Substrate Specificities of 3-Oxoacyl-CoA Thiolase A and Sterol Carrier Protein 2/3-Oxoacyl-CoA Thiolase Purified from Normal Rat Liver Peroxisomes

The two main thiolase activities present in isolated peroxisomes from normal rat liver were purified to near homogeneity. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the first enzyme preparation displayed a single band of 41 kDa that was identified as 3-oxoacyl-CoA thiolase A (thiolase A) by N-terminal amino acid sequencing. The second enzyme preparation consisted of a 58- and a 46-kDa band. The 58-kDa polypeptide reacted with antibodies raised against either sterol carrier protein 2 or the thiolase domain of sterol carrier protein 2/3-oxoacyl-CoA thiolase (SCP-2/thiolase), formerly also called sterol carrier protein X, whereas the 46-kDa polypeptide reacted only with the antibodies raised against the thiolase domain. Internal peptide sequencing confirmed that the 58-kDa polypeptide is SCP-2/thiolase and that the 46-kDa polypeptide is the thiolase domain of SCP-2/thiolase. Thiolase A catalyzed the cleavage of short, medium, and long straight chain 3-oxoacyl-CoAs, medium chain 3-oxoacyl-CoAs being the best substrates. The enzyme was inactive with the 2-methyl-branched 3-oxo-2-methylpalmitoyl-CoA and with the bile acid intermediate 24-oxo-trihydroxycoprostanoyl-CoA. SCP-2/thiolase was active with medium and long straight chain 3-oxoacyl-CoAs but also with the 2-methyl-branched 3-oxoacyl-CoA and the bile acid intermediate. In peroxisomal extracts, more than 90% of the thiolase activity toward straight chain 3-oxoacyl-CoAs was associated with thiolase A. Kinetic parameters (K m and V max) were determined for each enzyme with the different substrates. Our results indicate the following: 1) the two (main) thiolases present in peroxisomes from normal rat liver are thiolase A and SCP-2/thiolase; 2) thiolase A is responsible for the thiolytic cleavage of straight chain 3-oxoacyl-CoAs; and 3) SCP-2/thiolase is responsible for the thiolytic cleavage of the 3-oxoacyl-CoA derivatives of 2-methyl-branched fatty acids and the side chain of cholesterol.

A first peroxisomal 3-oxoacyl-CoA thiolase was purified from livers of rats treated with the peroxisome proliferator di (2ethylhexyl)phthalate by Hashimoto and co-workers (10). It is active with short, medium, and long straight chain 3-oxoacyl-CoAs, medium chain CoA esters being the best substrates (11). The enzyme, which was subsequently cloned, consists of two identical subunits of 41 kDa. The subunit is synthesized as a 44-kDa precursor containing a 26-amino acid N-terminal leader sequence that functions as a peroxisome targeting signal and that is cleaved off after import of the polypeptide in the peroxisome (13). Some years later Hashimoto and co-workers (14) and independently Bodnar and Rachubinski (15) found that the rat genome contains not one but two closely related thiolase genes, gene B encoding the precursor of the previously purified peroxisomal thiolase (thiolase B) 1 and gene A encoding a thiolase (thiolase A) precursor with a 10-residue longer Nterminal presequence. In their mature form thiolases A and B differ in only 6 (14) or 9 (15) amino acid residues. mRNA analysis revealed that the A gene is constitutively transcribed albeit at a low level and that transcription of the B gene is strictly dependent on activation of the gene by a peroxisome proliferator (14,15). Thiolase A has not been purified yet, so it is not known how far its properties differ from those of the inducible thiolase B.
Peroxisomes also contain a 58-kDa protein that cross-reacts with antibodies raised against SCP-2. Molecular cloning of the protein revealed that it consists of an N-terminal 404 amino acid domain that shows homology with a number of thiolases and a C-terminal 143 amino acid domain that is identical to the N-terminal presequence (20 amino acids) and mature sequence (123 amino acids) of SCP-2 (16). The 58-kDa protein, which was called SCP-X, and SCP-2 are transcribed from a single gene that contains two independent promoters (17). SCP-X has not been purified in its active form but the recombinant protein possessed 3-oxoacyl-CoA thiolase activity and displayed a straight chain length specificity that is roughly similar to that of thiolase B (18). In view of its thiolase activity, Seedorf et al. (18) proposed to rename the protein SCP-2/thiolase. Related to SCP-2/thiolase is a peroxisomal 46-kDa protein that crossreacts with antibodies raised against various epitopes of the thiolase domain of SCP-2/thiolase but not with antibodies raised against SCP-2 (19). The data suggest that the 46-kDa protein may originate from SCP-2/thiolase by intraperoxisomal proteolytic cleavage, possibly at the SCP-2 presequence-mature sequence transition (19).
The present study aimed at elucidating the functional role of the different peroxisomal 3-oxoacyl-CoA thiolases catalyzing the last step in ␤-oxidation. We purified the two main thiolases present in peroxisomal fractions from normal rat liver and comparatively investigated their substrate specificity. The enzymes proved to be thiolase A and SCP-2/thiolase. Using various substrates, we could demonstrate that thiolase A cleaves straight chain 3-oxoacyl-CoAs, whereas SCP-2/thiolase is responsible for the cleavage of the 3-oxoacyl-CoAs of 2-methylbranched fatty acids and the bile acid intermediates.
Medium chain (C 6 -C 14 ) 3-oxoacyl-CoAs were synthesized enzymatically from the corresponding acyl-CoAs as described (7) with some modifications. The incubation mixtures contained (final concentrations) 50 mM Tris-Cl, pH 8.0, 1 mM pyruvate, 0.5 mM NAD ϩ , lactate dehydro-genase (1 unit/ml), acyl-CoA oxidase (0.02 units/ml), crotonase (0.5 units/ml), 3-hydroxyacyl-CoA dehydrogenase (0.5 units/ml), 0.01% (w/v) bovine serum albumin, and 60 M acyl-CoAs. After conversion of more than 70% of the acyl-CoAs to the corresponding oxocompounds (monitored at 303 nm by registration of the Mg 2ϩ -enolate complex formation, see below), the incubation media were adjusted to pH 5.5 by 1 M KH 2 PO 4 and the substrates and products were separated from the synthesizing enzymes by ultrafiltration over Amicon Centriprep membranes. Partial purification of the oxocompounds was achieved by using C 18 cartridges eluted with methanol (recovery 70 -80%). After evaporation of the methanol under a stream of nitrogen, the residues were dissolved in 50 mM Tris-Cl, pH 8.0. The same conditions were used for the synthesis of 3-oxohexadecanedioyl-CoA from 25 M hexadecanedioyl-CoA. The yield of the oxocompound was in the range of 25-30%. 3-Oxopalmitoyl-CoA was usually prepared by the same method from 25 M palmitoyl-CoA (yield 25%). In some experiments 3-oxopalmitoyl-CoA was made from 25 M racemic 3-hydroxypalmitoyl-CoA in the presence of 0.01% (w/v) albumin, the NAD ϩ -regenerating system, and 0.5 units/ml 3-hydroxyacyl-CoA dehydrogenase (yield 25-30%). Similar conditions were used for producing 3-oxo-2-methylpalmitoyl-CoA from 25 M racemic 3-hydroxy-2-methylpalmitoyl-CoA (yield 22-25%). For the synthesis of 24-oxo-THC-CoA from 25 M racemic varanoyl-CoA (yield 17-20%), the incubation medium contained 50 mM Tris-Cl, pH 8.0, 50 mM KCl, 0.01% (w/v) albumin, 0.5 mM oxaloacetate, 0.2 units/ml purified MFP-1, and 0.5 units/ml malate dehydrogenase. The use of malate dehydrogenase instead of lactate dehydrogenase for the regeneration of NAD ϩ is mandatory in this case because lactate dehydrogenase is able to reduce NAD ϩ in the presence of varanoyl-CoA reacting with one of the hydroxyl groups of the steroid nucleus. 2 The low yields of 3-oxopalmitoyl-CoA, 3-oxo-2-methylpalmitoyl-CoA, and 24oxo-THC-CoA after synthesis (monitored by the hydroxylamine/DTNB test, see below) prompted us to use an additional step for the purification of these compounds. After elution from the C 18 cartridges and evaporation of the methanol as described above, the CoA derivatives were dissolved in 50 mM potassium phosphate, pH 5.5 (buffer A), and injected onto an Econosphere C 18 column (150 ϫ 4.6 mm, 80 Å, 5 m, Alltech) attached to a Waters gradient HPLC system. The oxocompounds were eluted with a linear gradient of acetonitrile in buffer A. The adenine content of the CoA derivatives was determined at 259 nm (⑀, 16,400 M Ϫ1 cm Ϫ1 ) (27). Since 3-oxo-2-methyl-branched acyl-CoAs do not form Mg 2ϩ -enolate complexes at slightly alkaline pH values (Ref. 28, see also "Results"), concentrations of 3-oxo-2-methylpalmitoyl-CoA and 24-oxo-THC-CoA were measured by using the ability of hydroxylamine (25 mM) to cleave specifically the thioester linkage of 3-oxothioesters at mild conditions (28). The release of CoA was detected with DTNB (⑀, 14,100 M Ϫ1 cm Ϫ1 ). Straight chain 3-oxocompounds were assayed with the same test or in the presence of NADH (0.2 mM) and 3-hydroxyacyl-CoA dehydrogenase (0.5 units/ml).
Purification and Subfractionation of Peroxisomes-Male Wistar rats (body weight, approximately 200 g) maintained on a standard laboratory diet were used. Their livers were homogenized in 0.25 M sucrose, containing 10 mM MOPS, pH 7.4, 0.1% (v/v) ethanol, 1 mM EDTA, 1 mM EGTA, 2.5 mM benzamidine, 2 mM dithiothreitol, 0.5 mM Pefabloc, and 1 g/ml leupeptin. A light mitochondrial fraction, most enriched in lysosomes and peroxisomes, was isolated by differential centrifugation as described previously (29). This fraction was then further purified by isopycnic centrifugation in an iso-osmotic self-generating Percoll gradient (30). The purified peroxisomes were collected from the gradient, diluted in 20 mM Tris-Cl buffer, pH 8.0, 1 mM EDTA, and 2 mM dithiothreitol and sonicated in a pulsed regime (4 ϫ 15 s, output 4, 50% duty cycle; Branson sonifier B15) to release the matrix proteins from the particles. The cores and membrane fragments were sedimented by centrifugation at 100,000 ϫ g for 40 min, and the sediment was dissolved in the same buffer and subjected once again to sonication followed by centrifugation as described above. The supernatants of the two centrifugations were combined, concentrated, and used for further enzyme purification. This enzyme preparation contained 60 -80% of the total thiolase activities, found with the different substrates in the purified whole peroxisomes.
Enzyme Purification-The two main thiolase activities present in the concentrate of peroxisomal matrix proteins were purified to near homogeneity by four successive chromatographic steps on different matrices. First, the enzyme preparation was mixed with CoA (10 M, final concentration) and applied to a phosphocellulose column eluted with a linear gradient of KCl. Peroxisomal thiolases were not retained under these conditions, whereas traces of contaminating mitochondrial thiolases (3-oxoacyl-CoA thiolase and acetoacetyl-CoA thiolase) were bound to the column. 3 Separation of peroxisomal thiolases was achieved by DEAE-Sepharose chromatography. Thiolase A was eluted with the flow-through fractions, which were intensively dialyzed for the removal of CoA before reloading onto a phosphocellulose column. In the absence of CoA thiolase A bound to the column. The enzyme was eluted with a KCl gradient and passed through a Sephacryl-S-100-HR column for final purification. SCP-2/thiolase bound to the DEAE-Sepharose column and was eluted with increasing KCl concentrations. The enzyme preparation was than subjected to hydroxylapatite chromatography with KCl as the eluent. In the final purification step SCP-2/thiolase was passed through a Blue-Sepharose column, and the bound activity was eluted with a linear gradient of CoA (0 -100 M).
Enzyme purification was monitored by measuring the thiolase activities (acetoacetyl-CoA and 3-oxooctanoyl-CoA as the substrates) and by analyzing the eluates by SDS-PAGE followed by immunoblotting with antibodies raised against SCP-2 or the thiolase domain of SCP-2/thiolase (kindly provided by Prof. K. Wirtz). Final identification of the thiolases was performed by peptide sequencing of the purified enzymes. The complete purification procedure for peroxisomal thiolases, subunit structure of SCP-2/thiolase, and the effect of CoA at low concentrations on the chromatographic behavior of the thiolases will be discussed in full detail in a separate publication. 4 MFP-1 and MFP-2 were purified from rat liver peroxisomes as described previously (24).
Enzyme Assays-Two different methods were used for the detection of thiolase activity in the direction of 3-oxoacyl-CoA cleavage. First, the enzymes were assayed by measuring the increase in absorption at 233 nm due to the formation of new thioester bonds (⑀, 4,500 M Ϫ1 cm Ϫ1 ) (9). The reaction mixture (25°C) routinely consisted of 50 mM Tris-Cl, pH 8.0, 60 M CoA, and 10 -60 M oxocompound. Second, thiolase activity was also determined in terms of Mg 2ϩ -enolate complex disappearance (303 nm) as a result of the thiolytic cleavage (4, 7). The assay mixture (25°C) contained 50 mM Tris-Cl, pH 8.0, 5 mM MgCl 2 , albumin, CoA, and 3-oxoacyl-CoAs as indicated. The extinction coefficients for the Mg 2ϩ -enolate complexes were determined by measuring the increase in absorbance at 303 nm after addition of acyl-CoA oxidase to the incubation mixture for the synthesis of the oxocompounds (see above) and by measuring the concentration of the 3-oxoacyl-CoAs in the same samples by means of the hydroxylamine/DTNB test. Since measurements of the Mg 2ϩ -enolate complex disappearance resulted in an underestimation of the thiolytic cleavage rates when short and medium chain substrates were used 5 and since 2-methyl-branched oxocompounds such as 3-oxo-2-methylpalmitoyl-CoA and 24-oxo-THC-CoA do not form Mg 2ϩ -enolate complexes (28), the Mg 2ϩ -enolate method was used in only a limited number of experiments (see Fig. 7) with 3-oxopalmitoyl-CoA (⑀, 8,900 M Ϫ1 cm Ϫ1 ) and 3-oxohexadecanedioyl-CoA (⑀, 10,600 M Ϫ1 cm Ϫ1 ). 3-Hydroxyacyl-CoA dehydrogenase (24), crotonase (24), and acyl-CoA oxidase (30) activities were assayed as described.
Units of enzyme activity are expressed as mol of substrate utilized or product formed per min.
Analysis of Acyl-CoA Thioesters by HPLC-Products of the thiolytic cleavage of 3-oxo-2-methylpalmitoyl-CoA and 24-oxo-THC-CoA were analyzed at 25°C on a Waters dual-pump gradient system by using an Econosphere C 18 reverse-phase column (150 ϫ 4.6 mm). Separation of the acyl-CoA thioesters was achieved by linearly increasing the acetonitrile content of the 50 mM potassium phosphate, pH 5.5, elution buffer (see legends to figures for more details).
Preparation of Antibodies-Antibodies against thiolase A were prepared as follows: enzyme samples were subjected to preparative SDS-PAGE, and after transient visualization of the proteins (31), the 41-kDa polypeptide was excised and injected into rabbits according to standard procedures (32).
Amino Acid Sequencing-For N-terminal sequencing, purified thiolase A was dialyzed against 20 mM potassium phosphate, pH 7.8, and concentrated. The protein sample was then collected on a ProSorb membrane (Perkin-Elmer) and subjected to automated Edman degradation by using a Procise 492 sequenator (Applied Biosystems). Diffi-culties were encountered for the N-terminal sequencing of the 58-kDa SCP-2/thiolase and its related 46-kDa polypeptide due to an irregular transfer of both proteins to the ProBlot membrane (Applied Biosystems) during wet or semidry electroblotting after SDS-PAGE. Therefore, internal sequencing of these proteins was carried out following SDS-PAGE and in gel digestion of the bands of interest. The procedures for the in gel digestion were essentially based on those described by Rosenfeld et al. (33).
Other Methods-Electrophoresis in 10 -20% (w/v) acrylamide gradient gels, silver staining of the gels, and immunoblotting were performed as described previously (24). Protein was determined according to Peterson (34) or Bradford (35) with bovine serum albumin as standard.

RESULTS
Purification of Thiolase A and SCP-2/Thiolase-The two main thiolase activities present in an isolated peroxisomal fraction from normal rat liver were purified to near homogeneity. The first enzyme preparation showed a substrate spectrum similar to that of thiolase B (see below) and displayed a single band of 41 kDa on SDS-PAGE (Fig. 1, panel A). N-terminal sequencing of the enzyme yielded the following amino acid sequence: Phe-Pro-Gln-Ala-Ser-Ala-Ser-Asp-Val-Val-Val-Val-His-Gly-Arg-Arg-Thr-Pro-. This sequence starts at amino acid 4 of the predicted mature sequence of thiolase A and like thiolase A contains Arg in position 15 (position 18 of the predicted thiolase A sequence) instead of Gln, which is present at that position in the thiolase B sequence (see Refs. 13 and 15). These results demonstrate that the purified enzyme is indeed thiolase A and not thiolase B, the inducible enzyme that was purified from livers of rats treated with the peroxisome proliferator di-(2-ethylhexyl)phthalate (see Introduction). Our data also confirm earlier predictions from mRNA analyses that in normal rat liver thiolase A and not thiolase B is the prevailing enzyme (14,15). As far as we are aware, this is the first purification of thiolase A. Why our purified enzyme lacks the first three amino acids of the predicted sequence of mature thiolase A is not clear. Two possibilities can be envisaged: 1) during maturation thiolase A is not cleaved at the same site as thiolase B but three amino acids downstream, or 2) the mature thiolase A loses its first three amino acids by proteolysis during purification. The purified thiolase A preparation was also used for the production of polyclonal antibodies (see Fig. 1, panel B).
The second enzyme preparation contained two bands on SDS-PAGE, a 58-and a 46-kDa band (Fig. 1, panel A). Both bands cross-reacted with antibodies raised against the thiolase domain of SCP-2/thiolase (Fig. 1, panel B). Only the 58-kDa polypeptide reacted with antibodies raised against SCP-2 (data not shown). The sequences of two tryptic peptides of the 58-kDa protein (Phe-Met-Lys-Pro-Gly-Gly-Glu-Asn-Ser-Arg and Ile-Ala-Gly-Asn-Met-Gly-Leu-Ala-Met-Lys) could be located within the N-terminal thiolase part (amino acids 24 -33) and C-terminal SCP-2 part (amino acids 525-534) of SCP-2/thiolase, respectively. Peptide sequencing of the 46-kDa protein yielded the sequence Gly-His-Pro-Leu-Gly-Ala-Thr-Gly-Leu-Ala, which starts at Gly-354 of the thiolase domain of SCP-2/ thiolase. These data confirm that the 58-kDa polypeptide is SCP-2/thiolase and that the 46-kDa polypeptide is its N-terminal thiolase part. Molecular sieving experiments indicated that the enzyme may be present in the cell as a mixture of three dimeric isoforms consisting of homo-and heterodimeric combinations of the 58-and 46-kDa subunits. 4 The 58-kDa polypeptide has been purified from rat liver also by others but under denaturing conditions (16). The recombinant 58-kDa protein expressed in Escherichia coli possessed thiolase activity with straight chain 3-oxoacyl-CoAs (18). Like the recombinant protein, our purified enzyme was fully active as a thiolase (see below).
Thiolase Activities with Straight Chain 3-Oxoacyl-CoAs-The short chain acetoacetyl-CoA was a good substrate for the purified thiolase A (see Table I) but a poor substrate for the purified SCP-2/thiolase (1.3 mol/min⅐mg protein; n ϭ 3). Further measurements with substrates of increasing chain length showed that the two thiolases display optimum activity with medium chain 3-oxoacyl-CoAs (C 10 for thiolase A; C 8 for SCP-2/thiolase; Fig. 2). At chain lengths of C 12 and longer, substrate inhibition occurred at elevated 3-oxoacyl-CoA concentrations with both enzymes. The substrate inhibition, which was particularly marked with 3-oxopalmitoyl-CoA, could be abolished by the addition of albumin. The more polar 3-oxohexadecanedioyl-CoA did not induce substrate inhibition of either of the thiolases (data not shown).
Thiolase Activity with 2-Methyl-branched 3-Oxoacyl-CoA-In rat peroxisomes straight chain acyl-CoAs are oxidized via palmitoyl-CoA oxidase and MFP-1, which displays enoyl-CoA hydratase and L-3(3S)-hydroxyacyl-CoA dehydrogenase activities (23,24), whereas 2-methyl-branched acyl-CoAs are oxidized via pristanoyl-CoA oxidase (23) and MFP-2, which displays enoyl-CoA hydratase and D-3(3R)-hydroxyacyl-CoA dehydrogenases activities (26). One of the aims of this study was to investigate whether straight chain acyl-CoAs and 2methyl-branched acyl-CoAs would be metabolized also by separate thiolases. The main obstacle for revealing the thiolase involved in branched chain fatty acid breakdown is the chemical synthesis of the appropriate substrates. However, we found that commercially available mitochondrial 3-hydroxyacyl-CoA dehydrogenase is capable of catalyzing the synthesis of 3-oxo-2-methylpalmitoyl-CoA from racemic 3-hydroxy-2-methylpalmitoyl-CoA. During incubation of the 3-hydroxyacyl-CoA with the enzyme, NAD ϩ reduction went hand in hand with the appearance of a new CoA derivative as revealed by reverse phase HPLC analysis (Fig. 3, panel A). The compound was rapidly cleaved by low concentrations (25 mM) of neutral hydroxylamine, whereas other CoA derivatives were unaffected

FIG. 2. Chain length specificities of thiolase A (panel A) and SCP-2/thiolase (panel B) with medium straight chain 3-oxoacyl-CoAs.
Enzyme activity was measured at 233 nm with 0.02% (w/v) albumin (q) or without (E). Concentrations of 3-oxoacyl-CoAs and CoA were fixed at 15 and 60 M, respectively. In choosing the 3-oxoacyl-CoA concentrations, a compromise was made between the saturation of the enzyme by the substrates and the inhibiting effect of C 12 -C 14 oxocompounds at higher concentrations. by these conditions. The presumed dehydrogenation product possessed still other characteristic features of a 3-oxo-2-methylacyl-CoA (28) such as the inability, contrary to straight chain 3-oxoacyl-CoAs, to form a Mg 2ϩ -enolate complex at pH Ͼ8.0 (data not shown) and an absorbance maximum at 315 nm at pH 12 (Fig. 4). Finally, after purification by HPLC, the new compound was completely re-converted into two peaks with the same retention time as the stereoisomers of 3-hydroxy-2-methylpalmitoyl-CoA, when incubated with 3-hydroxyacyl-CoA dehydrogenase and NADH (data not shown). The action of thiolase A and SCP-2/thiolase on the 3-oxo-2-methylpalmitoyl-CoA was then analyzed. As shown in Fig. 3, panel A, thiolase A in the presence of CoA did not react with the substrate. On the other hand, the oxocompound completely disappeared when SCP-2/thiolase and CoA were added. Also one of the cleavage products, myristoyl-CoA, could be identified in this experiment. In another experiment, carried out with purified 3-oxo-2-methylpalmitoyl-CoA and a modified gradient of acetonitrile, we could also identify the second cleavage product, propionyl-CoA (Fig. 3, panel B).
As was the case for 3-oxopalmitoyl-CoA (see above), elevated concentrations of 3-oxo-2-methylpalmitoyl-CoA caused a strong substrate inhibition of SCP-2/thiolase, which could be prevented by the addition of albumin (Fig. 5).
Thiolase Activity with 24-Oxo-trihydroxycoprostanoyl-CoA-In rat liver peroxisomes the bile acid intermediate trihydroxycoprostanoyl-CoA is metabolized by, successively, trihydroxycoprostanoyl-CoA oxidase and MFP-2 (23)(24)(25). Like for the branched fatty acids, we also wanted to investigate which of the peroxisomal thiolases is responsible for the thiolytic cleavage of the bile acid intermediates. For the enzymatic synthesis of 24-oxo-THC-CoA from racemic varanoyl-CoA (the 3-hydroxyacyl-CoA derivative of trihydroxycoprostanic acid), we used the peroxisomal MFP-1 or MFP-2, purified from normal rat liver. Upon incubation of varanoyl-CoA with MFP-1 in the presence of the NAD ϩ -regenerating system but without NAD ϩ , followed by reverse phase HPLC, the appearance of a new UV-absorbing compound was evident (Fig. 6, panel A), corre-sponding to 24-trans-trihydroxycoprostenoyl-CoA. 6 The appearance of the new compound went hand in hand with a decrease in one of the varanoyl-CoA stereoisomers. Addition of NAD ϩ resulted in the appearance of a broad absorbance peak that partially coincided with the peaks of the varanoyl-CoA stereoisomers. After further purification of the newly formed compound by HPLC, it was identified as 24-oxo-THC-CoA on the basis of its sensitivity to hydroxylamine (see also Fig. 6, panel A) and the other lines of evidence given above for 3-oxo-2-methylpalmitoyl-CoA (data not shown). Addition of thiolase A and CoA to the above mentioned reaction mixtures containing the newly synthesized oxocompound did not result in any changes in the HPLC pattern (Fig. 6, panel A). However, addi- tion of SCP-2/thiolase and CoA caused a dramatic decrease in the peak height of the oxocompound together with an increase in a more polar CoA ester, the mobility of which coincided with that of one of the stereoisomers of varanoyl-CoA as well as that of choloyl-CoA, one of the cleavage products (Fig. 6, panel A). These data were confirmed with partially purified 24-oxo-THC-CoA (Fig. 6, panel B). Incubation of the oxocompound with SCP-2/thiolase and CoA resulted in two new peaks, whose retention time corresponded to that of the cleavage products propionyl-CoA and choloyl-CoA.
The substrate curve for SCP-2/thiolase with 24-oxo-THC-CoA revealed a weak inhibition at elevated concentrations, which could be prevented by the addition of albumin (data not shown). 7 Substrate Specificity of Thiolase A and SCP-2/Thiolase- Table I summarizes the kinetic constants obtained for the purified enzymes with the different substrates. As shown in the table both enzymes are optimally active with medium chain 3-oxocompounds. Interestingly, 3-oxohexadecanedioyl-CoA is a better substrate for both thiolases than its monocarboxylic counterpart 3-oxopalmitoyl-CoA. Acetoacetyl-CoA is a good substrate for thiolase A but only a poor substrate for SCP-2/ thiolase. More importantly, the 2-methyl-branched compounds 3-oxo-2-methylpalmitoyl-CoA and 24-oxo-THC-CoA are good substrates for SCP-2/thiolase but no substrate for thiolase A, implying that SCP-2/thiolase is responsible for the thiolytic cleavage of 2-methyl-branched fatty acids and the side chain of the bile acid intermediates di-and trihydroxycoprostanic acids. The role of thiolase A and SCP-2/thiolase in the cleavage of straight chain mono-and dicarboxylates will be addressed in the next paragraph.
Thiolase A displayed a pH optimum of 8.0 when acetoacetyl-CoA and 3-oxooctanoyl-CoA were used as the substrates. The pH optimum for SCP-2/thiolase was near 7.6 with 3-oxooctanoyl-CoA and 3-oxo-2-methylpalmitoyl-CoA. The pH profile with 24oxo-THC-CoA was complex, showing a shoulder at pH 7.2-8.2 but increasing further at higher pH.

Contribution of Thiolase A and SCP-2/Thiolase to the Peroxisomal Cleavage of Straight Chain 3-Oxoacyl-CoAs-Since
both thiolase A and SCP-2/thiolase catalyze the cleavage of straight chain 3-oxoacyl-CoAs, we wanted to determine the contribution of each enzyme to the overall reaction. Therefore, peroxisomal matrix proteins released by sonication of a purified peroxisomal fraction were passed through a phosphocellu- 7 The experiments described in the text were carried out with 24-oxo- lose column that binds traces of contaminating mitochondrial thiolases, 3 and the flow-through fraction containing the peroxisomal thiolases was separated on a DEAE-Sepharose column. As illustrated by immunoblot analysis, thiolase A was eluted first with the flow-through volume, whereas SCP-2/thiolase, consisting of the 58-and 46-kDa polypeptides, was eluted with increasing KCl concentrations (Fig. 7, upper panel). As could be expected, thiolytic activity with 3-oxo-2-methylpalmitoyl-CoA was recovered exclusively in the fractions containing SCP-2/ thiolase, whereas the activity with acetoacetyl-CoA was confined to the fractions containing thiolase A (Fig. 7, lower panel). Thiolytic activity with 3-oxooctanoyl-CoA, 3-oxopalmitoyl-CoA, and 3-oxohexadecanedioyl-CoA displayed a bimodal distribution. By far the major (Ͼ90%) portion of the activity was eluted with the thiolase A peak, whereas the small remaining part was eluted with the SCP-2/thiolase peak. Since recoveries versus the original whole peroxisome preparation were essentially the same for both enzymes as illustrated by the comparable recoveries obtained with 3-oxooctanoyl-CoA (40%) and 3-oxo-2methylpalmitoyl-CoA (44%) and since the two enzymes display similar K m values for the different substrates (Table I), one can conclude that the activity profile of the DEAE column reflects the situation in the intact liver cell and that thiolase A is responsible for the bulk of the thiolytic cleavage of straight chain 3-oxoacyl-CoAs. DISCUSSION Our present results indicate that in normal rat liver, thiolase A is responsible for the peroxisomal thiolytic cleavage of straight chain mono-and dicarboxylic 3-oxoacyl-CoAs, whereas SCP-2/thiolase cleaves the 3-oxoacyl-CoAs of 2-methylbranched fatty acids (the synthetic 2-methylpalmitic acid and, as a consequence, most probably also the naturally occurring pristanic acid) and of the bile acid intermediates di-and trihydroxycoprostanic acids, which also possess a 2-methyl branch in their side chain. These data complete our earlier results obtained for the preceding steps of peroxisomal ␤-oxidation (23-26, 36, 37) and allow for an overall description of the ␤-oxidation pathways involved in the degradation of straight chain fatty acids, 2-methyl-branched fatty acids, and the side chain of cholesterol. This overall picture is summarized in Fig.  8.
MFP-2 and SCP-2/thiolase metabolize not only the branched chain fatty acids and the bile acid intermediates but also straight chain fatty acids, which because of their relative excess in concentration might seriously hinder the efficient breakdown of the branched carboxylates. One might speculate, therefore, that the ␤-oxidation pathways described above are catalyzed by multienzyme complexes, consisting of an association of the appropriate oxidase, MFP and thiolase, and in which the intermediates are channeled from one enzyme to the other without being released into the surrounding matrix. If this picture holds, the substrate specificity of the complex would be determined by the substrate specificity of the oxidase, and the straight chain fatty acid intermediates would be prevented from having access to MFP-2 or SCP-2/thiolase.
Our results also confirm the earlier contention (from mRNA analysis, see Refs. 14 and 15) that in normal rat liver thiolase A is the prevailing enzyme. A very recent comparison of the substrate spectrum of thiolase A with that of the closely related inducible thiolase B, which is the prevailing enzyme in livers from rats treated with peroxisome proliferators, revealed that there are no essential differences. 4 Finally, a portion of SCP-2/thiolase appears to be present as cleavage products: the 46-kDa N-terminal thiolase domain and, presumably, the C-terminal domain that is identical to SCP-2. Interestingly, two other peroxisomal ␤-oxidation enzymes also occur in a partially cleaved form. Palmitoyl-CoA oxidase con-sists of a mixture of subunits (A 2 , ABC, B 2 C 2 ), in which the B (52 kDa) and C (20 kDa) subunits originate by intraperoxisomal proteolytic cleavage of the A (72 kDa) subunit (39,40). Likewise, a portion of MFP-2 (79 kDa) is cleaved in the peroxisome in an N-terminal 34-kDa polypeptide that comprises the D-3-hydroxyacyl-CoA dehydrogenase (and 17-␤-hydroxysteroid dehydrogenase) domain and a C-terminal 45-kDa polypeptide that consists in its N-terminal part of the 2-enoyl-CoA hydratase domain and in its C-terminal part of a domain that displays similarity with SCP-2 (41,42). It is presently unknown whether the parent polypeptides and their cleavage products display differences in substrate specificity. Separation of the cleavage products from the parent molecules or separate expression of the cleavage products and parent molecules will be required to obtain such knowledge. SCP-2 is known to bind sterols (reviewed in Ref. 43) and acyl-CoAs (44). This raises the question as to whether the C-terminal SCP-2 domain of SCP-2/thiolase and the C-terminal domain of MFP-2, which is similar to SCP-2, are involved in the binding of certain substrates or in intermediate channeling from one enzyme to the other.