ACP isoform

PE than for DGD, PG and PI. A possible explanation for this matter could be the existence of ACP-isoforms with different Km for acyl transferase and thioesterase reactions, as could be shown for spinach (Guerra et al. 1986). 

Two forms of ACP have been purified from spinach and barley leaf, but, only one ACP Isoform predominates In spinach seed tissue (2, 3). Oleic acid Is the major product of fatty acid synthesis by Isolated chloroplasts (4). The regulation of plant lipid metabolism Is believed to Involve the export of oleic acid from Its site of synthesis (plastld) to sites of complex lipid metabolism (eg. ER). This pathway requires the Initial release of oleic acid from ACP by oleoyl-ACP thloesterase (5). In addition, the oleoyl moiety may also enter glycerollpld synthesis within the plastld through the action of acyl-ACP acyl-transferase (6). The preferred substrate for both the thloesterase and glycerol—3-phosphate acyl transferase reactions Is 18 1-ACP (6,7). 

The occurrence of two forms of ACP raises the question of their metabolic function. Because 18 1—ACP Is the substrate for two competing reactions, ACP Isoforms might facilitate acyl chain distribution within the cell. He have analyzed the reactivity and kinetics of oleoyl-ACP thloesterase and oleoyl-ACP glycerol-3—phosphate acyl transferase with oleoyl-ACP-I and oleoyl-ACP-II. Our results suggest that ACP Isoforms may partially regulate the flow of oleic acid from Its site of de novo synthesis In higher plants. 

The enzymatic synthesis of [ C]18 1-ACP Isoforms was according to a published procedure (9), and was purified through the octyl sepharose step. Oleoyl-ACP thloesterase was partially purified from spinach tissue by methods which Involved acid precipitation and either Ion-exchange or affinity chromatography (7,8). Glycerol-3-phosphate acyl transferase was purified by ammonium sulfate fractionation, DEAE and... 

Suh et al. (1999) studied the isoforms of acyl carrier protein involved in seed-specific fatty acid synthesis in coriander seed, ft produces unusual monoenoic fatty acids which constitute over 80% of the total fatty acids of the seed oil. The initial step in the formation of these fatty acids is the desaturation of palmitoyl-ACP (acyl carrier protein) at the DELTA4 or DELTA6 positions to produce DELTA4-hexadecenoic acid (16 1DELTA4) or DELTA6-hexadecenoic acid (16 1DELTA6), respectively. 

Malonyl-CoA condenses with an acyl carrier protein (ACP). This reaction is catalyzed by malonyl-CoA ACP transacylase, an enzyme that has been purified from several plants (Ohlrogge et al., 1993). ACP is a relatively small heat-stable protein of 9000 molecular weight that occurs in several isoforms (Browse and Somerville, 1991 Lehninger, 1982). Both ACP and coenzyme A have a 4 -phosphopan-tetheine prosthetic group (Ohlrogge et al., 1993). Amino acid sequences for ACP molecules from more than 15 plants, including both monocots and dicots, are available. The... 

Isoforms of ACP have different activity In acyl transferase and thloesterase reactions. 

Figure IB shows the effect of [ C]oleoyl-ACF Isoforms on the reaction velocity of spinach oleoyl-ACF thloesterase. Oleoyl-ACF-I was a more efficient substrate for the reaction at all concentrations tested while oleoyl-ACF-11 was a relatively poor substrate. The Km for oleoyl-ACP-1 was 4.8 yM while that for oleoyl-ACF-II was 50 yM. For comparison, the Km for JE. coll oleoyl-ACF was 1.4 yM.

Depending on the AGP Isoform esterlfled to oleic acid within the plastld, the oleoyl moiety may enter Into either plastld glycerollpld biosynthesis or may leave the plastld and enter lipid metabolism at sites such as the ER. The acyl transferase selectively transfers the oleoyl moiety from ACP-II and directs the synthesis of 1-oleoyl-lysophosphatldlc acid. The thloesterase preferentially hydrolyzes the oleoyl moiety from ACF-1 and directs free oleic acid to the outer envelope where It Is subsequently esterlfled to CoA. The oleoyl-CoA thus formed Is utilized outside the plastld for assembly of eukaryote type glycerollplds at the ER. The lysophosphatldlc acid remains within the plastld as substrate for prokaryote-type glycerollpld synthesis. 

EXPRESSION OF mRNA AND STEADY-STATE LEVELS OF PROTEIN ISOFORMS OF ENOYL-ACP REDUCTASE FROM Brassica napus. 

The de novo biosynthesis of fatty acids in plants occurs in the plastid and is cataiysed by a type II, dissociable fatty acid synthetase(FAS). Enoyl-ACP reductase (ER) catalyses the second reductive step in the synthetic cycle, converting a trans 2,3 moiety into a saturated acyl chain. The protein from B. napus has been purified[1], extensively sequenced[2] and the absolute mass of the enzyme in developing rape seed determined[3]. The cDNA which encodes ER has been cloned and fully sequenced Southern hybrisization with this cDNA shows the presence of four genes in B. napu A]. This study investigates the expression of ER mRNA and the steady-state levels of ER protein isoforms by Northern and two-dimensional Western analyses. 

Isoforms of Enoyl-ACP Reductase. Protein extracts from seeds and leaf were subjected to two-dimensional electrophoresis and Western analysis. The same four isoforms of ER were present in the leaf and seed material(Fig. 2), indicating that all four genes in 8. napus show increased expression in seeds, rather than switching on a seed specific gene. The denatured isoelectric point of the four isoforms was determined by a comparison with carbamylated creatine kinase(BDH) markers. The relative pi s of the polypeptides are ER I, 5.9 ER II, 6.1 ER III, 6.4 and ER IV, 6.8. 

It is well known that the two-carbon units for condensation in de novo fatty acid synthesis are provided as malonate. A specific transacylase transfers the malonate from coenzyme A to ACP. Malonyl-CoA ACP transacyl-ases have been purified from a number of sources including leaf and seed tissues as well as the cyanobacterium Anabaena variabilis. The enzymes usually have masses of about 40 kDa and appear to use a random sequence mechanism. Tissue-specific isoforms have been reported in some instances though their significance is not known. 

Once condensation has taken place, the /3-ketoacyl-ACP undergoes a cycle of reduction, dehydration, and reduction to yield a new fatty acid. The enzymes concerned are )8-ketoacyl-ACP reductase, )8-hydroxyacyl-ACP dehydrase, and enoyl reductase, and detailed information is provided in recent reviews. Both of the reductases have been reported in isoforms from several tissues. Thus, in avocado, an NADH form of )8-ketoacyl-ACP reductase has been resolved from an NADPH form. In seed tissues, two forms of enoyl reductase appear to be present they differ in the chain-length activity and also in whether they use NADH only or use NADPH in preference to NADH only the NADH form has been purified from leaves. ... 

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