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Query: UNIPROT:P06889 (Mol)
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The level of two thioesterases, acyl-CoA thioesterase and acyl-ACP thioesterase was determined during seed maturation in oil seed rape. Both thioesterase activities rose markedly prior to the onset of lipid accumulation, but the induction kinetics suggest that the activities reside on distinct polypeptides. Acyl-ACP thioesterase (EC 3.1.2.14) was purified 2000-fold using a combination of ion exchange, ACP-affinity chromatography, chromatofocusing and gel filtration. Using native gel electrophoresis, and assays for enzymic activity, two polypeptides were identified on SDS-PAGE as associated with the activity. Cleveland mapping of these polypeptides, of 38 kDa component and 33 kDa respectively, demonstrated that they are related. An antibody was prepared against the 38 kDa component, and this also recognises the 33 kDa polypeptide in highly purified preparations. Western blotting of a crude extract identifies one band at 38 kDa consistent with the 33 kDa component being a degradation product generated during purification. The native molecule has a M(r) of 70 kDa indicating a dimeric structure. The enzyme has a pH optimum of 9.5 and shows strong preference for oleoyl-ACP as substrate. The intact enzyme has an N-terminus blocked to protein sequencing. We also found that two other polypeptides co-purify with acyl-ACP thioesterase under native conditions. The N-terminal amino-acid sequence of these polypeptides is shown and their possible identity is discussed.
Plant Mol Biol 1992 Dec
PMID:Induction, purification and characterisation of acyl-ACP thioesterase from developing seeds of oil seed rape (Brassica napus). 130 Oct 73

Acyl-(acyl-carrier-protein) hydrolase (EC 3.1.2.14) releases fatty acids from the end-product of fatty acid synthesis in plastids for the subsequent synthesis of glycerolipids in the cytoplasm. Isoelectric focusing of chloroplast stroma proteins from squash cotyledons suggested that there were at least three isomeric forms of acyl-(acyl-carrier-protein) hydrolase having pI values of 4.5, 5.3 and 7.8. The pI 4.5 and pI 5.3 forms showed maximum activity at pH 9.8 whereas the activity of the pI 7.8 form increased within the range 6.2 to 10.2 but no optimum was seen. The pI 4.5 form was purified 100,000-fold from squash cotyledons. The highly purified fraction contained two polypeptides, whose molecular masses were estimated to be 35 kDa and 33 kDa by SDS-PAGE. It is suggested that the 33 kDa polypeptide was a degradation product of the 35 kDa polypeptide. Oleoyl-(acyl-carrier protein) was the preferred substrate of this enzyme over palmitoyl- and stearoyl-(acyl-carrier protein), whereas lauroyl-(acyl-carrier protein) was nearly inactive. These results indicate the enzyme is specific for long-chain acyl-(acyl-carrier protein).
Plant Mol Biol 1992 Oct
PMID:Acyl-(acyl-carrier protein) hydrolase from squash cotyledons specific to long-chain fatty acids: purification and characterization. 139 66

Acyl-ACP thioesterases are involved in regulating chain termination of fatty acid biosynthesis in plant systems. Previously, acyl-ACP thioesterase purified from Brassica napus seed tissue has been shown to have a high preference for hydrolysing oleoyl-ACP. Here, oligonucleotides derived from B. napus oleoyl-ACP thioesterase protein sequence data have been used to isolate two acyl-ACP thioesterase clones from a B. napus embryo cDNA library. The two clones, pNL2 and pNL3, contain 1642 bp and 1523 bp respectively and differ in the length of their 3' non-coding regions. Both cDNAs contain open reading frames of 366 amino acids which encode for 42 kDa polypeptides. Mature rape thioesterase has an apparent molecular weight of 38 kDa on SDS-PAGE and these cDNAs therefore encode for precursor forms of the enzyme. This latter finding is consistent with the expected plastidial location of fatty acid synthase enzymes. Northern blot analysis shows thioesterase mRNA size to be ca. 1.6 kb and for the thioesterase genes to be highly expressed in seed tissue coincident with the most active phase of storage lipid synthesis. There is some sequence heterogeneity between the two cDNA clones, but overall they are highly homologous sharing 95.7% identity at the DNA level and 98.4% identity at the amino acid level. Some sequence heterogeneity was also observed between the deduced and directly determined thioesterase protein sequences. Consistent with the observed sequence heterogeneity was Southern blot data showing B. napus thioesterase to be encoded by a small multi-gene family.
Plant Mol Biol 1993 Nov
PMID:Isolation and characterization of two Brassica napus embryo acyl-ACP thioesterase cDNA clones. 825 30