separation, properties, and regulation of acyl coenzyme a dehydrogenases from bovine heart and liver

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSKS Vol. 213, No. 1, January, pp. 155-162,1982

Separation, Properties, and Regulation of Acyl Coenzyme A Dehydrogenases from Bovine Heart and Liver’

BRUCE DAVIDSON AND HORST SCHULZ

Llepartment of Chemistry, City College of the City University of New York, New York, New York 10031

Received May 22, 1981, and in revised form July 29, 1981

A simple two-step procedure has been developed for the separation of bovine liver butyryl-CoA dehydrogenase (EC 1.3.99.2), medium chain acyl-CoA dehydrogenase (EC 1.3.99.3), which is most active with octanoyl-CoA as a substrate, and long-chain aeyl-CoA dehydrogenase (EC 1.3.99.3) which acts preferentially on dodecanoyl-CoA. The same method was used for separating the acyl-CoA dehydrogenases present in bovine heart. Three acyl-CoA dehydrogenases were thus identified and found to be identical with the bovine liver enzymes as judged by their chromatographic behaviors, chain length specificities, and kinetic properties. Medium-chain and long-chain acyl-CoA dehydrogenases are active with a wide range of substrates from butyryl-CoA to stearoyl-CoA. However, their chain length specificities are clearly different from each other although insuffi- ciently so to draw a firm conclusion regarding their physiological functions. The effects of mitochondrial coenzymes and metabolites, including intermediates of @ oxidation, on the activities of the bovine acyl-CoA dehydrogenases, were investigated. Most noteworthy are the inhibitions of butyryl-CoA dehydrogenase by acetoacetyl-CoA (4 = 10m6 M) and of medium-chain and long-chain acyl-CoA dehydrogenases by 3-ketodecanoyl-CoA. The KI values for 3-ketodecanoyl-CoA with medium-chain and long-chain acyl-CoA dehydrogenases were found to be 8 X 10m7 and 7.5 X lo-* M, respectively. We propose that the rate of fatty acid oxidation in heart is tuned to the energy demand of the tissue by a sequence of feedback controls which include the regulation of acyl-CoA dehydrogenases by 3-ketoacyl-CoA compounds.

The first step of /3 oxidation is catalyzed by a group of flavoenzymes named acyl- CoA dehydrogenases. Beinert and co- workers, who studied these enzymes ex- tensively, identified, purified, and charac- terized three acyl-CoA dehydrogenases in pig liver which differ in their chain length specificities (1). One of the three dehydrogenases with a narrow specificity for short- chain substrates is named butyryl-CoA dehydrogenase (EC 1.3.99.2). The other two acyl-CoA dehydrogenases (EC 1.3.99.3) exhibit wider chain length specificities and are believed to act preferentially on medium-chain and long-chain substrates, respectively. More limited studies of

1 This investigation was supported in part by Grant HL 18089 of the National Heart, Lung and Blood In- stitute and by a City University of New York Faculty Research Award.

the bovine heart and liver acyl-CoA dehydrogenases have resulted in the iden- tification of only butyryl-CoA dehydrogenase and one other acyl-CoA dehydrogenase (2, 3).

During the course of studying the con- trol of fatty acid oxidation in heart it be- came necessary for us to identify and char- acterize the acyl-CoA dehydrogenases of heart muscle. Since established purification procedures are not useful for rapidly and quantitatively separating all acyl-CoA dehydrogenases, we have developed a simple two-step procedure by which the acyl- CoA dehydrogenases present in mitochondria of bovine liver and heart can be com- pletely separatd from each other and can be partially purified. A study of the effects of various mitochrondrial coenzymes and metabolites on the activities of the three acyl-CoA dehydrogenases led to the ob-

155 0003-9861/82/010155-08$02.00/O Copyright 0 1982 by Academic Press. Inc. All rights of reproduction in any form reaerwd.

156 DAVIDSON AND SCHULZ

servation that all three enzymes are inhibited by 3-ketoacyl-CoA compounds. However, most severely inhibited is the acyl-CoA dehydrogenase believed to catalyze the first step in the degradation of long-chain fatty acids.

EXPERIMENTAL PROCEDURES

Materials. CoASH and CoA derivatives of saturated fatty acids were purchased from P-L Biochem- icals, Inc. Sigma Chemical Company was the source of DL-3-hydroxybutyric acid, phenazine methosulfate, 2,6-dichlorophenolindophenol, and L-3-hydroxyacyl-CoA dehydrogenase. tran.s-2-Decenoic acid was obtained from Aldrich Chemical Company. Fresh bovine heart and liver were bought from Max Insel Cohen Company, Livingston, New Jersey. DL-3-Hy- droxydecanoic acid was synthesized by reduction with NaBH, of ethyl 3-ketodecanate, prepared according to an established procedure (4), followed by hydrolysis. The CoA derivatives of DL-hydroxybutyric acid, 2-decenoic acid, and DL-3-hydroxydecanoic acid were synthesized from the corresponding free acids and CoA by the method of Goldman and Va- gelos (5). 3-Ketodecanoyl-CoA was prepared enzy- matically from trans-2-decenoyl-CoA according to the procedure of Seubert et al. (6). Acetoacetyl-CoA (7) and crotonyl-CoA (8) were prepared by established procedures. The concentration of 3-ketodecanoyl-CoA was estimated by recording at 340 nm the oxidation of NADH in the presence of 3-hydroxyacyl- CoA dehydrogenase. The concentations of all other acyl-CoA compounds were determined by the method of Ellman (9) after cleaving the thioester bond with hydroxylamine at pH 7.

AC&CoA dehydrogenase and protein assags. All three acyl-CoA dehydrogenases were assayed spec- trophotometrically by following at 600 nm the acyl- CoA-dependent reduction of 2,6-dichlorophenolindophenol in the presence of phenazine methosulfate as outlined by Hoskins (10). The assay mixture con- tained 0.1 M potassium phosphate (pH 7.6) 28 gM 2,6- dichlorophenolindophenol, 0.65 mM phenazine methosulfate, 20 pM acyl-CoA, 0.2 mM N-ethylmaleimide, and enzyme to obtain a A A/min of approximately 0.06. The reaction was initiated by the addition of enzyme when partially purified acyl-CoA dehydrogenase was assayed. Phenazine methosulfate was added last when mitochondria or a mitochondrial extract was used as an enzyme source. Additionally added were 0.09% Triton X-100 to dissolve the mitochondria and 0.45 mM KCN to prevent the nonspe- cific reoxidation of phenazine methosulfate. When general acyl-CoA dehydrogenase was assayed, the concentration of phenazine methosulfate was increased to 3.3 mM. N-Ethylmaleimide was present in

the assay mixture to reduce the background rate due to the reaction of 2,6-dichlorophenolindophenol with sulfhydryl groups. N-Ethylmaleimide at the eoncen- tration used had no effect on the activities of the acyl-CoA dehydrogenases. Assays were performed at 25’C and an extinction coefficient of 21,300 Mm1 was used to calculate rates which were corrected for non- specific reactions. A unit of enzyme activity is defined as the amount of enzyme that catalyzes the dehydrogenation of 1 pmol of substrate per minute. KI values were determined graphically from Dixon plots. Lines were drawn based on least-squares treat- ment of the data. Protein concentrations were determined according to Lowry et al. (11).

Separation and partial purification of a&-CoA dehydrogenases from bovine Ziver and heart. Mitoehon- dria from fresh bovine liver were prepared essen- tially by the method of Fleischer et al. (12). Mitochondria from fresh bovine heart were prepared by the method of Crane et al. (13). Frozen mitochondria (5-10 g) were quickly thawed and diluted with 10 vol of 10 mM potassium phosphate (pH 7.6) and subjected to sonic oscillation in an ice-cooled rosette cell for 90 s at 50 W with a Branson sonifier (Model W185D) equipped with a microtip. Soluble mitochondrial proteins were isolated by centrifugation at 100,OOOg for 60 min at 4°C. The sonic supernatant containing 100-500 mg of protein was applied to a DEAE-cellulose column (2.5 X 30 cm) which had previously been equilibrated with 50 mM potassium phosphate (pH 7.6). The column was washed with 3 column vol of the same buffer until material absorbing light at 280 nm ceased to be eluted. The resulting column was washed with 6 column vol of 100 mM potassium phosphate (pH 7.6) and fractions of 19 ml were collected until all butyryl-CoA dehydrogenase activity had been eluted. The most active fractions were combined and concentratd to approximately 1 ml in an Amicon concentrator (PM-10 membrane) and stored at -76°C. The column was then washed with 3 column vol of 0.5 M potassium phosphate (pH 7.6). Fractions of 18 ml were collected and assayed with decanoyl-CoA and palmitoyl-CoA as substrates. The most active fractions were combined and concentrated to approximately 10 ml and dialyzed against 6 liters of 10 mM postassium phosphate (pH 7.6) for 6 h with two changes of buffer. The dialyzate was centrifuged at 20,000~ for 15 min to remove small amounts of precipitated material. The resulting supernatant was diluted with 2.5 v01 of dialysis buffer and applied to a hydroxylapatite column (2.5 X 7 cm) which had previously been equilibrated with the dialysis buffer. The column was washed wih 1.5 column vol of dialysis buffer and 2 column vol of 50 mM potassium phosphate (pH 7.6) until material absorbing light at 280 nm ceased to be eluted. The resulting column was developed with

BOVINE ACYL COENZYME A DEHYDROGENASE REGULATION 157

a gradient made up of 5 column vol each of 50 mM potassium phosphate (pH 7.6) and 300 mM potassium phosphate (pH 7.6) at a flow rate of 1.5-2 ml/min. Fractions of 5 ml were collected and assayed with butyryl-CoA, decanoyl-CoA, and palmitoyl-Coil as substrates. Fractions of highest activity representing either medium-chain or long-chain acyl-CoA dehydrogenase were separately combined, concentrated to approximately 1 ml, and stored at -76°C.

RESULTS

Separatim and identijcatim of acyl- CoA dehydrogenases of bovine liver and heart. When a soluble extract of either bovine liver or bovine heart mitochondria was subjected to chromatography on DEAE-cellulose, a complete separation of butyryl-CoA dehydrogenase from medium-chain and long-chain acyl-CoA dehydrogenase activities was achieved by a simple stepwise elution procedure (see Fig. 1). The separation of the two acyl-CoA dehydrogenases cochromatographing on DEAE-cellulose was accomplished by chromatography on hydroxylapatite (see Fig. 2). Development of the hydroxylapatite column with a linear potassium phosphate gradient resulted in the elution of first an acyl-CoA dehydrogenase that was most active with decanoyl-CoA as a substrate, less active with palmitoyl-CoA, and almost inactive with butyryl-CoA (see Fig.

FIG. 1. Separation of butyryl-CoA dehydrogenase from medium-chain and long-chain acyl-CoA dehydrogenases by chromatography on DEAE-cellulose. For experimental details see under Experimental Procedures. Substrates used for assaying acyl-CoA dehydrogenases were: A, butyryl-CoA; 0, decanoyl- CoA; n , palmitoyl-Cot\.

FIG. 2. Separation of medium-chain and long-chain acyl-CoA dehydrogenases on hydroxylapatite. For experimental details see under Experimental Pro- cedures. Substrates used for assaying acyl-CoA dehydrogenase were: A, butyryl-CoA, 0, decanoyl-CoA; n , palmitoyl-CoA.

2). Following the first acyl-CoA dehydrogenase, but well separated from it, ap- peared a second dehydrogenase that was highly active with both decanoyl-CoA and palmitoyl-CoA as substrates but was significantly less active toward butyryl-CoA. Based on their relative activities toward these three substrates we were unable to match the two bovine liver acyl-CoA dehydrogenases with the corresponding pig liver enzymes described by Beinert (1). Most important to us was the finding that extracts from bovine liver and heart mitochondria yielded identical purification patterns for the acyl-CoA dehydrogenases. Thus both organs contain possibly identical sets of three acyl-CoA dehydrogenases. The three acyl-CoA dehydrogenases isolated from bovine liver and heart were subjected to polyacrylamide disc gel elec- trophoresis and were found to contain several proteins. The most abundant of these proteins, which accounted for approximately 30% of the material, was identified as acyl-CoA dehydrogenase by assaying extracts of gel segments. Furthermore, these experiments proved that the corresponding acyl-CoA dehydrogenases from bovine liver and bovine heart have the same relative mobilities on polyacrylamide gels.

Chain length speci$cities and sme kinetic properties of acykCoA dehydroge-


FIG. 3. Chain length specificities of bovine acyl-CoA dehydrogenases. n , Butyryl-CoA dehydrogenase; 0, medium-chain acyl-CoA dehydrogenase; A, long-chain acyl-CoA dehydrogenase. Solid lines represent activities obtained at optimal concentrations of phenazine methosulfate; broken lines represent activities observed at a suboptimal concentration of 0.65 mM phenazine methosulfate. The activities of long-chain acyl-CoA dehydrogenase with myristoyl-CoA or shorter-chain substrates were unaffected by a change in the concentration of phenazine methosulfate from 0.65 to 6.5 m&l.

rimes. The chain length specificities of the three acyl-CoA dehydrogenases isolated from bovine liver and bovine heart were determined at 20 pM substrate concentrations (see Fig. 3). The patterns of activity versus substrate chain length for corre- ponding dehydrogenases from heart and liver were found to be identical. Butyryl- CoA dehydrogenase is most active with butyryl-CoA and only slightly less active with hexanoyl-CoA. However, it was found to be virtually inactive with longer chain substrates (see Fig. 3). The acyl-CoA dehydrogenase which was eluted last from the hydroxylapatite column, was found to be most active with octanoyl-CoA as a substrate. When this dehydrogenase was assayed at the standard concentration of phenazine methosulfate of 0.65 mM, its

activity varied little with the substrate chain lengths as long as the acyl chain was

between 6 and 16 carbons long (see Fig. 3). However, at an optimal concentration of phenazine methosulfate of 6.5 mM the enzyme is highly active with medium- chain substrates, moderately active with long-chain substrates (C&-Cr6), and virtually inactive with butyryl-CoA (see Fig. 3). Consequently, this enzyme is best char- acterized as a medium-chain acyl-CoA dehydrogenase. The acyl-CoA dehydrogenase, that was eluted first from the hydroxylapatite column, was found to be most active with dodecanoyl-CoA as a substrate but it acts effectively on medium- chain and long-chain substrates including stearoyl-CoA. It is virtually inactive with butyryl-CoA and marginally active with hexanoyl-CoA (see Fig. 3). Consequently, it is best described as a long-chain acyl- CoA dehydrogenase. Its activity with palmitoyl-CoA or longer-chain substrates was significantly increased when the concentration of phenazine methosulfate was raised from the standard 0.65 to 6.5 mM

(see Fig. 3). Some kinetic constants (K, and relative

V values) were determined for all three bovine acyl-CoA dehydrogenases. All i(, values were found to be in the low micro- molar range except for values determined with medium-chain acyl-CoA dehydrogenase for butyryl-CoA and with long-chain acyl-CoA dehydrogenase for hexanoyl-CoA (see Table I). These two high K, values explain why medium-chain and long-chain acyl-CoA dehydrogenases are nearly inactive toward butyryl-CoA and hexanoyl- CoA, respectively, when assayed at 20 PM

substrate concentrations used to deter- mine the chain length specificities of these enymes (see Fig. 3). The Km values obtained with both medium-chain and long- chain acyl-CoA dehydrogenase were found to decrease with increasing chain lengths of the substrates. An opposite trend was observed for butyryl-CoA dehydrogenase. In all cases tested the K, values for substrates of medium-chain acyl-CoA dehydrogenase were lower by approximately a facor of 10 than the values obtained for the same substrates with long-chain acyl- CoA dehydrogenase. The decrease of V values with increasing substrate chain


TABLE I

KINETIC PROPERTIES OF BOVINE Acyl-CoA DEHYLIROGENASES

Substrate

Butyryl-CoA dehydrogenase

KWZ rel. V

(PM) (%I

Medium-chain acyl- CoA dehydrogenase

rel. V

(%)

Long-chain acyl- CoA dehydrogenase

KR rel. V

(PM) (%I

Butyryl-CoA 3 100 ~100 31 - - Hexanoyl-CoA 12 100 3.5 loo 80 50 Octanoyl-CoA 2 100 15 46 Decanoyl-CoA 0.7 67 10 166 Dodecanoyl-CoA 4 106 Palmitoyl-CoA 2 16

length observed both with medium-chain and long-chain acyl-CoA dehydrogenase may be partially due to the detergent properties of the substrates.

The effects of various mitochundrial coenx~mes and metabolites cm the activities of bovine a&CoA dehydrogenuse. Several mitochondrial coenzymes and metabolites were tested for their ability to either in- hibit or stimulate the three bovine acyl- CoA dehydrogenases. None of the following coenzymes tested at the indicated concentations affected the activities of the dehydrogenases: ATP (5 InM), ADP (5 mM), GTP (5 IrIM), GDP (5 mM), FAD (10 mM), NAD (1 mM), NADP (1 mM), carni- tine (5 mM), acetyl-CoA (1 mM), and suc- cinyl-CoA (0.1 mM). Also the tricarboxylic acid cycle intermediates citrate, isocitrate, succinate, a-ketoglutarate, malate, and fu- marate at 10 mM concentrations were without effect. However, several intermediates of fatty acid oxidation were found to be effective inhibitors of these dehydrogenases. Tested for their inhibitory potential were the short-chain fatty acid oxidation intermediates crotonyl-CoA, DL-3-hydroxylbutyryl-CoA, and acetoacetyl-CoA as well as the medium-chain intermediates 2-decenoyl-CoA, DL-3-hydroxydecanoyl-CoA, and 3-ketodecanoyl- CoA. The results obtained with butyryl- CoA dehydrogenase are presented in Fig. 4A. The most effective inhibitor is acetoacetyl-CoA. This finding agrees with a pre- vious report in which the inhibition of the same enzyme from Peptostreptococcus els-

denii by acetoacetyl-CoA was demonstrated (14). In contrast, 3-ketodecanoyl- CoA is a weak inhibitor possibly because its long alkyl chain does not fit into the substrate binding site. Crotonyl-CoA, the product of the butyryl-CoA-catalyzed reaction, is less inhibitory than acetoacetyl- CoA for which a KI of 10 -6 M was determined. Both 3-hydroxyacyl-CoA compounds had no effect on the activity of butyryl-CoA dehydrogenase. Medium- chain acyl-CoA dehydrogenase is inhibited only by 2-decenoyl-CoA and more severely by 3-ketodecanoyl-CoA (see Fig. 4B) for which a KI of 8 X lo-? M was obtained. Acetoacetyl-CoA, which has been reported to be an inhibitor of pig liver general acyl- CoA dehydrogenase (15), has virtually no effect on the bovine liver enzyme over the concentation range tested. The most in- teresting result was obtained when the inhibition of long-chain acyl-CoA dehydrogenase was studied (see Fig. 4C). 3-Ke- todecanoyl-CoA was found to be an ex- tremely effective inhibitor of this enzyme with a KI value of 7.5 X lOWa M. As expected, 2-decenoyl-CoA is also inhibitory, although to a lesser degree. Surprisingly, even DL-3-hydroxydecanoyl-CoA causes a significant inhibition. Crotonyl-CoA is a very weak inhibitor whereas both acetoacetyl-CoA and DL-3-hydroxybutyryl-CoA are virtually without effect.

DISCUSSION

By use of a simple two-step procedure described in this publication we have been

T-IAVTTKWN ANTI SC!HTll,Z


the assay procedure, especially the use of electron-transferring flavoprotein as a primary electron acceptor by Beinert and co-workers and phenazine methosulfate by us may account for some of the observed differences in chain length specificities.

Although butyryl-CoA dehydrogenase and the two acyl-CoA dehydrogenases identified in pig and bovine tissues are assumed to function in fatty acid oxidation, their specific roles under physiological conditions have not been established. An evaluation of the chain length specificities of the dehydrogenases from pig liver suggests that these enzymes may complement each other to assure a high rate of dehydrogenation of all common fatty acyl compounds. However, the co- operation of butyryl-CoA dehydrogenase and acyl-CoA dehydrogenase (C6-C,r6) alone should result in the efficient dehydrogenation of all fatty acids. The chain length specificities of the bovine enzymes determined by us also suggest that butyryl-CoA dehydrogenase together with long-chain acyl-CoA dehydrogenase would catalyze effectively the dehydrogenation of all fatty acids with 4 to 18 carbons. However, the relatively high ri, values for medium-chain substrates observed with long-chain acyl-CoA dehydrogenase may result in a low capacity to metabolize medium-chain fatty acids if they are present at low concentrations unless medium-chain acyl-CoA dehydrogenase participates in this process. Since the effective concentrations of /3 oxidation intermediates under physiological conditions are not known, the question concerning the necessary co- operation of two or three dehydrogenases in the degradation of saturated fatty acids cannot yet be answered. Another function for medium-chain acyl-CoA dehydrogenase has recently been proposed by Kunau and Dommes (16) who showed that &s-4- decenoyl-CoA, an intermediate in the degradation of linoleic acid, can only be de- hydrogenated by medium-chain acyl-CoA dehydrogenase. Thus it is possible that the degradation of saturated fatty acids re- quires the presence of butyryl-CoA de-

TCA cyc1* /

NAOH nw+g I Tr,pl”F,,,d,s - FW” ACOd. x N&O UP

FIG. 5. Proposed regulation of fatty acid oxidation in heart.

hydrogenase and long-chain acyl-CoA dehydrogenase whereas the medium-chain dehydrogenase is essential for the break- down of polyunsaturated fatty acids.

The strong inhibitory effect of S-ketodecanoyl-CoA on long-chain acyl-CoA dehydrogenase, which presumably catalyzes the initial step in p oxidation, leads us to suggest that 3-ketoacyl-CoA compounds may function in vivo as feedback inhibitors of fatty acid oxidation. Although some intermediates of ,f3 oxidation have been found to accummulate in mitochondria under normal conditions as well as in the presence of rotenone (1’7), no at- tempt has been made to identify and quan- titate medium-chain and long-chain 3-ketoacyl-CoA compounds, which may be effective regulators at nanomolar concentrations. On the basis of findings reported here and elsewhere (18) we propose that the rate of fatty acid oxidation is tuned to the energy demand of heart via several connecting feedback controls as outlined in Fig. 5. It has been demonstrated both with isolated mitochondria (19) and in the perfused heart (20) that a decrease in the energy demand leads to an increase in the concentration of acetyl-CoA and to a corresponding decrease of CoASH. Increases in the acetyl-CoA/CoASH ratio of the magnitude observed in mitochondria and the perfused heart result in the inhibition of 3-ketoacyl-CoA thiolase (18) which catalyzes one of the slow steps of /3 oxidation (21). The expected accumulation of long- chain 3-ketoacyl-CoA compounds may lead to the inhibition of long-chain acyl-CoA dehydrogenase and consequently to a de- creased rate of fatty acid oxidation.


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separation, properties, and regulation of acyl coenzyme a dehydrogenases from bovine heart and liver

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