control [24]. Similarly, particulate enzyme activities were found to be increased under the influence of triiodothyronine treatment, but there was no change in the soluble phosphohydrolase (unpublished observation). Consequently, evidence obtained so far supports a view that each subcellular fraction contains individual, different phosphohydrolases.
Acknowledgement
This work was supported by grants from the Medical Research Council and Ontario Heart Foundation. M.S.L. is a recipient of Scholarship by the Alcoholism and Drug Addiction Research Foundation of Ontario, and K. J.K. is an MRC Research Associate. The valuable assistance of Miss Dale Patterson is gratefully acknowledged.
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