Acetate, active incorporation into fatty acids

Evidence has been presented that citrate is the source of acetyl-CoA in the soybean cotyledon. Nelson and Rinne (1975, 1977a,b) have described the occurrence of citrate lyase in the cytosol of developing soybean seeds. Furthermore, [1,5-1 ] citrate was an effective donor of [ KI ]acetyl-CoA for the synthesis of fatty acids in these extracts. However, Weaire and Kekwick (1975) did not find [l,5- K ]citrate to be an acetyl donor for fatty acid synthesis in avocado extracts. In addition, Yamada and Nakamura (1975) showed with isolated spinach chloroplasts that, whereas pyruvate was effectively incorporated into fatty acid, citrate, malate, and oxeiloacetate were far less active. Apparently different tissues may supply acetyl-CoA from different precursors, i.e., pyruvate, citrate, and acetate. 

Many studies have revealed that thyroid hormones markedly affect lipid metabolism in man and in several species of animals. Concerning fatty acid biosynthesis it was demonstrated that the administration of thyroxine stimulates the incorporation of l-l c acetate into fatty acids in rats and mice (Dayton et al, I960) (Gompertz and Greenbaum, 1966) (March and Mayer, 1959). According to Gompertz and Greenbaum (1966) these observations appear to be associated with an increase of stearyl-CoA desaturase activity. Moreover Myant and Iliffe (1963) found that rats treated with thyroxine showed an inhibition of acetate incorporation, but not of malonate incorporation, into fatty acids by mitochondria free, subcellular liver preparations. Other authors have shown that the thyrotoxic state was accompanied by an increased incorporation of acetyl-CoA to fatty acid and a rise in the activity of fatty acid synthetase in rat livers (Diamant et al, 1972) (Roncari and Murthy, 1975). However, in vitro studies of fatty acid synthesis in which liver supernatant of 105,000 xg and microsomal preparations were incubated with the hormone showed that thyroxine inhibits de novo synthesis of palmitate and stimulates the desaturation reactions (Faas et al, 1972). 

Howard (1968b) studied fatty acid synthetic systems in cell-free preparations from squirrel monkey aortas, and the data were similar to those for the rabbit aorta with regard to the mitochondrial system. Acetate or acetyl-CoA was a more efficient precursor than malonyl-CoA, and the Schmidt degradation data indicated that it was primarily an elongation system. The cytosol or HSS was examined, and malonyl-CoA was found to be incorporated into fatty acids 55-200 times more actively than acetyl-CoA, a finding that had been noted previously in liver HSS by Abraham et al. (1962a). Majerus and Lastra (1967) noted that malonyl-CoA was incorporated into fatty acids six or seven times as fast as acetyl-CoA by human leukocytes. The latter authors were unable to find any acetyl-CoA carboxylase activity in leukocytes and reasoned that these cells possess only the fatty acid synthetase. As they pointed out, in the absence of any acetyl-CoA carboxylase, the synthetase alone uses 1 mole of acetyl-CoA plus 7 moles of malonyl-CoA to make 1 mole of palmitate (Wakil... 

Matsuhashi et al. (1962), and Martin and Vagelos (1962) demonstrated that citrate and isocitrate stimulate acetate incorporation into fatty acids by activation of the acetyl-CoA carboxylase. Later, Vagelos et al. (1963) showed that activation by citrate was associated with aggregation of monomeric units of the enz)ane. 

Chloroplasts were incubated with [ " C]acetate to isolate MCTE activity from p-oxidation. Radiolabelled fatty acids were extracted and separated using successive argentation thin layer chromatography (TLC) and Cig-reverse phase TLC. The Brassica chloroplasts did not synthesise fatty acids well whereas control spinach chloroplasts incorporated [ " CJacetate into fatty acids at a rate of 100-300nmol/hour/mg chlorophyll, the Brassica chloroplasts had a specific activity of only approximately 20nmol/hour/mg chlorophyll. 

R(+) enantiomer is herbicidally active (23.24). Hoppe and Zacher (12) showed that the R(+) enantiomer of diclofop was more effective than the S(-) enantiomer in reducing acetate incorporation into free fatty acids in isolated maize chloroplasts. ACCase activity is inhibited by R(+) (98% enantiomeric excess) haloxyfop acid but not by the S(-) (94% enantiomeric excess) enantiomer (Fig. 5). The inhibition caused by the S(-) enantiomer could be accounted for by the 3% contamination in the S(-) preparation by the R(+) enantiomer. 

When cells of H. cutirubrum were grown in the presence of [ C]acetate, over 98% of the label was incorporated into the isopranyl (phytanyl) groups of the polar lipids, and less than 0.5% was found in long-chain fatty acids [86]. Cell-free studies subsequently demonstrated the presence of a fatty acid synthetase (FAS) which is largely ( 80%) inhibited by 4M NaCl or KC1[87]. However, sufficient FAS activity remains for the formation of saturated fatty acids (14 0, 16 0, 18 0) recently found to be esterified to proteins of the red membrane but not to bacteriorhodopsin in the purple membrane of extreme halophiles nor to the polar lipids (Pugh and Kates, unpublished data). 

Fluorophenylalanine acts in a manner different from other inhibitors, since it permits protein synthesis. This difference in behavior results in incorporation into proteins (Westhead and Boyer, 1%1) that often possess decreased or altered activity (G and Millard, 1971). Further studies showed that the addition of fluorophenylalanine caused extensive inhibition of fatty acid and phenol (acetate-derived) synthesis shortly after administration (Ward and Packter, 1974), presumably after incorporation into the polypeptides comprising fatty acid synthetase. In contrast, cycloheximide halved... 

The pivotal role of aleprolic acid (cyclopentenylcarboxylic acid) as primer in the biosynthesis of cyclopentenyl fatty acids was examined in a variety of tissues. In seeds of anthelminthica and C. echinata [l- C]aleprolic acid was almost exclusively incorporated into cyclopentenyl fatty acids, and in cells of I. polycarpa suspension cultures at a level of well over 60% (Cramer and Spener, 1976 Buchholz and Spener, 1980). In the cell cultures used, where endogenous cyclopentenyl fatty acids occurred only at a minor level, the substrate was taken up and activated for both anabolic and catabolic reactions. The acetate thus formed was refunneled and used for the de novo synthesis predominantly of straight-chain fatty acids (Buchholz and Spener, 1980). 

Fig. 1 shows the specific activities of the different fatty acids. In fact, linoleic acid is not only converted to arachidonic acid and higher o)6 polyenoic acids but is also degraded to acetate which is incorporated into the common fatty acids. 

Feeding a fat-free diet to rats results not only in the fatty acid changes cited previously but also in increased synthesis of palmitic acid in rats injected intratesticularly with C-acetate or in slices of testes incubated with C-acetate. There was relatively less incorporated into the polyenes in the tissue from the deficient rats (Whorton Coniglio, 1977). It was observed that there was no effect on fatty acid synthetase or on microsomal elongation enzymes, but there was an increase in the acetyl CoA carboxylase activity of testes of the fat-deficient rats. 

An active enzyme fraction was isolated according to H0j and Mikkelsen (1982) with some modifications. Details of plant cultivation, enzyme isolation, and enzyme assay were described before (Focke and Lichtenthaler 1987, Kobek et al. 1988). 6-day-old etiolated barley (var. Alexis) were illuminated for six hours. The 40-70% ammonium sulphate saturation fraction of the chloroplast stroma was dialyzed and concentrated against polyethyleneglycol. Enzymic activity was measured by the incorporation of acetate, acetyl-CoA, malonate, or malonyl-CoA into the fatty acid fraction. Herbicides were added in a... 

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