Induction, purification and characterisation of acyl-ACP thioesterase from developing seeds of oil seed rape (Brassica napus).
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.
Hellyer
A
A
Unilever Research, Sharnbrook, Bedford, UK.
Leadlay
P F
PF
Slabas
A R
AR
D000595:Amino Acid Sequence; D001937:Brassica / Q000201:enzymology*; D019730:Carbon-Sulfur Ligases; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008025:Ligases / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012639:Seeds / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013869:Thiolester Hydrolases / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism
D013869:Thiolester Hydrolases; C018748:oleoyl-(acyl-carrier-protein) hydrolase; D008025:Ligases; D019730:Carbon-Sulfur Ligases; C018749:long-chain-fatty-acid-(acyl-carrier-protein) ligase