Comparison of acetate- and pyruvate-dependent fatty-acid synthesis by spinach chloroplasts

In recent studies using intact chloroplasts of spinach (Spinacia oleracea L.) to investigate the accumulation of acetyl-CoA produced by the activity of either acetyl-CoA synthetase (EC 6.2.1.1) or the pyruvate-dehydrogenase complex, this product was not detectable. These results in combination with new information on the physiological levels of acetate and pyruvate in spinach chloroplasts (H.-J. Treede et al. 1986, Z. Naturforsch. 41 C, 733–740) prompted a reinvestigation of the incorporation of 1-¹⁴C] acetate and 2-¹⁴C] pyruvate into fatty acids at physiological concentrations.The K _m for the incorporation into fatty acids was about 0.1 mM for both metabolites and thus agreed with the values obtained by H.-J. Treede et al. (1986) for acetyl-CoA synthetase and the pyruvate dehydrogenase complex. However, acetate was incorporated with a threefold higher V _max. Saturation for pyruvate incorporation into the fattyacid fraction was achieved only at physiological pyruvate concentrations (<1.0 mM). The diffusion kinetics observed at higher concentrations may be the result of contamination with derivates of the labeled substrate. Competition as well as double-labeling experiments with ³H]acetate and 2-¹⁴C]pyruvate support the notion that, at least in spinach, chloroplastic acetate is the preferred substrate for fatty-acid synthesis when both substrates are supplied concurrently (P.G. Roughan et al., 1979 b, Biochem. J. 184, 565–569).Experiments with spinach leaf discs confirmed the predominance of fatty-acid incorporation from acetate. Radioactivity from 1-¹⁴C]acetate appeared to accumulate in glycerolipids while that from 2-¹⁴C]pyruvate was apparently shifted in favor of the products of prenyl metabolism.Abbreviations Chl chlorophyll - TLC thin-layer chromatography