Crude spinach leaf extract readily forms the stearoyl derivative ofacyl-carrier-protein (ACP) when acetyl-ACP and malonyl-ACP are incubated together. Palmitoyl-ACP is also elongated by malonyl-ACP to stearoyl-ACP. When P-ketoacyl-ACP synthase {3-oxoacyl- [ACP] synthase; acyl-[ACP]:malonyl- [ACP] C-acyltransferase (decarboxylating), EC 2.3.1.41} is purified with decanoyl-ACP as the assay substrate, palmitoyl-ACP elongation activity is lost. When palmitoyl-ACP is the assay substrate, another protein is isolated that specifically elongates pal-.mitoyl-ACP to P-ketostearoyl-ACP but has no. activity towards decanoyl-ACP. The first protein is designated -ketoacyl-ACP synthase I and participates in the conversion of acetyl-ACP to palmitoyl-ACP, whereas the second-protein is designated (3-ketoacyl-ACP synthase II, and its substrate specificity is highly restricted to myristoyl-ACP and palmitoyl-ACP. The purification ofsynthase II is described, and its activity is compared to synthase L Reconstitution experiments with the highly purified nonassociated enzymes in fatty acid synthesis plus synthases I and II clearly demonstrate the roles of these two proteins in fatty acid synthesis. Vma than did synthase I with palmitoyl-ACP. The E. coli synthase I is a dimer with -a molecular weight of 80,000, whereas synthase II is' a dimer with a molecular weight of 85,000 (2). It was proposed that synthase II played a role in modulating fatty acid synthesis and, hence, fatty acid composition when E. coli' were grown at different temperatures (2).In 1974, Jaworski et al. (3) reported on the conversion ofpalmitoyl-ACP to stearoyl-ACP by an extract ofmaturing safflower seeds. The system appeared to differ from the de novo fatty acid synthesis (FAS) enzyme system (with acetyl-ACP as the primer substrate) in that (i) it was inactivated at 370C, whereas the de novo system that formed palmitoyl-ACP remained fully active, and (ii) the elongation system was relatively insensitive to cerulenin, whereas the de novo-system was highly sensitive. These and other data suggested a de novo system responsible for the conversion of C2 to C16 fatty acids, and a restrictive elongation system that specifically converted C16 to C18 fatty acids.Because the plant FAS (PFAS) enzyme system in the spinach chloroplast is of a nonassociated type (4), the opportunity to separate the de novo and the elongation systems was quite feasible. Therefore, this investigation reports on (i) the isolation oftwo distinct synthases I and II from spinach leafextracts with unusual substrate specificities and (ii) reconstitution experiments with highly purified enzymes of the PFAS system to which synthases I and II alone and together were added to determine their role in. FAS. While this work. was in progress, Schfiz et at (5) reported on the isolation of a &k-etoacyl-ACP synthase for suspension cells of parsley, which appears to be identical to synthase I of spinach leaf. MATERIALS AND METHODSPurification ofSpinach f-Ketoacyl-ACP Synthases I and II.Methods for preparin...

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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...