The fractionation of the fatty acid synthetase activities of avocado mesocarp plastids

Weaire, P J; Kekwick, R. G. O.

doi:10.1042/bj1460439

Cited by 39 publications

(15 citation statements)

References 11 publications

(14 reference statements)

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“…Based on the location of the N-terminal peptide sequence, SQ830, the 2-1 cDNA encodes a transit peptide of 60 amino acids. This is consistent with the expected localization of 18:1-ACP thioesterase to the seed proplastid (31). The calculated mol wt for the mature protein is 37,254.…”

Section: Purification and Sequencing Of 18:1 -Acp Thioesterasesupporting

confidence: 75%

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Isolation and Characterization of Two Safflower Oleoyl-Acyl Carrier Protein Thioesterase cDNA Clones

Knutzon¹,

Bleibaum²,

Nelsen³

et al. 1992

Plant Physiol.

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Oleoyl-acyl carrier protein (18:1-ACP) thioesterase has been partially purified from developing safflower (Carthamus tinctorius) seeds. Protein species with molecular masses of 34 and 40 kD associated with thioesterase activity were identified and partially sequenced. Analysis of amino-terminal and internal cyanogen bromide peptide sequences revealed no differences in the primary structure of the two species. Amino acid sequence was used to design degenerate oligonucleotides for primers in a polymerase chain reaction (PCR) using safflower embryo cDNA as a template. A 380-base pair PCR product was used to isolate two classes of cDNA clones, designated 2-1 and 5-2, from the embryo cDNA library. Clone 2-1 encodes a 389-amino acid protein including a 60-amino acid transit peptide, and contains all of the protein sequence determined from the 34-and 40-kD proteins. Clone 5-2 encodes a 385-amino acid protein with 80% identity to that encoded by 2-1. Expression of the two safflower cDNA clones in Escherichia coli resulted in a 50-to 100-fold increase in the level of 18:1-ACP thioesterase activity. Both thioesterases are most active on 18:1-ACP; however, the enzyme encoded by 5-2 shows less discrimination against saturated 16-and 18-carbon acyl-ACP substrates.Fatty acids are important components of membrane phospholipids in all plant tissues and are also stored in the form of triacylglycerols in the seeds of many species for use as an energy source upon germination. Fatty acid biosynthesis in plants is catalyzed by a series of soluble, discrete enzymes (23) that are localized in the chloroplasts of leaves (16) nia bay (Umbellularia californica), which accumulates high levels of lauric acid (12:0) in its seed triacylglycerols. Recently, a 12:0-ACP-specific thioesterase has been isolated from this species and shown to redirect fatty acid synthesis to the production of laurate in transgenic plants (6,18,30). In plants that accumulate the longer-chain fatty acids, the specificity of the acyl-ACP thioesterase may also play a role in determining the final fatty acid composition of the storage oil.We are interested in studying acyl-ACP thioesterases with specificities for 16-and 18-carbon fatty acids. Acyl-ACP thioesterase activities have been characterized at various stages of purification from a number of plant species that accumulate 16-and 18-carbon fatty acids in their triacylglycerols, including avocado (17, 24), safflower (Carthamus tinctorius) (12), squash (8), and rapeseed (7). The enzymes show distinct preferences for 18:1-ACP over 18:0-ACP and 16:0-ACP substrates and are relatively inactive on acyl-CoAs.To investigate further the role of acyl-ACP thioesterase in the determination of seed oil composition, we have purified and obtained partial amino acid sequence of an 18:1-ACP thioesterase from developing safflower seeds. Two different classes of cDNA clones encoding 18:1-ACP thioesterase have been isolated and characterized. The substrate specificity of the protein encoded by each clone has been examined by ...

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Section: Purification and Sequencing Of 18:1 -Acp Thioesterasesupporting

confidence: 75%

“…Fatty acid biosynthesis in plants is catalyzed by a series of soluble, discrete enzymes (23) that are localized in the chloroplasts of leaves (16) and the proplastids of developing seed tissue (14, 25,31). Fatty acids up to 18 carbon atoms in length are synthesized in twocarbon increments as ACP2 thioesters (15).…”

mentioning

confidence: 99%

Isolation and Characterization of Two Safflower Oleoyl-Acyl Carrier Protein Thioesterase cDNA Clones

Knutzon¹,

Bleibaum²,

Nelsen³

et al. 1992

Plant Physiol.

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“…The 99 % of the fas activity which was precipitated by 40-70 % (NH4)2SO4 was totally dependent upon addition of exogenous ACP. In accord with these results, the ACP dependent fases from potato tubers (23), avocado mesocarp plastids (52) and Euglena gracilis (10) were precipitated at 40-65, 35-65 and 35-70% (NH4)2SO4 saturations, respectively. The multifunctional, A'CP independent fas from E. gracilis (10), by contrast, was precipitated preferentially by 0-25 % (NHahSO4 saturation.…”

Section: Partial Purification Of the Chloroplastsupporting

confidence: 71%

Partial separation of individual enzyme activities of an ACP-dependent fatty acid synthetase from barley chloroplasts

Høj

Mikkelsen

1982

Carlsberg Res. Commun.

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An acyl carrier protein (ACP) dependent fatty acid synthetase (fas) from barley chloroplast stroma was purified five-fold by ammonium sulphate precipitation and gel filtration on Sephacryl S-300. The {~-ketoacyl-ACP reductase, ~-ketoacyl-ACP synthetase, acetyl-CoA:ACP transacylase and malonyl-CoA:ACP transacylase activities were resolved on the Sephacryl S-300 column with apparent molecular weights of respectively 125, 92, 82 and 41 kilodalton. The fas activity exhibited an apparent molecular weight of 87 kilodalton resulting from the overlapping portions of the component activities. A fifth component of the active fas, ACP, was separated completely from the other four individual enzyme activities by the ammonium sulphate precipitation. When the fas purified by gel filtration was applied to a Ma-trex Gel Blue B column, the component activities were separated into two groups. A bound fraction contained all the malonyl-CoA:ACP transacylase whereas the [~-ketoacyl synthetase activity was exclusively present in the non-bound fraction. Neither the bound nor the non-bound fraction showed any fas activity alone, but complete reconstitution of fas activity was obtained when both protein fractio~ were combined. The barley chloroplast fas is therefore not a multifunctional protein but consists of at least five separable components. Characterization with respect to cofactor requirements was also performed. Variation of certain cofactor concentrations markedly altered the pattern of fatty acid synthesis.Abbreviations: ACP = acyl carrier protein; DTNB = 5",5-dithiobis(2-nitro benzoic acid); DT1 ~ = 1,4-dithiotreitol; Hepes = N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; HPLC = high pressure liquid chromatography; kD = kilodalton; Tricine = N-(Tris(hydroxy-methyl)-methyl)-glycin; SDS = sodium dodecyl sulphate.

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“…Unlike procaryotic and eucaryotic cells, in which the FAS system is solely localized in the cytosol, the FAS system in the C3 leaf cell is wholly associated with chloroplasts (10) and, in developing and germinating seeds, with proplastids (18,19). With the exception of ACP (12), no attempt has been made yet to isolate and purify all of the enzymes responsible for the synthesis of fatty acids in plants.…”

Section: Introductionmentioning

confidence: 99%

“…In bacteria, with the exception of mycobacteria (21), which represents a more advanced procaryote, the overall biosynthesis of fatty acids is carried out by a series of reactions catalyzed by discrete enzymes which are not associated with a polyfunctional peptide. In addition, ACP is easily separated from the other proteins involved in the FAS system.Unlike procaryotic and eucaryotic cells, in which the FAS system is solely localized in the cytosol, the FAS system in the C3 leaf cell is wholly associated with chloroplasts (10) and, in developing and germinating seeds, with proplastids (18,19). With the exception of ACP (12), no attempt has been made yet to isolate and purify all of the enzymes responsible for the synthesis of fatty acids in plants.…”

mentioning

confidence: 99%

Fatty Acid Synthetase of Spinacia oleracea Leaves

Shimakata¹,

Stumpf²

1982

Plant Physiol.

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The molecular organization of fatty acid synthetase system in spinach (Spinacia oleracea L. var. Viroflay) leaves was examined by a procedure similar to that employed for the safflower system (Carthamus tinctorius var. UC-1). The crude extract contained all the component activities (acetylCoA:ACP transacylase, malonyl-CoA:ACP transacylase, 8-ketoacyl-ACP synthetase, 8-ketoacyl-ACP reductase, p8-hydroxyacyl-ACP dehydrase, and enoyl-ACP reductase III) involved in the synthesis of fatty acids, but enoyl-ACP reductase (II) present in safflower seeds extract could not be detected spectrophotometrically. By polyethylene glycol fractionation followed by several chromatographic procedures, ie. Sephadex G-200, hydroxyapatite, and blue-agarose, the component enzymes were clearly separated from one another. Properties of ,B-ketoacyl-ACP reductase, ,Bhydroxyacyl-ACP dehydrase, and enoyl-ACP reductase (I) from spinach were compared with the same enzymes in safflower seeds and Escherichia coli.From these results, it was concluded that the fatty acid synthetase system of spinach leaves, as well as that of safflower seeds, was nonassociated and similar to the Escherichia coli system.There are two types of FAS2 systems, which differ in organizational complexity in a variety of organisms. In yeast (9,15) and in animals (13,14), the steps of fatty acid biosynthesis from malonyl-CoA and acetyl-CoA are catalyzed by a polyfunctional polypeptide containing covalently bound ACP. In bacteria, with the exception of mycobacteria (21), which represents a more advanced procaryote, the overall biosynthesis of fatty acids is carried out by a series of reactions catalyzed by discrete enzymes which are not associated with a polyfunctional peptide. In addition, ACP is easily separated from the other proteins involved in the FAS system.Unlike procaryotic and eucaryotic cells, in which the FAS system is solely localized in the cytosol, the FAS system in the C3 leaf cell is wholly associated with chloroplasts (10) and, in developing and germinating seeds, with proplastids (18,19). With the exception of ACP (12), no attempt has been made yet to isolate and purify all of the enzymes responsible for the synthesis of fatty acids in plants. Recently, this laboratory, in examining the FAS system in safflower seeds, obtained evidence that this system consisted of separable component enzymes (11) similar to the Escherichia coli (17) and Euglena gracilis (5) systems. Therefore, similar methods were applied to the FAS system in spinach leaves.Evidence will be presented that the FAS system from spinach leaves is very similar to the nonassociated system in safflower seeds (11), Escherichia coli (17) (10), except that antibody was omitted. Elongation of added acyl-ACPs was assayed by using [I-'4CJpalmitoyl-ACP, as described previously (6). Assays of acetyl-CoA:ACP and malonyl-CoA:ACP transacylases, fl-ketoacyl-ACP synthetase, ,8ketoacyl-ACP reductase, 8i-hydroxyacyl-ACP dehydrase, and enoyl-ACP reductases (I and II) were performed as described by Shimak...

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The fractionation of the fatty acid synthetase activities of avocado mesocarp plastids

Cited by 39 publications

References 11 publications

Isolation and Characterization of Two Safflower Oleoyl-Acyl Carrier Protein Thioesterase cDNA Clones

Isolation and Characterization of Two Safflower Oleoyl-Acyl Carrier Protein Thioesterase cDNA Clones

Partial separation of individual enzyme activities of an ACP-dependent fatty acid synthetase from barley chloroplasts

Fatty Acid Synthetase of Spinacia oleracea Leaves

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