Biochemistry 1983, 22, 5641-5646
5641
Purification of Rabbit Myocardial Cytosolic Acyl-CoA Hydrolase, Identity with Lysophospholipase, and Modulation of Enzymic Activity by Endogenous Cardiac Amphiphilest Richard W. Gross*
ABSTRACT:
Rabbit myocardial cytosolic acyl coenzyme A (acyl-CoA) hydrolase activity was purified to near-homogeneity by ammonium sulfate precipitation and ion-exchange, gel filtration, chromatofocusing, and hydroxylapatite chromatographies. Kinetic analysis of the purified protein demonstrated a maximum velocity of 24 pmol/(mgmin) and an apparent Michaelis constant of 50 pM. Cytosolic acyl-CoA hydrolase and lysophospholipase activities cochromatographed in every fraction of every step. The purified protein was a single band ( M , 23 000) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. These results suggest that cytosolic lysophospholipase and palmitoyl-CoA hydrolase activities are catalyzed by a single polypeptide with
dual activities. Palmitoyl-CoA competitively inhibited lysophospholipase activity (Ki = 4 pM). Low concentrations (20 pM) of lysophosphatidylcholine or L-palmitoylcarnitine increased palmitoyl-CoA hydrolase activity at low palmitoylCoA concentrations but had little effect at high concentrations of palmitoyl-CoA. In contrast, high concentrations (100pM) of lysophosphatidylcholine or L-palmitoylcarnitine inhibited palmitoyl-CoA hydrolase activity. The results suggest that interactions between endogenous cardiac amphiphiles and palmitoyl-CoA hydrolase contribute to the regulation of intracellular long-chain acyl-CoA concentrations and therefore potentially modulate fluxes of fatty acid through several biochemical pathways.
xioesterification of long-chain fatty acids is the first step of fatty acid utilization. The subsequent metabolic fate of activated fatty acids in myocardial cytosol can be grouped into three main pathways: (1) transfer to carnitine by palmitoylcarnitine acyltransferase; (2)transfer to glycerol phosphate as the first committed step toward the synthesis of lipid metabolites; and (3) hydrolysis by acyl coenzyme A (acyl-CoA)' hydrolase to regenerate free fatty acid. Little is known of the physiologic significance of the role of the net futile cycle of thioesterification and hydrolysis, but cytosolic acyl-CoA occupies an ideal position to provide control over several metabolic pathways (Holroyde et al., 1976;Bloch & Vance, 1977; Weinhouse, 1976). Recently acyl-CoA was demonstrated to be an allosteric modulator of glucokinase (Tippett & Neet, 1982),and thus cytosolic acyl-CoA levels are likely to modify rates of glycolysis, P-oxidation, and lipid synthetic pathways. Thus, enzymes which modulate cystolic acyl-CoA concentration, such as acyl-CoA hydrolase, could potentially provide significant metabolic control. Acyl-CoA hydrolase activity has previously been identified in rabbit myocardial cytosol, but its low reported specific activity (Kako & Patterson, 1975) had placed the physiological significance of hydrolysis of cytosolic thio esters in myocardium in doubt. This study demonstrates the presence of palmitoyl-CoA hydrolase activity in rabbit myocardial cytosol 2 orders of magnitude higher than previously reported, the purification of cytosolic palmitoyl-CoA hydrolase to near-homogeneity and its identity with cytosolic lysophospholipase, and also presents kinetic evidence which demonstrates that endogenous cardiac amphiphiles can be either positive or negative modulators of acyl-CoA hydrolase activity.
Materials and Methods Preparation of Subcellular Fractions. New Zealand White rabbits were sacrificed by cervical dislocation, their hearts promptly removed after a left thoracotomy, and subcellular fractions prepared after Polytron homogenization by differential centrifugation as previously described (Gross & Sobel, 1982). The lOOOOg,, pellet (mitochondria) was washed with homogenization buffer and resuspended by utilizing a Dual1 apparatus (5.5 mg of protein/mL). The 105000gm,, pellet (microsomes) was resuspended in homogenization buffer (2 mg/mL). The supernatant after the 105000gm.centrifugation, cytosol (7 mg/mL), was utilized for the subsequent protein purification. Purification of Cytosolic Acyl-CoA Hydrolase Activity. Palmitoyl-CoA hydrolase activity from rabbit myocardial cytosol was precipitated by slowly adding solid ammonium sulfate (to 45% saturation), stirred for 10 min, and centrifuged at 140O0gm for 5 min. The pellet was resuspended in 20 mM phosphate, 10 mM HSEtOH, 10% glycerol, and 10 pM lyso-PC, pH 7.6,and dialyzed for 14 h against 100 volumes of the same buffer. The purification scheme was nearly identical with that previously reported for rabbit myocardial cytosolic lysophospholipase (Gross & Sobel, 1983) with the following modifications: (a) AcA-54 resin was utilized for both gel filtration columns, resulting in improved resolution of the low molecular, weight polypeptide; (b) active fractions from hydroxylapatite chromatography were dialyzed against pH 6.7 imidazole buffer (20mM) containing 10 pM lyso-PC, 10 mM HSEtOH, and 10% glycerol, pH 6.7 (adjusted with 6 N HCl), and loaded onto a chromatofocusing column packed with Polybuffer exchanger 94 (0.9 X 18 cm). The retained proteins were washed with 1 column volume of starting buffer, and the column was subsequently developed with Polybuffer 74 (1:8
From the Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110. Received March 28, 1983. Supported in part by National Institutes of Health Grant HL17646 SCOR in Ischemic Heart Disease and by a Missouri Heart Association Grant-in-Aid. * Address correspondence to this author at the Cardiovascular Division, Washington University School of Medicine.
0006-2960/83/0422-5641$0lSO/O
I Abbreviations: CK, creatine kinase; CoA, coenzyme A; HSEtOH, 6-mercaptoethanol; LDH, lactate dehydrogenase; lyso-PC, 1 -palmitoylsn-glycero-3-phosphocholine; SDS, sodium dodecyl sulfate; TLC, thinlayer chromatography; DEAE, diethylaminoethyl.
0 1983 American Chemical Society
5642
BIOCHEMISTRY
dilution) containing 10% glycerol, 10 mM HSEtOH, and 10 pM lyso-PC, at pH 5.3 (adjusted with 6 N HCl). Assay of Palmitoyl-CoA Hydrolase and Lysophospholipase Activities. Palmitoyl-CoA hydrolase activity from column eluates was quantitated by incubating 50 pL of column fractions in 700 pL of 0.1 M phosphate buffer (pH 7.0) containing 140 pM [ l-14C]palmitoyl-CoA(specific activity approximately 2000 dpm/nmol) at 37 OC for 15 min. Reaction products were extracted into 500 pL of butanol by vortexing twice for 30 s and centrifuged at 2000g- for 2 min to separate layers. Thirty microliters of the butanol layer (upper phase) was applied to a silica OF TLC plate (Analabs). Reaction products were separated by utilizing a 3:4:1: 1:0.5 CHC13:acetone:MeOH:HOAc:H20 solvent system. Regions corresponding to fatty acid (Rf= 1) were scraped into scintillation vials, 10 mL of Aquasol I1 was added, and radioactivity was quantitated by scintillation spectrometry. Kinetic assays were performed similarly except that a 10-min incubation in 1 mL of buffer was used. Palmitoyl-CoA hydrolase activity assayed with this procedure was linear with respect to incubation time and mass of protein under the conditions utilized in this study (
