Journal of Neurochemistry Raven Press, Ltd., New York © 1995 International Society for Neurochemistry Purification, Properties, and Specificity of Rat Brain Cytosolic Fatty Acyl Coenzyme A Hydrolase Constantinos G. Broustas and Amiya K. Hajra Department of Biological Chemistry and Mental Health Research Institute, University of Michigan, Ann Arbor, Michigan, U.S.A. Abstract : Rat brain Cytosolic acyl-CoA hydrolase has and Srere et al. (1959). The latter authors reported that been purified 3,500-fold to apparent homogeneity using the enzyme is also present in liver and kidney but that heat treatment, ammonium sulfate fractionation followed brain had the highest activity. Kurooka et al. (1972) by anion exchange, hydrophobic interaction, and hy- studied the distribution and properties of the Cytosolic droxyapatite chromatography . The purified enzyme re- enzyme in various mammalian organs and showed that high concentration mains stable only in the presence of a brain had the highest activity, followed by testis. (30%, vol/vol) of ethylene glycol . On sodium dodecyl sul- fate-polyacrylamide gel electrophoresis the purified en- Though normal mammalian liver has comparatively zyme shows a single band of 40.9 kDa. However, on high- low activity of the soluble enzyme, it is highly induced performance size-exclusion chromatography the migra- in rodent liver after administration of peroxisome-pro- tion rate of the enzyme corresponds with an apparent liferating agents (Kawashima et al., 1981 ; Miyazawa molecular mass of 148 kDa, indicating that the native et al., 1981). enzyme may be a tetramer. The enzyme catalyzes the A number of laboratories have studied the properties hydrolysis of fatty acyl-CoAs from six to 18 carbon chains and regulation of the crude and purified Cytosolic en- long, having the highest activity for lauroyl (12 :0)-CoA . zyme from various sources. However, the physiologi- For the purified enzyme the Km for palmitoyl-CoA is 5.8 cal function of the enzymes is not clear. In rat and protein . The pM and the Vm ~ is 1,300 pmol/min/mg of rabbit mammary gland, where the hydrolase is specific enzyme is inhibited by bovine serum albumin, various detergents, lysophosphatidylcholine, and palmitoyl carni- for medium chain acyl-CoAs, it probably regulates the tine .Among the sulfhydryl agents, only p-hydroxymercu- end product of fatty acid biosynthesis by hydrolyzing ribenzoate inhibited the enzyme. The enzyme is also inac- the medium chain fatty acids thioesterified to the fatty tivated by treatment with a high concentration of diethyl acid synthase complex (Knudsen et al., 1976; Libertini pyrocarbonate, an active center histidine-reacting agent, and Smith, 1978). However, in most other systems, but not by phenylmethylsulfonyl fluoride (10 mM), a serine the thioester hydrolase that catalyzes the terminal step esterase inhibitor . The purified enzyme does not appear of fatty acid biosynthesis (i.e., release of the product, to possess any O-ester hydrolase, lysophospholipase, free fatty acid) is a member of a fatty acid synthase transacylase, or acyltransferase activity . Key Words: Pal- complex and is different from the enzyme studied here. mitoyl-CoA hydrolase-Acyl-CoA hydrolase-Purifica- It has been speculated that the soluble acyl-CoA hy- tion-Rat brain-Cytosol . drolase regulates the concentration of Cytosolic free J. Neurochem. 64, 2345-2353 (1995). acyl-CoAs, which are inhibitory to many enzymes (Ta- keta and Pogell, 1966). However, the increase in activ- ity of this enzyme during times of high lipid biosyn- Long chain acyl-CoA hydrolase (palmitoyl-CoA hy- thetic activity, such as during myelination or induction drolase, EC 3 .1 .2.2) is a ubiquitous enzyme present in in liver by peroxisome-proliferating (hepatomegaly) all living organisms. The enzyme catalyzes the hydro- agents, points toward a different regulatory role for lysis of fatty acyl-CoAs to free fatty acids and CoASH. this enzyme. In vertebrate tissues, this enzyme is present both as a soluble form in the cytosol (Kurooka et al., 1972) and Received July 18, 1994 ; revised manuscript received October 17, as a membrane-bound form in microsomes (Berge, 1994 ; accepted November 9, 1994. 1979), mitochondria (Berge and Farstad, 1979), and Address correspondence and reprint requests to Dr. A. K. Hajra probably peroxisomes (Berge et al., 1984). The proper- at Neuroscience Laboratory, University of Michigan, 1103 E. Huron, different Ann Arbor, MI 48104-1687, U.S .A . ties of the membrane-bound enzymes are Abbreviations used: CMC, critical micellar concentration; DTNB, from those of the Cytosolic enzyme. The Cytosolic en- dithio-bis(nitrobenzoic acid); lysoPtdCho, 1-acyl-sn-glycerol-3-phos- zyme was first discovered to be present, in mammalian phocholine ; PAGE, polyacrylamide gel electrophoresis; PtdCho, 1,2- brain and other organs, by Vignais and Zabin (1958) diacyl-sn-glycerol-3-phosphocholine; SDS, sodium dodecyl sulfate. 2345 2346 C. G. BROUSTAS AND A. K. HAJRA The enzyme has been reported to be purified to ho- Methods mogeneity from several sources such as rat liver mito- Acyl-CoA hydrolase activity was assayed spectrophoto- chondria (Berge and Farstad, 1979), microsomes metrically by measuring the release of free CoASH from (Berge, 1979) and cytosol (after induction) (Miyazawa acyl-CoA in the presence of excess' S,5'-dithio-bis(2-nitro- et al., 1981 ; Yamada et al., 1994), rabbit heart cytosol benzoic acid) (Ellman, 1959). One milliliter of the reaction (Gross, and duck uropygial gland (Cheesbrough mixture contained 100 mM potassium phosphate, pH 7.5, 0.2 1983), mM dithio-bis(nitrobenzoic acid) (DTNB), 60 ~M palmitoyl- and Kolattukudy, 1985). The properties and subunit CoA, and enzyme at 37°C. The assay was started by adding molecular weight of the purified enzyme from each enzyme and the increase in absorbante at 412 nm was mea- source are quite different, indicating that these are iso- sured for 3 min. A molar absorbante of 1 .36 x 104 M- ' cm ` zymes. Brain cytosolic enzyme has been only partially for the 2-nitro-5-thiobenzoate anion was used to calculate purified as reported by different investigators. Srere et activity. al. (1959) partially purified the enzyme from pig brain One unit of enzyme activity was defined as that hydrolyz- and reported that there were probably two acyl-CoA ing 1 /cmol of palmitoyl-CoA to free fatty acid and CoASH. hydrolyzing enzymes, each specific for the hydrolysis A radiometric assay was also used to measure the enzyme of acyl-CoAs of different chain lengths. Anderson and activity, especially when the effect of thiol compounds Erwin (1971) reported that the activity of the enzyme (CoASH and dithiothreitol) was studied. Hydrolase activity was determined by measuring the release of [1-'4C]palmitic was greater in medulla, pons, and midbrain than in 4 cerebral cortex and caudate nucleus. These authors pre- acid from [1-' C]palmitoyl-CoA, which were separated from each other using Dole's extraction method (Dole, 1955). sented results that show that the partially purified en- The reaction mixture (0.4 ml) containing 100 mM phosphate zyme (60-fold) is present as a dimer with a subunit buffer, pH 7.5, 60 ~M [1-'4C]palmitoyl-CoA (2,600 dpm/ molecular mass of ^~46 kDa. Knauer (1979) also pre- nmol), and enzyme was incubated at 37°C for 5 min. Two sented evidence that the rat brain soluble hydrolase milliliters of isopropanoUheptane/concentrated sulfuric acid exists in monomerkc (70 kDa) and dimeric (130 kDa) (40:10:1) was added to the reaction mixture, mixed, and an forms . Lin et al. (1984) purified rat brain cytosolic additional 0.8 ml of heptane and 1.2 ml of 0.5 M sulfuric enzyme by ^-20-fold and tentatively identified the en- acid was added. The mixture was mixed well and was left zyme to be a 65-kDa polypeptide that showed no evi- to stand for 10 min and then centrifuged at 600 g for 5 min dence of associated or dissociated forms. in a tabletop Beckman TJ-6 centrifuge. Aliquots from the upper phase, containing the free palmitic acid, were trans- In this study, we report the purification of the rat ferred to scintillation vials, the solvent was removed by brain cytosolic acyl-CoA hydrolase to apparent homo- blowing a stream ofair, and the residue was dissolved in 2 ml geneity and present data on its properties. of scintillation liquid (Universol) and counted in a Beckman scintillation counter. Palmitoyl L-carnitine hydrolase activity was determined MATERIALS AND METHODS by measuring the release of [1-`4C]palmitic acid from [1- '4C]palmitoyl L-carnitine . The assay conditions were the Materials same as for the hydrolase assay with the exception that [1- `4C]palmitoyl L-carnitine (20,000 dpm/nmol; 10, 50, and 100 Frozen rat brains were obtained from Pel-Freez (Rogers, 4 AR, U.S.A.). Phenyl-Sepharose CL-4B was purchased from ~M) was used instead of [1-` C]palmitoyl-CoA. Pharmacia Biotech (Piscataway, NJ, U.S.A.). Biocel HTP, The ability of the enzyme to hydrolyze other sulfhydryl low molecular weight protein standards, N,N,N',N'-tetra- compounds such as palmitoyl glutathione and palmitoyl cys- methylenediamine, sodium dodecyl sulfate (SDS), and acryl- teine was examined by using the spectrophotometric hy- amide/bisacrylamide were from Bio-Rad (Melville, NY, drolase assay and adding palmitoyl glutathione or palmitoyl cysteine to the reaction mixture instead of palmitoyl-CoA. U.S.A.). TSK-G3000SW HPLC column was from TosoHaas 3Z (Montgomeryville, PA, U.S.A.). Centricon-30 filters were 1-Acyl-sn-glycerol-3-[3ZP]phosphocholine ([ P]lysoPtd- from Amicon (Beverly, MA, U.S.A.). Reagent grade ethyl- Cho) was prepared by enzymatic hydrolysis of 1,2-diacyl- ene glycol, sodium fluoride, and ß-mercaptoethanol were sn-glycerol-3-[ 3ZP]phosphocholine ([32P]PtdCho) with phos- pholipase AZ. The labeled PtdCho was prepared by overnight from Fisher Scientific (Pittsburgh, PA, U.S.A.).