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1.
Thiolase is part of the fatty acid oxidation machinery which in plants is located within glyoxysomes or peroxisomes. In cucumber cotyledons, proteolytic modification of thiolase takes place during the transfer of the cytosolic precursor into glyoxysomes prior to the intraorganellar assembly of the mature enzyme. This was shown by size comparison of the in vitro synthesized precursor and the 45 kDa subunit of the homodimeric glyoxysomal form. We isolated a full-length cDNA clone encoding the 48 539 Da precursor of thiolase. This plant protein displayed 40% and 47% identity with the precursor of fungal peroxisomal thiolase and human peroxisomal thiolase, respectively. Compared to bacterial thiolases, the precursor of the plant enzyme was distinguished by an N-terminal extension of 34 amino acid residues. This putative targeting sequence of cucumber thiolase shows similarities with the cleavable presequences of rat peroxisomal thiolase and plant peroxisomal malate dehydrogenase. 相似文献
2.
Catalase activity was inhibited by aminotriazole administration to rats in order to evaluate the influence of catalase on the peroxisomal fatty acyl-CoA beta-oxidation system. 2 h after the administration of aminotriazole, peroxisomes were prepared from rat liver, and the activities of catalase, the beta-oxidation system and individual enzymes of beta-oxidation (fatty acyl-CoA oxidase, crotonase, beta-hydroxybutyryl-CoA dehydrogenase and thiolase) were determined. Catalase activity was decreased to about 2% of the control. Among the individual enzymes of the beta-oxidation system, thiolase activity was decreased to 67%, but the activities of fatty acyl-CoA oxidase, crotonase and beta-hydroxybutyryl-CoA dehydrogenase were almost unchanged. The activity of the peroxisomal beta-oxidation system was assayed by measuring palmitoyl-CoA-dependent NADH formation, and the activity of the purified peroxisome preparation was found to be almost unaffected by the administration of aminotriazole. The activity of the system in the aminotriazole-treated preparation was, however, significantly decreased to 55% by addition of 0.1 mM H2O2 to the incubation mixture. Hydrogen peroxide (0.1 mM) reduced the thiolase activity of the aminotriazole-treated peroxisomes to approx. 40%, but did not affect the other activities of the system. Thiolase activity of the control preparation was decreased to 70% by addition of hydrogen peroxide (0.1 mM). The half-life of 0.1 mM H2O2 added to the thiolase assay mixture was 2.8 min in the case of aminotriazole-treated peroxisomes, and 4 s in control peroxisomes. The ultraviolet spectrum of acetoacetyl-CoA (substrate of thiolase) was clearly changed by addition of 0.1 mM H2O2 to the thiolase assay mixture without the enzyme preparation; the absorption bands at around 233 nm (possibly due to the thioester bond of acetoacetyl-CoA) and at around 303 nm (due to formation of the enolate ion) were both significantly decreased. These results suggest that H2O2 accumulated in peroxisomes after aminotriazole treatment may modify both thiolase and its substrate, and consequently suppress the fatty acyl-CoA beta-oxidation. Therefore, catalase may protect thiolase and its substrate, 3-ketoacyl-CoA, by removing H2O2, which is abundantly produced during peroxisomal enzyme reactions. 相似文献
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4.
V D Antonenkov K Croes E Waelkens P P Van Veldhoven G P Mannaerts 《European journal of biochemistry》2000,267(10):2981-2990
Acetoacetyl-CoA specific thiolases catalyse the cleavage of acetoacetyl-CoA into two molecules of acetyl-CoA and the synthesis (reverse reaction) of acetoacetyl-CoA. The formation of acetoacetyl-CoA is the first step in cholesterol and ketone body synthesis. In this report we describe the identification of a novel acetoacetyl-CoA thiolase and its purification from isolated rat liver peroxisomes by column chromatography. The enzyme, which is a homotetramer with a subunit molecular mass of 42 kDa, could be distinguished from the cytosolic and mitochondrial acetoacetyl-CoA thiolases by its chromatographic behaviour, kinetic characteristics and partial internal amino-acid sequences. The enzyme did not catalyse the cleavage of medium or long chain 3-oxoacyl-CoAs. The enzyme cross-reacted with polyclonal antibodies raised against cytosolic acetoacetyl-CoA thiolase. The latter property was exploited to confirm the peroxisomal localization of the novel thiolase in subcellular fractionation experiments. The peroxisomal acetoacetyl-CoA thiolase most probably catalyses the first reaction in peroxisomal cholesterol and dolichol synthesis. In addition, its presence in peroxisomes along with the other enzymes of the ketogenic pathway indicates that the ketogenic potential of peroxisomes needs to be re-evaluated. 相似文献
5.
A novel, cleavable peroxisomal targeting signal at the amino-terminus of the rat 3-ketoacyl-CoA thiolase. 总被引:59,自引:6,他引:53
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B W Swinkels S J Gould A G Bodnar R A Rachubinski S Subramani 《The EMBO journal》1991,10(11):3255-3262
Several peroxisomal proteins do not contain the previously identified tripeptide peroxisomal targeting signal (PTS) at their carboxy-termini. One such protein is the peroxisomal 3-ketoacyl CoA thiolase, of which two types exist in rat [Hijikata et al. (1990) J. Biol. Chem., 265, 4600-4606]. Both rat peroxisomal thiolases are synthesized as larger precursors with an amino-terminal prepiece of either 36 (type A) or 26 (type B) amino acids, that is cleaved upon translocation of the enzyme into the peroxisome. The prepieces are necessary for import of the thiolases into peroxisomes because expression of an altered cDNA encoding only the mature thiolase, which lacks any prepiece, results in synthesis of a cytosolic enzyme. When appended to an otherwise cytosolic passenger protein, the bacterial chloramphenicol acetyltransferase (CAT), the prepieces direct the fusion proteins into peroxisomes, demonstrating that they encode sufficient information to act as peroxisomal targeting signals. Deletion analysis of the thiolase B prepiece shows that the first 11 amino acids are sufficient for peroxisomal targeting. We conclude that we have identified a novel PTS that functions at amino-terminal or internal locations and is distinct from the C-terminal PTS. These results imply the existence of two different routes for targeting proteins into the peroxisomal matrix. 相似文献
6.
S Miura M Takiguchi H Matsue Y Amaya M Tatibana K Shigesada T Osumi T Hashimoto M Mori 《European journal of biochemistry》1986,154(2):479-484
Messenger RNA of rat 3-oxoacyl-CoA thiolase (acetyl-CoA acyltransferase), a mitochondrial matrix enzyme involved in fatty acid beta-oxidation, was enriched by immunoprecipitation of rat liver free polysomes and recombinant plasmids were prepared from the enriched mRNA by a modification of the vector-primer method of Okayama and Berg. The transformants were initially screened for 3-oxoacyl-CoA thiolase cDNA sequences by differential colony hybridization with [32P]cDNAs, synthesized from the immunopurified and unpurified mRNAs. The cDNA clones for 3-oxoacyl-CoA thiolase were identified by hybrid-arrested translation and hybrid-selected translation. One of the clones, designated pT1-1, contained a 700-base insert and hybridized to a mRNA species of 1.6 X 10(3) bases in rat liver. The transformants were rescreened using the cDNA insert of pT1-1 as a hybridization probe and a clone (pT1-19) with a 1.5 X 10(3)-base insert was obtained. Activity and concentration of 3-oxoacyl-CoA thiolase mRNA were quantified by in vitro translation and dot-blot analysis using the cDNA insert as a hybridization probe. The level of translatable and hybridizable mRNA in rat liver was increased about 5.1-fold and 4.6-fold, respectively, after administration of di-(2-ethylhexyl)phthalate, a potent inducer of the enzyme. The 3-oxoacyl-CoA thiolase mRNA levels thus determined correlated closely with levels of the activity and amount of this enzyme. 相似文献
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The nucleotide sequence of complementary DNA and the deduced amino acid sequence of peroxisomal catalase of the yeast Candida tropicalis pK233 总被引:4,自引:0,他引:4
We report the isolation and nucleotide (nt) sequence determination of cDNA encoding peroxisomal catalase (Cat) from the yeast Candida tropicalis pK233. The catalase cDNA (Cat) has a single open reading frame (ORF) of 1455 nt, encoding a protein of 484 amino acids (aa), not including the initiator methionine. The Mr of the protein is 54767. Codon use in the gene is not random, with 90.9% of the aa specified by 25 principal codons. The principal codons used in the expression of Cat in C. tropicalis are similar to those used in the expression of the fatty acyl-CoA oxidase gene of C. tropicalis and of highly expressed genes in Saccharomyces cerevisiae. Cat shows 48.0%, 49.7%, and 48.3% aa identity with human, bovine, and rat catalases, respectively, and 44.3% aa identity with catalase T of S. cerevisiae. The 3 aa of bovine liver catalase previously postulated to participate in catalysis and 79.5% of those aa in the immediate environment of hemin, the prosthetic group of catalase, are conserved in Cat of C. tropicalis. 相似文献
9.
《Archives of biochemistry and biophysics》1981,209(1):100-110
β-Ketothiolase was found in rat liver peroxisomes that were isolated by sucrose density gradient centrifugation. The presence of dithiothreitol was essential for measuring the peroxisomal thiolase, but dithiothreitol had little effect upon the thiolase activity in the mitochondria or cytosol. Dithiothreitol was not used during the isolation procedures. Acetoacetyl CoA and β-ketolauryl CoA, which was synthesized chemically, were used as substrates. The peroxisomal thiolase was active with β-ketolauryl CoA but had almost no activity with acetoacetyl CoA as the substrate. Thiolase activities in the mitochondrial and cytosolic fractions utilized both long- and short-chain substrates. The thiolases in the various fractions bound to and were purified by chromatography on calcium phosphate gel cellulose columns. The peroxisomal thiolase did not bind to phosphocellulose, whereas the mitochondrial and cytosolic activities could be chromatographed on phosphocellulose. Peroxisomal β-ketolauryl CoA thiolase activity was inhibited about 20% by 25 mm Mg2+, and more activity was measured in a phosphate than in a Tris buffer. In control rat livers, the total activity of β-ketolauryl CoA thiolase was 3.4 μmol/min per gram of liver in the peroxisomes and 4.9 μmol/min per gram of liver in the mitochondria, which indicates that peroxisomes contain the capacity for 40% of the total thiolase activity associated with β-oxidation systems. In addition, 6.3 μmol/min per gram of liver of β-ketolauryl CoA thiolase was found in the soluble fraction and chromatographed differently from the peroxisomal enzyme on phosphocellulose. Clofibrate in the rat diet for 6 or 21 days resulted in about a 15-fold increase in peroxisomal thiolase when assayed with β-ketolauryl CoA and somewhat less of an increase in the other fractions. 相似文献
10.
cDNA-derived amino acid sequence of rat mitochondrial 3-oxoacyl-CoA thiolase with no transient presequence: structural relationship with peroxisomal isozyme. 总被引:13,自引:2,他引:11
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The sorting of homologous proteins between two separate intracellular organelles is a major unsolved problem. 3-Oxoacyl-CoA thiolase is localized in mitochondria and peroxisomes, and provides a good system for the study on the problem. Unlike most mitochondrial matrix proteins, mitochondrial 3-oxoacyl-CoA thiolase in rats is synthesized with no transient presequence and possess information for mitochondrial targeting and import in the mature protein. Two overlapping cDNA clones contained an open reading frame encoding a polypeptide of 397 amino acid residues (predicted Mr = 41,868), a 5' untranslated sequence of 164 bp, a 3' untranslated sequence of 264 bp and a poly(A) tract. The amino acid sequence of the mitochondrial thiolase is 37% identical with that of the mature portion of rat peroxisomal 3-oxoacyl-CoA thiolase precursor. These results suggest that the two thiolases have a common origin and obtained information for targeting to respective organelles during evolution. Two portions in the mitochondrial thiolase that may serve as a mitochondrial targeting signal are presented. 相似文献
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S Guerroui P Aubourg W W Chen T Hashimoto J Scotto 《Biochemical and biophysical research communications》1989,161(1):242-251
Immunoblot analysis of peroxisomal beta-oxidation enzymes proteins was carried on liver samples from 15 patients with peroxisomal disorders in which accumulation of very long chain fatty acids was always observed in plasma. In 11 cases including 4 cerebro-hepatorenal syndrome (CHRS), 4 neonatal adrenoleukodystrophy (NALD) and 3 infantile Refsum's disease, the liver peroxisomes could not be detected by electron microscopy. Immunoblot analysis revealed the absence, or presence in weak amounts, of the 72-kDa subunit of acyl-CoA oxidase, and the complete absence of the 52-kDa and 21-kDa subunits which are processed from the 72-kDa. The bifunctional protein (78-kDa) was absent or very reduced, as was the mature form of peroxisomal 3-ketoacyl-CoA thiolase (41-kDa). Multiple defects of peroxisomal beta-oxidation enzymes may be caused by an absence of synthesis or an inability to import proteins into peroxisomes in these patients. One patient, diagnosed as NALD, had no detectable liver peroxisomes but the presence, in normal amounts, of the three peroxisomal beta-oxidation enzyme proteins suggests that the transport of these enzymes into "peroxisomal ghosts" was still intact. The last 3 patients, clinically diagnosed as NALD, had normal liver peroxisomes. One patient had an isolated deficiency of the bifunctional protein and the 2 others had normal amounts of the 3 peroxisomal beta-oxidation enzymes, as shown by immunoblotting. This suggests that import and translocation of some peroxisomal proteins had occurred and that a mechanism is therefore required to explain the defect in these patients. 相似文献
13.
Acyl-CoA synthetase and the peroxisomal enzymes of beta-oxidation in human liver. Quantitative analysis of their subcellular localization. 总被引:4,自引:1,他引:3
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The presence of acyl-CoA synthetase (EC 6.2.1.3) in peroxisomes and the subcellular distribution of beta-oxidation enzymes in human liver were investigated by using a single-step fractionation method of whole liver homogenates in metrizamide continuous density gradients and a novel procedure of computer analysis of results. Peroxisomes were found to contain 16% of the liver palmitoyl-CoA synthetase activity, and 21% and 60% of the enzyme activity was localized in mitochondria and microsomal fractions respectively. Fatty acyl-CoA oxidase was localized exclusively in peroxisomes, confirming previous results. Human liver peroxisomes were found to contribute 13%, 17% and 11% of the liver activities of crotonase, beta-hydroxyacyl-CoA dehydrogenase and thiolase respectively. The absolute activities found in peroxisomes for the enzymes investigated suggest that in human liver fatty acyl-CoA oxidase is the rate-limiting enzyme of the peroxisomal beta-oxidation pathway, when palmitic acid is the substrate. 相似文献
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The peroxisomal localization of 3-ketoacyl-CoA thiolase (hereafter referred to as thiolase) was characterized in five Chinese hamster ovary (CHO) mutant cell lines each harboring a dysfunction in the PEX2 protein. PT54 (Pex2pN100) cells carry a nonsense mutation that results in the PEX2 protein truncated at amino acid position 100. SK24 (Pex2pC258Y) cells carry a missense mutation resulting in the amino acid substitution of a cysteine residue by a tyrosine residue at amino acid position 258 of the PEX2 protein. The WSK24 (Pex2pC258Y/+wild) cell line is a stable transformant of SK24 (Pex2pC258Y) cells transfected with wild-type rat PEX2 cDNA. The SPT54 (Pex2pN100/+Pex2pC258Y) and WPT54 (Pex2pN100/+wild) cell lines are stable transformants of PT54 (Pex2pN100) cells transfected with the mutant PEX2 cDNA from SK24 (Pex2pC258Y) cells and wild-type rat PEX2 cDNA, respectively. In these cell lines, except PT54 (Pex2pN100), thiolase appeared to be localized in peroxisomes, as it is in the wild-type cells. When the molecular size of the enzyme was examined on SDS-polyacrylamide gel electrophoresis, the peroxisome-localized enzyme exhibited a larger precursor form in these mutant cells. The characterizations with salt wash, sodium carbonate extraction and proteinase K digestion indicated that the precursor forms of the enzyme were accumulated at different states in peroxisomes of these mutant cells. The dispositions on the peroxisomal membrane were further sustained by differential permeabilization using digitonin, followed by immunocytochemical fluorescence. These results suggest that PEX2 protein functions differently on two processes of the maturation and the disposition in the import pathway of thiolase. 相似文献
16.
At least three different subcellular compartments, including peroxisomes, are involved in cholesterol synthesis. The peroxisomal targeting signals for phosphomevalonate kinase and isopentenyl diphosphate isomerase have been identified. In the current study we identify the peroxisomal targeting signals required for four other enzymes of the cholesterol biosynthetic pathway: acetoacetyl-CoA (AA-CoA) thiolase, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase, mevalonate diphosphate decarboxylase (MPPD), and farnesyl diphosphate (FPP) synthase. Data are presented that demonstrate that mitochondrial AA-CoA thiolase contains both a mitochondrial targeting signal at the amino terminus and a peroxisomal targeting signal (PTS-1) at the carboxy terminus. We also analyze a new variation of PTS-2 sequences required to target HMG-CoA synthase and MPPD to peroxisomes. In addition, we show that FPP synthase import into peroxisomes is dependent on the PTS-2 receptor and identify at the amino terminus of the protein a 20-amino acid region that is required for the peroxisomal localization of the enzyme.These data provide further support for the conclusion that peroxisomes play a critical role in cholesterol biosynthesis. 相似文献
17.
Quantitative immunoelectron microscopy in conjunction with quantitative analysis of immunoblots have been used to study the effects of bezafibrate (BF), a peroxisome-proliferating hypolipidemic drug, upon six different enzyme proteins in rat liver peroxisomes (Po). Antibodies against following peroxisomal enzymes: catalase, urate oxidase, alpha-hydroxy acid oxidase, acyl-CoA oxidase, bifunctional enzyme (hydratase-dehydrogenase) and thiolase, were raised in rabbits, and their monospecificities were confirmed by immunoblotting. Female Sprague-Dawley rats were treated for 7 days with 250 mg/kg/day bezafibrate and liver sections were incubated with the appropriate antibodies followed by the protein A-gold complex. The labeling density for each enzyme was estimated by automatic image analysis. In parallel experiments immunoblots prepared from highly purified peroxisome fractions of normal and BF-treated rats were incubated with the same antibodies. The antigens were visualized by an improved protein A-gold method including an anti-protein A step and silver amplification. The immunoblots were also quantitated by an image analyzer. The results revealed a selective induction of beta-oxidation enzymes by bezafibrate with thiolase showing the most increase followed by bifunctional protein and acyl-CoA oxidase. The labeling density for catalase and alpha-hydroxy acid oxidase was reduced, confirming fully the quantitative analysis of immunoblots which in addition revealed reduction of uricase. These observations demonstrate that hypolipidemic drugs induce selectively the beta-oxidation enzymes while other peroxisomal enzymes are reduced. The quantitative immunoelectron microscopy with automatic image analysis provides a versatile, highly sensitive and efficient method for rapid detection of modulations of individual proteins in peroxisomes. 相似文献
18.
An impaired peroxisomal targeting sequence leading to an unusual bicompartmental distribution of cytosolic epoxide hydrolase. 总被引:3,自引:0,他引:3
To gain an understanding of the mechanism by which the subcellular distribution of cytosolic epoxide hydrolase (cEH) is directed, we have analyzed the carboxy terminal region of rat liver cEH by means of cDNA cloning to define the structure of its possible peroxisomal targeting sequence (PTS). Purified cEH was subjected to peptide analysis following endoproteinase Glu-C digestion and HPLC-separation of the fragments. The obtained sequence information was used to perform PCR experiments resulting in the isolation of a 680 bp cDNA clone encoding the carboxy terminus of cEH. The deduced amino acid sequence displays a terminal tripeptide Ser-Lys-Ile which is highly homologous to the PTS (Ser-Lys-Leu) found in other peroxisomal enzymes. This slight difference appears to be sufficient to convert the signal sequence into an impaired and therefore ambivalent PTS, directing the enzyme partly to the peroxisomes and allowing part to reside in the cytosol. 相似文献
19.
Cloning and sequence of a cDNA encoding human carbamyl phosphate synthetase I: molecular analysis of hyperammonemia. 总被引:1,自引:0,他引:1
Carbamyl phosphate synthetase I (CPSI) is the first enzyme involved in urea synthesis. CPSI deficiency is an autosomal recessive disorder characterized by hyperammonemic coma in the neonatal period. To analyze the enzyme and gene structures, and to elucidate the nature of mutations in CPSI deficiency, we isolated cDNA clones encoding human liver CPSI. Oligo(dT)-primed and random primer human liver cDNA libraries in lambda gt11 were screened using 5', middle, and 3' fragments of the rat CPSI cDNA as probes. Seven positive clones covered the full-length cDNA sequence with an open reading frame encoding a precursor polypeptide of 1500 amino acids (aa) (deduced Mr, 164,828) with a putative N-terminal presequence of 38 or 39 aa, a 5'-untranslated sequence of 118 bp and a 3'-untranslated sequence of 597 bp. Comparison with the rat CPSI cDNA showed that the deduced aa sequence of the human liver CPSI precursor is 94.4% identical to the rat enzyme precursor. A molecular analysis was made of the genomic DNA from three patients with CPSI deficiency. Heterogeneity of hybridized fragments that may or may not be the cause of the deficiency was apparent on the DNA blots from tissues from one patient. 相似文献
20.
Primary structure of rat secretory acid phosphatase and comparison to other acid phosphatases 总被引:5,自引:0,他引:5
Overlapping cDNA clones encoding rat prostatic acid phosphatase (rPAP) were isolated by using two human prostatic acid phosphatase (hPAP)-encoding cDNAs to screen rat prostatic cDNA libraries. The isolated cDNAs encompassed a total of 1626 nucleotides (nt), of which 1143 nt corresponded to the protein coding sequence encoding a mature polypeptide of 350 amino acids (aa) and a 31-aa long signal peptide-like sequence. The deduced Mr of the mature rPAP was 40,599. RNA blot analysis indicated the presence of three mRNA species (4.9, 2.3 and 1.5 kb in size) in the rat prostate. The deduced aa sequences of rPAP and hPAP show 75% identity, whereas the similarity between rPAP and human lysosomal acid phosphatase (hLAP) is only 45%. Furthermore, the sequence similarity between rPAP and rat lysosomal acid phosphatase (rLAP) is 46% at the aa level. Similar to hPAP, but unlike hLAP and rLAP, the rPAP sequence lacks a membrane-anchoring domain indicating the secretory character of this phosphatase. All six cysteines present in the overlapping areas of the mature rPAP, hPAP, rLAP and hLAP proteins are positionally conserved, suggesting that these residues are important for the tertiary structure of acid phosphatases (APs). The previously reported active site residues, two arginines and one histidine, are also conserved in these APs. 相似文献