Temperature effects on malonyl-CoA inhibition of carnitine palmitoyltransferase I

BiochimicaL et Biophysics &ta

ELSEVIER

Biochimica

et Biophysics

Acta 1257 (1995) 133-139

Temperature effects on malonyl-CoA inhibition of carnitine palmitoyltransferase I Khosrow Kashfi Department

of Pharmacology,

*‘l,

George A. Cook

College of Medicine, University of Tennessee-Memphis, 38163,

Received 26 October

The Health Science

Center, 874 Union Avenue, Memphis,

TN,

USA

1994; revised 13 February

1995; accepted 6 March 1995

Abstract Malonyl-CoA inhibition of hepatic mitochondrial carnitine palmitoyltransferase I and malonyl-CoA binding were measured at temperatures ranging from 0°C to 37°C. Protease treatment of mitochondria resulted in greatly diminished malonyl-CoA binding, indicating that the method used detected malonyl-CoA binding sites located on the outer surface of the mitochondrial outer membrane as expected. The apparent K, for malonyl-CoA inhibition was found to increase with increasing temperature. Arrhenius plots for the initial velocity of the enzymatic reaction and for the Ki for malonyl-CoA both indicated a transition temperature between 20 and 25” C with the transition for the malonyl-CoA interaction being more pronounced. Total specific binding of malonyl-CoA to mitochondrial proteins increased with increasing temperature, and K, values decreased. The opposite effect of temperature on K, values and K, values was surprising because it was expected that these equilibrium constants would be identical. These observations indicate that K, values for malonyl-CoA binding and K, values for inhibition of camitine palmitoyltransferase I by malonyl-CoA represent two significantly different binding phenomena. These data suggest that either: (a) malonyl-CoA binding measurements are unrelated to malonyl-CoA inhibition, or (b) inhibition of carnitine palmitoyltransferase I by malonyl-CoA involves more complex relationships than binding of malonyl-CoA to a single protein. Keywords:

Carnitine

palmitoyltransferase;

Fatty acid oxidation;

Malonyl-CoA;

1. Introduction

Camitine palmitoyltransferase I (CPT-I; EC 2.3.1.21) is the primary regulatory enzyme in the hepatic mitochondrial fatty acid oxidation pathway which is located in the mitochondrial outer membrane [ 11 and is regulated through inhibition by malonyl-CoA, its physiological inhibitor [2]. Starvation and diabetes bring about increased rates of hepatic fatty acid oxidation through increased substrate supply and by changes in CPT-I that include increased activity and decreased sensitivity to inhibition by malonylCoA [3-81. Quantitatively, the most significant change that occurs with the onset of diabetes [7,8] or fasting [9,10] is a IO-fold increase in its apparent K, for malonyl-CoA.

* Corresponding author. Fax: + 1 (901) 448 7300. ’Present address: Department of Medicine, Division of Digestive Diseases, The New York Hospital-Cornell Medical Center, New York, NY. USA. 0005.2760/95/$09.50 SSDI 0005-2760(95)00063-l

0

1995 Elsevier

Science B.V. All rights reserved

Enzyme inhibition;

Temperature

effect; Mitochondrion;

Liver

Since malonyl-CoA is a competitive inhibitor of CPT-I, its Ki for malonyl-CoA inhibition is defined as the equilibrium constant for the dissociation of the enzymemalonyl-CoA complex. It would be expected, therefore, that Ki values would be identical with K, values derived from radioligand binding experiments using radiolabelled malonyl-CoA. There is substantial evidence to associate closely the binding of [ ‘“Clmalonyl-CoA to intact mitochondria and mitochondrial outer membranes with the regulatory properties of CPT-I [ II- 171. Diabetes decreases the K, (indicating increased binding affinity) for malonylCoA binding to CPT-I in a manner that closely correlates with increased sensitivity of CPT-I to malonyl-CoA inhibition [ 1I]. L-Camitine can displace malonyl-CoA from its binding sites on mitochondria while D-camitine has no effect [ 121, and the specific inhibitor of CPT-I, 2-bromopalmitoyl-CoA, displaces malonyl-CoA bound to mitochondria [ 131. In skeletal muscle [ 141 and in heart and liver [15] the occupancy of high-affinity binding sites by malonyl-CoA has been closely correlated with regulation of

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CPT-I activity. It has been hypothesized that binding of palmitoyl-CoA at the active site is distinct from that which occurs at a second, malonyl-CoA binding site [ 161. There appear to be at least two malonyl-CoA binding sites, which have a greater than IO-fold difference in affinity for malonyl-CoA [ 15,161. However, Kolodziej and Zammit [ 171, using isolated mitochondrial outer membranes, have suggested that there is only one binding site for malonyl-CoA. Using Yonetani-Theorell kinetic analysis of CPT-I inhibition we recently reported two independent sites for inhibition of this enzyme, but only one site is specific for malonyl-CoA [ 181. CPT-I sensitivity to malonyl-CoA inhibition is greatly enhanced as the incubation temperature is lowered from 37” to 20” C [19]. Unfortunately, neither Ki values for malonyl-CoA inhibition of CPT-I nor K, values for CPT-I substrates have ever been measured and compared at different temperatures, so it is not known which of these factors is responsible for increased sensitivity. Therefore, we wanted to examine the effects of incubation temperature on K, values for inhibition of CPT-I by malonyl-CoA and on K, values for malonyl-CoA binding. We obtained some rather surprising results which indicate that temperature has totally different effects on K, and on K,. These results are discussed in relation to their implications for the regulation of CPT-I.

2. Materials

and methods

2. I. Animals Male Sprague-Dawley rats (200-250 g) obtained from Harlan Industries (Indianapolis, IN, USA) were fed on Purina rat chow (Ralston Purina, Richmond, IN, USA) and water ad libitum. On the day of the experiment, rats were killed by decapitation and their livers were removed rapidly for preparation of mitochondria. 2.2. Isolation of mitochondria Intact mitochondria were isolated by the method of Johnson and Lardy [20], with the modifications previously published [9], except that the isolation medium contained 210 mM-mannitol, 70 mM-sucrose, 0.1 mM EDTA and 10 mM Tris-HCI (pH 7.4). In some experiments, mitochondria (5 mg/ml) were incubated at 37” C in isolation medium with the protease subtilisin BPN’ at 5 pg/ml. After 20 min, proteinolysis was stopped by addition of 200 ~1 of 20% (w/v) BSA/ml of incubation volume followed by addition of 40 ml of ice-cold isolation medium, and mitochondria were subsequently sedimented by centrifugation at 5600 X g for 10 min and then resuspended in the isolation medium to a concentration of 20 mg/ml. Protein was determined by a biuret method [21].

rt Biophwictr

Actu 1257 (19951 133-134,

2.3. Carnitine palmitoyltransferase

assay

The method of Bremer [4] was used as modified and reported previously [7]. Each assay contained in a total volume of 1 ml:82 mM sucrose, 70 mM KC], 70 mM imidazole, I Fg of antimycin A, 1 mM EGTA, 2 mg of BSA, 40 PM palmitoyl-CoA and 0.5 mM t-camitine (0.4 mCi/mmol of L-[methyl-“Hlcarnitine). The assay pH was adjusted at each assay temperature according to Ellis and Morrison [22] to maintain identical ionic strength at each temperature. 2.4. Binding of [‘4C~malon~l-CoA

to mitochondria

Malonyl-CoA binding experiments were performed in 1.5 ml plastic stoppered centrifuge tubes. The binding cocktail for incubations at 0” C, IO” C and 20” C contained in a total volume of 1 ml:82 mM sucrose, 70 mM imidazole, 1 pg of antimycin A, 1 mM EGTA and enough KC1 to give the same ionic strength as that in the CPT assay after the pH was adjusted to 7.0 with 6 N HCl at each temperature [22]. Albumin was omitted from the binding cocktail since it had been shown to influence malonyl-CoA binding [23]. The concentration of [‘“C]malonyl-CoA (specific activity 51 mCi/mmol) was varied from 5 to 660 nM, although several higher concentrations were used initially to ensure that malonyl-CoA binding sites were saturable (data not shown). The incubation medium was allowed to equilibrate at the test temperature for approx. 1 h and checked before beginning experiments. The experiments were started by the addition of mitochondria to a concentration of 1 mg protein per ml. After equilibrium was reached, the time for which varied according to temperature, the tubes were centrifuged for 2 min at 12 000 X g using an Eppendorf microcentrifuge at O-4” C. The supernatant solution was aspirated and the walls of the tubes were dried with tissue paper. The mitochondrial pellets were solubilized with 0.5 ml of 10% Triton X-100 and transferred to scintillation vials. Radioactivity in the pellet and supernate was measured by scintillation spectrometry. Non-specific binding was estimated by inclusion of 250 nmol unlabeled malonyl-CoA in the incubation medium. Non-specific binding was proportional to the concentration of labeled malonyl-CoA and was non-saturable (data not shown); specific binding was calculated by subtracting non-specific binding from total binding at each concentration of ligand. lnitial experiments were carried out at 0” C using 3 mg/ml of mitochondrial protein. Under these conditions equilibrium was reached within 20 min as suggested by others [ 14,151. However, on closer examination. it was discovered that using such a high protein concentration caused about 30% of the labelled malonyl-CoA to be bound at equilibrium which indicated that the concentration of the free ligand would have changed too greatly to estimate binding constants accurately. One of the basic

Khosrow Kashji, George A. Cook/Biochimica

et Biophysics Acta 1257 (lYY5l 133-139

OS-

0

100

200

300

400

[ Malonyl-CoA

500

600

0

700

1, nM

135

B

10

30

20

Malonyl-CoA

40

Bound, pmol/mg

Fig. 1. Specific binding of malonyl-CoA in protease-treated mitochondria. Specific binding of [‘4C]malonyl-CoA was measured at 0” C in control (0) and protease-treated (0) mitochondria as described in Section 2. Results are mean f SE. for three different preparations of mitochondria. Panel A shows specific binding, and panel B illustrates Scatchard analysis of specific binding. Binding parameters derived from these plots are given in Table 1.

assumptions made in the development of the theory for calculation of K, is that the concentration of free ligand does not change [24]. Hence the mitochondrial concentration was lowered to 1 mg/ml which resulted in less than 10% of the ligand bound at equilibrium. It took 80 min to reach binding equilibrium with control mitochondria, 80 min for controls plus 5 PM palmitoyl-CoA, and 120 min for protease-treated mitochondria. All binding assays at 0” C were hence made after 120 min of equilibration. At higher temperatures, 40 min were used for binding assays at 10” C, and 20 min were used for assays conducted at 20” C; however, hydrolysis of malonyl-CoA was observed at this temperature and these data were not used for further analysis (see Results). 2.5. Analysis

of ~‘“Clmalonyl-CoA

multiple binding sites exist with unchanging and different affinities; (b) negative cooperativity exists, such that the affinity of the overall binding population decreases with increasing occupancy of the binding sites [26]. In order to distinguish between these two possibilities, “infinite dilution” experiments were carried out according to DeMeyts et al. [27]. From a plot of amount of malonyl-CoA bound at zero time as a function of the time elapsed after the dilution of the system, it was concluded that negative cooperativity did not exist since the rate of dissociation after dilution of the labeled ligand alone and after dilution in presence of excess unlabelled malonyl-CoA both followed the same time course (data not shown). After establishing that the Scatchard plots represented multiple binding sites, estimates of binding parameters were obtained using the computer programs EBDA [28] and LIGAND [29,30]. The analysis of the data fitted the two site models (P < 0.05) better than the one site (P 2 0.1) model when the residual sum of squares were compared. Hence, all parameters reported here are for a two site model. These data clearly suggest that malonyl-CoA

binding data

The binding data were plotted according to the method of Scatchard [25]. This plot gave a curve which was upward concave (downward convex) in shape. Two possible biological interpretations of these data are possible: (a)

Table 1 Malonyl-CoA

binding parameters

for binding to intact hepatic mitochondria

Binding conditions

K,t fnM)

$0

Control, 0” C Control, 10” C Control + 5 PM palmitoyl-CoA, Protease

63+ 13 38*4” 102+ 1 il 47 f 4

257 * 20 166 f 10 11 2321 + 124 ’ 366 + 50

0” C

Nl (pmol/mg)

N2

19 + 3 25 + 5 _

52 f 5 68* 10 _

4+0.2

a

fpmol/mg)

20+4

a

Malonyl-CoA binding was measured under the conditions indicated as described in Section 2. Protease treatment was carried out using the protease subtilisin BPN’ at 5 wg/ml for 20 min. Binding parameters were calculated using the LIGAND program. Results are mean + SE. for three different preparations of mitochondria. ’ Statistically different from control values at 0” C (P < 0.05).

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et Biophxsica Acta 1257 (1995) 133-139

binds to more than one site in hepatic mitochondrial membranes, although binding data alone cannot indicate whether these sites are on distinct proteins. 2.6. Materials Palmitoyl-CoA, imidazole, BSA, malonyl-CoA, EGTA, and Subtilisin BPN’ were purchased from Sigma Chemical (St. Louis, MO, USA). L-[methyl-3H]Carnitine hydrochloride and [2-‘4C]malonyl-CoA were obtained from Amersham (Arlington Heights, IL, USA).

3. Results

01

3.1. Effects of protease to mitochondria

treatment on malonyl-CoA

10

binding

Treatment of intact mitochondria with the protease subtilisin BPN’ resulted in substantial loss in the ability of malonyl-CoA to bind to mitochondria (Fig. 1). Scatchard analysis indicated that the specific binding to all binding sites for malonyl-CoA was substantially reduced by the protease. Based on our experience with incubation of intact mitochondria with this protease in experiments which have been published previously [ 181, it appears that the bulk of malonyl-CoA binding is to the external surface of the mitochondria, i.e., outside the mitochondrial outer membrane. Data presented in Table 1 derived from these analyses indicate that the binding sites having the highest affinity for malonyl-CoA were decreased by about 80%, while lower affinity binding sites were decreased by 60%. These data indicate that the measurement of malonyl-CoA binding to whole, intact mitochondria correlates closely with the known location of CPT-I.

I

I

30

40

I

0

20 Temperature,

I

“C

Fig. 2. Effect of temperature on CPT-I activity. Intact hepatic mitochondria were incubated with 40 FM palmitoyl-CoA and 0.5 mM carnitine at different temperatures after determining incubation times that would yield linear assays with respect to time. The inset shows an Arrhenius plot of the data.

Fig. 3, which indicates that the apparent Ki values for malonyl-CoA increased as a function of increasing temperature. When the data were replotted as an Arrhenius plot, a sharp bend in the plot was apparent. The exact transition temperature could not be assessed because of the small number of points used. Only three temperatures were used because of the large number of assays contributing to each determination of Ki and because it was not expected that the Arrhenius plot would be nonlinear. The bend in the Arrhenius plot for malonyl-CoA inhibition was noticeably greater than that for CPT-I activity, however, suggesting there may be a much greater effect of membrane lipids on

3.2. Effects of temperature on CPT-I activity, malonyl-CoA inhibition and the apparent Ki for malonyl-CoA The activity of CPT-I increased with increasing temperature as expected (Fig. 2), but a replot of these data produced a non-linear Arrhenius plot having a transition temperature between 20” C and 25” C. Although several explanations have been presented to explain non-linear Arrhenius plots, the most likely explanation is a phase change in the mitochondrial outer membrane lipid associated with the enzyme [3 1,321. The Arrhenius plot indicates that the activation energy for the CPT-I reaction increases at temperatures above the lipid phase transition temperature. As shown previously [ 191, malonyl-CoA inhibition increased greatly as the temperature decreased. Dixon plots were constructed at 10” C, 20” C and 37” C using several concentrations of malonyl-CoA at each of three different substrate concentrations for each temperature. From these plots apparent Ki values were calculated and are shown in

I

0

10

20

Temperature,

30

40

“C

Fig. 3. Effect of temperature on apparent K, values for malonyl-CoA. CPT activity was measured in intact hepatic mitochondria in presence and absence of malonyl-CoA (l-5 FM) at different temperatures from which Dixon plots were constructed and apparent K, values determined. Palmitoyl-CoA and carnitine concentrations were 40 FM and 0.5 mM, respectively. Results are meanfS.E. for three different preparations of mitochondria.

Khosmw

Kashji, George A. Cook/Biochimica

et Biophysics Acta 1257 (1995) 133-139

137

0.6

0 0

20

40

60

80

100

120

-0

140

10

20

30

40

50

60

Malonyl-CoA Bound ( pmol/mg )

Time, min Fig. 4. Equilibrium time course of malonyl-CoA binding at different mitochondria were incubated with temperatures. Intact hepatic [‘4C]malonyl-CoA (220 nM) at 0” C (01, 10” C (W 1 and 20” C (A ). At the indicated times the assays were terminated and specific binding was determined as described in Section 2.

the CPT-I enzyme-inhibitor enzyme-substrate complex.

complex

compared

with the

3.3. Binding of malonyl-CoA

to hepatic mitochondria

Fig. 4 shows specific binding of malonyl-CoA as a function of time. Binding equilibrium required 2 h at 0” C, but equilibrium was reached in 1 h at 10” C. At 20” C malonyl-CoA binding appeared to be more rapid than at 10” C but decreased noticeably after 20 min, probably due to enzymatic hydrolysis of malonyl-CoA at the higher temperature [ 12-151. The data suggest that specific binding is greater at 10” C and 20” C than at 0” C. This effect is exactly the opposite of what is expected on the basis of malonyl-CoA inhibition studies. Specific binding as a

Fig. 6. Scatchard analysis of malonyl-CoA binding data. The concentrabound was determined after equilibrium had tion of [ “C]malonyl-CoA been reached. The concentration of free malonyl-CoA was calculated as described in Section 2. Symbols refer to control mitochondria at 0” C (01, control mitochondria at 0” C in the presence of 5 /.LM palmitoyl-CoA to show specificity of binding (A 1, and mitochondria incubated at 10” C (ml. Results are mean+S.E. for three different preparations of mitochondria.

function of malonyl-CoA concentration also indicated a greater extent of binding at 10” C and 20” C compared with that at 0” C (Fig. 5). Lower concentrations of malonyl-CoA seem to have promoted less binding at 20” C than higher concentrations, possibly because of malonyl-CoA metabolism. Scatchard plots were constructed from specific binding data at several concentrations of malonyl-CoA (Fig. 6). All Scatchard plots were curved, indicating multiple binding sites (see Methods). Data from experiments at 20” C were not used for Scatchard plots because of the loss due to metabolism. Palmitoyl-CoA at 5 PM competitively eliminated malonyl-CoA binding to all binding sites (Fig. 6), functionally demonstrating that malonyl-CoA binding was related to CPT-I. It can be seen that specific binding was greater at the higher temperature. Binding parameters were calculated as described in Section 2. Table 1 indicates that the dissociation constants K,, and K,,, associated with the putative high and low affinity binding sites, respectively, decreased as the temperature increased from 0” C to 10” C, just the opposite of the temperature effect on Ki values (Fig. 3). There were no changes in the number of binding sites with increasing temperature.

4. Discussion

[ Malonyl-CoA

I, nM

Fig. 5. Specific binding as a function of malonyl-CoA concentration at different temperatures. Specific binding of [‘4C]malonyl-CoA was measured at 0” C CO), 10°C (01, and 20°C (a), using intact hepatic mitochondria as described in Section 2. Results are mean + S.E. for three different preparations.

The fact that both activity and inhibition of CPT-I are characterized by a transition temperature between 20” C and 25” C which is probably associated with a membrane lipid phase transition is consistent with earlier reports associating alterations in malonyl-CoA sensitivity with changes in membrane fluidity [ 191. It is also consistent

I38

Khosmu

Km!@.

George A. Cook/Biochitnica

with changes in sensitivity of the enzyme as a result of changes in membrane phospholipids [39]. The very sharp bend in the Arrhenius plot for malonyl-CoA inhibition suggests that membrane fluidity had a greater influence on the malonyl-CoA-enzyme complex than on the enzymesubstrate complex. Membrane effects may also be responsible for the changes in activity and malonyl-CoA sensitivity produced by cholesterol [33,34]. Temperature effects producing discontinuities in Arrhenius plots for activity and inhibition are especially common with membrane-associated enzymes of the mitochondria; they are most often caused by changes in the physical state of the lipid phase of the membrane and occur between 20” C and 25” C [32]. Kinetic data reported here have shown that the decrease in malonyl-CoA inhibition of CPT-I that occurs with increasing temperature [19] is caused by a change in the CPT-I K, for malonyl-CoA. The Ki values for malonylCoA increase as the temperature increases. Similar effects of temperature on K, values have been observed previously with other enzymes [35]. At 10” C the observed K, and Ki for CPT-I were different by approximately one order of magnitude (Table 1 and Fig. 2), and while Ki K, values decreased with increasing values increased temperature. These observations indicate that K, values for malonyl-CoA inhibition and K, values for malonylCoA binding do not represent the same equilibrium constants. The unknown component in this system is the protein component. It is possible that the equilibrium with which the K, is associated involves a more complex, temperature-dependent step which is not a component of malonyl-CoA binding, but these results seem to raise an important question: Is it possible that malonyl-CoA binding to mitochondria is not related to CPT-I regulatory properties? This has been thought to be unlikely because of the extensive data that have accumulated linking malonyl-CoA binding with CPT-I [ 1 l- 171. Two aspects linking malonyl-CoA binding to CPT-I inhibition that are demonstrated in this paper are the very potent effect of palmitoyl-CoA competition with both high and low affinity binding and the destruction of malonyl-CoA binding sites with protease. Decreased binding of malonyl-CoA would be expected in the presence of palmitoyl-CoA since malonyl-CoA is a competitive inhibitor of CPT-I with respect to palmitoyl-CoA [9]. It is interesting in this regard that palmitoyl-CoA produced a decreased binding at both putative malonyl-CoA binding sites. Recent data have suggested that it is possible to discriminate two components of liver CPT-I, a catalytic component and a regulatory component that binds malonyl-CoA [36-381. No malonyl-CoA binding regulatory protein has been isolated and purified so far nor has the hepatic CPT-I possessing catalytic capacity been isolated to show whether the catalytic component binds malonyl-CoA. If the hypothesis proposing a separate malonyl-CoA-binding regulatory component is correct, then our present data would suggest that K, values represent dissociation constants of a com-

et Biophpica

Acta 1257 (1995)

133-139

plex formed between malonyl-CoA and the binding protein, and K, represents a more complex dissociation constant of a ternary complex of malonyl-CoA, the binding component, and the catalytic component of CPT-I. The observance of two binding components having significantly different K, values may suggest that malonyl-CoA binds, with different affinities, to both the regulatory and the catalytic component. Additional support for malonylCoA binding at a site other than the active site has been provided by our recent finding that CPT-I possesses two sites for inhibition, the active site which does not bind malonyl-CoA but binds most other CPT-I inhibitors and another site that is specific for malonyl-CoA and is independent of the active site [ 181. It is not yet possible to determine whether the two inhibitory sites (the malonylCoA site and the non-malonyl-CoA site) reside on the same protein. Overall, the data presented in this paper suggests that either: (a> malonyl-CoA binding as it is currently being measured is not associated with the inhibition and regulation of CPT-I, or (b) inhibition of CPT-I by malonyl-CoA involves a more complex relationship than binding of malonyl-CoA to a single protein.

Acknowledgements This work was supported National Institutes of Health Service). Additional support in-Aid from the American Affiliate.

by grant HL-40929 from the (United States Public Health was received through a GrantHeart Association, Tennessee

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