| 1. |
- Engberg, ST, et al.
(författare)
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Peroxisome proliferator-induced acyl-CoA thioesterase from rat liver cytosol: molecular cloning and functional expression in Chinese hamster ovary cells
- 1997
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Ingår i: The Biochemical journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 323323 ( Pt 2), s. 525-531
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Tidskriftsartikel (refereegranskat)abstract
- We have isolated and cloned a cDNA that codes for one of the peroxisome proliferator-induced acyl-CoA thioesterases of rat liver. The deduced amino acid sequence corresponds to the major induced isoform in cytosol. Analysis and comparison of the deduced amino acid sequence with the established consensus sequences suggested that this enzyme represents a novel kind of esterase with an incomplete lipase serine active site motif. Analyses of mRNA and its expression indicated that the enzyme is significantly expressed in liver only after peroxisome proliferator treatment, but isoenzymes are constitutively expressed at high levels in testis and brain. The reported cDNA sequence is highly homologous to the recently cloned brain acyl-CoA thioesterase [Broustas, Larkins, Uhler and Hajra (1996) J. Biol. Chem. 271, 10470–10476], but subtle differences throughout the sequence, and distinct differences close to the resulting C-termini, suggest that they are different enzymes, regulated in different manners. A full-length cDNA clone was expressed in Chinese hamster ovary cells and the expressed enzyme was characterized. The palmitoyl-CoA hydrolysing activity (Vmax) was induced approx. 9-fold to 1 μmol/min per mg of cell protein, which was estimated to correspond to a specific activity of 250 μmol/min per mg of cDNA-expressed enzyme. Both the specific activity and the acyl-CoA chain length specificity were very similar to those of the purified rat liver enzyme.
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| 2. |
- Svensson, LT, et al.
(författare)
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Molecular cloning and characterization of a mitochondrial peroxisome proliferator-induced acyl-CoA thioesterase from rat liver
- 1998
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Ingår i: The Biochemical journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 329329 ( Pt 3), s. 601-608
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Tidskriftsartikel (refereegranskat)abstract
- We have previously reported the purification and characterization of the peroxisome proliferator-induced very-long-chain acyl-CoA thioesterase (MTE-I) from rat liver mitochondria [L. T. Svensson, S. E. H. Alexson and J. K. Hiltunen (1995) J. Biol. Chem. 270, 12177-12183]. Here we describe the cloning of the corresponding cDNA. One full-length clone was isolated that contained an open reading frame of 1359 bp encoding a polypeptide with a calculated molecular mass of 49707 Da. The deduced amino acid sequence contains a putative mitochondrial leader peptide of 42 residues. Expression of the cDNA in Chinese hamster ovary cells, followed by immunofluorescence, immunoelectron microscopy and Western blot analyses, showed that the product was targeted to mitochondria and processed to a mature protein of 45 kDa, which is similar to the molecular mass of the protein isolated from rat liver mitochondria. The recombinant enzyme showed the same acyl-CoA chain-length specificity as the isolated rat liver enzyme. Sequence analysis showed no similarity to known esterases, but a high degree (approx. 40%) of identity with bile acid-CoA:amino acid N-acyltransferase cloned from human and rat liver. A putative active-site serine motif (Gly-Xaa-Ser-Xaa-Gly) of several carboxylesterases and lipases was identified. Western and Northern blot analyses showed that MTE-I is constitutively expressed in heart and is strongly induced in liver by feeding rats with di(2-ethylhexyl)phthalate, a peroxisome proliferator, suggesting a role for the enzyme in lipid metabolism.
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