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A novel 3-oxoacyl-CoA thiolase was found in rat liver. This thiolase, mitochondrial general 3-oxoacyl-CoA thiolase and acetoacetyl-CoA thiolase were purified from the rat liver after the induction of these activities by the administration to rats of di(2-ethylhexyl)phthalate, which enhanced the peroxisomal P-oxidation activity. The new 3-oxoacyl-CoA thiolase was distinguished from mitochondrial and cytoplasmic thiolases by the following : DEAE-cellulose chromatography, phosphocellulose chromatography, immunochemical titration, and substrate specificity. Subcellular fractionation of liver and sucrose-density-gradient centrifugation of the light mitochondrial fraction revealed that the new thiolase was mainly in peroxisomes.Mammalian liver has three types of thiolases which differ in the intracellular localization [I 1. Cytoplasmic acetoacetyl-CoA thiolase plays a role in steroid biosynthesis. Mitochondria have two different thiolases. One of them is specific for acetoacetyl-CoA and is supposed to have a role for ketone body metabolism. The other, a general 3-oxoacyl-CoA thiolase, is involved in P-oxidation of fatty acid. Recently, it has been shown that rat liver peroxisomes have a new P-oxidation system and the activity of this system is markedly increased when a rat is fed a diet containing various hypolipidemic agents [2,3] or di(2-ethylhexy1)-phthalate [4]. Hepatic thiolase activities increased in the di(2-ethy1hexyl)phthalate-treated rats [5]. The increments of the activities for substrates with longer carbon-chain length were greater than that for acetoacetyl-CoA. The subcellular fractionation study suggests that peroxisomes have an unique thiolase different from cytoplasmic and mitochondrial thiolases [5].The present paper describes the purification of a new 3-oxoacyl-CoA thiolase and its location in peroxisomes. MATERIALS AND METHODS MaterialsAcetoacetyl-CoA was prepared by the reaction of diketene with CoA [6]. 2-Octenoyl-CoA was syn-

Postsynaptic membrane rafts are believed to play important roles in synaptic signaling, plasticity, and maintenance. However, their molecular identities remain elusive. Further, how they interact with the well-established signaling specialization, the postsynaptic density (PSD), is poorly understood. We previously detected a number of conventional PSD proteins in detergent-resistant membranes (DRMs). Here, we have performed LC-MS/MS (liquid chromatography coupled with tandem mass spectrometry) analyses on postsynaptic membrane rafts and PSDs. Our comparative analysis identified an extensive overlap of protein components in the two structures. This overlapping could be explained, at least partly, by a physical association of the two structures. Meanwhile, a significant number of proteins displayed biased distributions to either rafts or PSDs, suggesting distinct roles for the two postsynaptic specializations. Using biochemical and electron microscopic methods, we directly detected membrane raft-PSD complexes. In vitro reconstitution experiments indicated that the formation of raft-PSD complexes was not due to the artificial reconstruction of once-solubilized membrane components and PSD structures, supporting that these complexes occurred in vivo. Taking together, our results provide evidence that postsynaptic membrane rafts and PSDs may be physically associated. Such association could be important in postsynaptic signal integration, synaptic function, and maintenance.

Mitochondrial and peroxisomal enoyl-CoA hydratases were purified from rat liver. The mitochondrial enzyme, with a molecular weight of 161,000, was composed of 6 identical subunits. The molecular structure of the rat liver enzyme was very similar to that of the bovine liver enzyme. Acetoacetyl-CoA was a competitive inhibitor of the mitochondrial enzymes. The results of titration of the rat liver enzyme with acetoacetyl-CoA suggest that 3 subunits of the enzyme exhibit catalytic activity. The catalytic properties of the enzyme were studied. The peroxisomal enzyme was composed of one polypeptide with a molecular weight of 70,000-81,000. Some of the enzyme molecules were shown to be cleaved to two polypeptides in the cell by the following methods: amino acid analysis, peptide mapping and immunoprecipitin reaction. The catalytic properties of the peroxisomal enzyme were different from those of the mitochondrial enzyme. The peroxisomal enzyme is a bifunctional enzyme exhibiting 3-hydroxyacyl-CoA dehydrogenase activity. Studies on the titration with acetoacetyl-CoA, the effects of salts, SH titration and proteolytic inactivation suggest that the active centers for these two reactions are located at different sites.

We have isolated five cDNA clones for rat liver catalase (hydrogen peroxide:hydrogen peroxide oxidoreductase, EC 1.11.1.6). These clones overlapped with each other and covered the entire length of the mRNA, which had been estimated to be 2.4 kilobases long by blot hybridization analysis of electrophoretically fractionated RNA. Nucleotide sequencing was carried out on these five clones and the composite nucleotide sequence of catalase cDNA was determined. The 5' noncoding region contained 83 bases and was followed by 1581 bases of an open reading frame that encoded 527 amino acids. The 3' noncoding region was 831 bases long and contained long repeats of the unit AC. The amino acid sequence deduced from the nucleotide sequence of the cDNAs showed about 90% homology with the reported primary structure of bovine liver catalase. The molecular weight of rat liver catalase was calculated to be 59,758 from the predicted amino acid sequence. The amino acid residues in contact with the heme group are completely identical for bovine liver and rat liver catalases. The amino acid sequence at the COOH terminus was confirmed by the results ofcarboxypeptidase P treatment of the protein purified from rat liver in the presence of leupeptin. Rat liver catalase has no cleavable signal peptide for translocation of the enzyme into peroxisomes.