This communication demonstrates that all de novo fatty acid biosynthesis in spinach leaf cells requires acyl carrier protein (ACP) and occurs specifically in the chloroplasts. Antibodies raised to purified spinach ACP inhibited at least 98% of malonyl CoA-ependent fatty acid synthesis by spinach leaf homogenates. Therefore, the presence of ACP in a compartment of the spinach leaf cell would serve as a marker for de novo fatty acid biosynthesis. A radioimmunoassa capable of detecting 10-'S mol (10-" g) of spinach ACP was deve oped to measure the levels of ACP in leaf cell components isolated by sucrose gradient centrifugation of a gentle lysate of spinach leaf protoplasts. All of the ACP of the leaf cell could be attributed to the chloroplast. Less than 1% of the ACP associated with chloroplasts resulted from binding of free ACP to chloro lasts. Of interest, ACP from Escherichia coli, soybean, and sunflower showed only partial crossreactivity with spinach ACP by the radioimmunoassay. These results strongly suggest that, in the leaf cell, chloroplasts are the sole site for the degnovo synthesis of C16 and C18 fatty acids. These fatty acids are then transported into the cytoplasm for further modification and are either inserted into extrachloroplastic membrane lipids or returned to the chloroplast for insertion into lamellar membrane lipids. In plants, acyl carrier protein (ACP) plays an essential role in both the synthesis and the subsequent metabolism of the C16 and C18 fatty acids (1). Whereas de novo fatty acid synthesis in isolated chloroplasts has long been known (1), the site of synthesis of fatty acids that are required for the formation of plasma, mitochondrial, and other extrachloroplast membranes in the leaf cell is not clear. In sharp contrast, in both animal and yeast cells, synthesis occurs in the cytoplasm (2). In Escherichi colA cells, it has been shown that ACP is localized on or near the inner face of the plasma membrane and this implies that the nonassociated fatty acid synthetase enzymes may be organized in the same area in vivo (3).Although earlier studies have provided evidence that fatty acid biosynthesis occurs in chloroplasts (1) and in proplastids (4-8), until recently the methods used for the isolation of these organelles led to substantial breakage and release of enzymes into the cytoplasmic fraction. Consequently, it has been difficult to assign a precise site for an enzyme in the leaf cell. In addition, after cell disruption, attempts to localize the complex set of reactions comprising fatty acid synthesis can be further complicated by cofactor dilution (particularly ACP), enzyme dilution, and inactivation. Hence, it has been difficult to conclude from these earlier studies whether de novo fatty acid synthesis takes place only in the chloroplast (or plastid) or in the cytoplasm or other organelles as well. Damage to organelles can be greatly reduced in the isolation procedure by using protoplasts as the starting material (9). Gentle lysis of isolated protoplasts gives an incre...

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...

In addition to acyl‐CoA, purified glycerol‐3‐phosphate acyltransferases from pea and spinach chloroplasts can also use acyl‐(acyl‐carrier protein), acyl‐ACP, as a substrate for glycerol 3‐phosphate acylation. The enzyme fractions showed absolute specificity for glycerol 3‐phosphate as acyl acceptor. Dihydroxyacetone phosphate was ineffective. Glycerol 3‐phosphate was almost exclusively acylated at the C‐1 position. If mixtures of palmitoyl‐ACP, stearoyl‐ACP and oleoyl‐ACP were offered, the oleoyl group was preferred. These data fully agree with previous experiments on these enzymes carried out with various acyl‐CoA thioesters. Kinetic data determined with different acyl‐ACPs as substrates are consistent with the observed fatty acid selectivity for the oleoyl group. Double labelling experiments with mixtures of oleoyl‐ACP and oleoyl‐CoA demonstrated a preference for ACP‐thioesters. Monoacylglycerol‐3‐phosphate acyltransferase, localized in the envelope of chloroplasts, can also utilize acyl‐ACP as substrate. Envelope fractions of spinach as well as of pea showed a high specificity for the palmitoyl group when ACP‐thioesters or CoA‐thioesters were offered and directed this acyl group to the C‐2 position of the glycerol backbone. Results from competition experiments with [14C]palmitoyl‐ACP and [3H]palmitoyl‐CoA indicate that the membrane‐bound acyltransferase preferably uses ACP‐thioesters for the acylation of 1‐acylglycerol 3‐phosphate. According to fatty acid selectivities and specificities the main product of the recombined acyltransferase systems in chloroplasts of 16:3 plants as well as 18:3 plants is a phosphatidic acid with the oleoyl group at C‐1 and the palmitoyl group at C‐2 whereas molecules with the oleoyl group at both positions are not synthesized. Under appropriate conditions the phosphatidic acid formed by the soluble acyltransferase and spinach envelope was rapidly converted to monogalactosyl diacylglycerol in the presence of UDP‐galactose. In analogous assays with acyltransferase and envelope from pea only a low proportion of labelled phosphatidic acid was converted via diacylglycerol to monogalactosyl diacylglycerol. In both systems monogalactosyl diacylglycerol synthesized in the presence of C16:0‐thioesters and C18:1‐thioesters carried C18:1 at C‐1 and C16:0 at C‐2 in agreement with the fatty acid selectivity of the acyltransferase systems.

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A bstract. In the maturing castor bean seed (Ricinus communis), maximum $3-oxidation appears at 28 days after flowering and in the germinating seed, 4 days after germination. Highest specific activities for both 1-oxidation systems and their component enzymes are associated with cytosomal particles banding at a density of 1.25 g/ml in a sucrose gradient. Substrate specificity studies indicate that of several fatty acidis, ricinoleate is oxidized most rapidly by the preparation from the maturing seed (28 days after flowering) while palmitate and linoleate are oxidized most rapidly by extracts obtained from tissue germinated for 4 days. The :-oxidation activities observed in both systems reflect the expression of activity of at least 3 of the component enzymes, crotonase, ,3-hydroxyacyl dehydrogenase and /3-keto-thiolase, which rise and fall coordinately. Acyl thiokinase does not appear to play a limiting role in regulating :-oxidation per se under the conditions employed here.

The following enzyme activities were demonstrated in cell‐free homogenates from developing jojoba cotyledons: 1) elongation of long chain acyl‐CoAs in the presence of malonyl‐CoA and NADPH (or NADH), 2) NADPH‐dependent reduction of long chain acyl‐CoAs to the corresponding alcohols, 3) esterification of long chain acyl‐CoAs and the alcohols produced from them into wax, 4) elongation of stearoyl‐ACP to eicosanoate and docosanoate as well as reduction to stearyl alcohol, 5) desaturation of stearoyl‐ACP to oleate in the presence of reduced ferredoxin, and 6) incorporation of malonyl‐CoA into long chain fatty acids and alcohols in the presence of added acyl carrier protein. These activities were associated entirely with the floating wax pad after centrifugation of the cell‐free homogenate at 12,000 g for 20 min. The relevance of the above reactions (1–6) to wax biosynthesis in vivo is discussed. Production of oleate from acetate by enzymes utilizing ACP‐thioesters as substrates followed by conversion of oleyl‐ACP to oleoyl‐CoA (via free oleic acid) for subsequent elongation, reduction, and esterification, is presented as the most probable in vivo pathway, for wax biosynthesis. The substrate specificities of the elongation and reduction reactions utilizing acyl‐CoAs as substrates are examined in terms of wax composition.

The storage triacylglycerols of meadowfoam (Limnanthes alba) (10). The triacylglycerol fraction of the mature seed is composed principally of 20: 1 (Sc), 22:1(13c), and 22:2(5c13c) fatty acids (13,20). Our interest in this plant seed stems from the fact that the developing seed contains the enzymes necessary for the biosynthesis of C20 and C22 acids. The fatty acids also contain an unusual A5-cis double bond. This study complements our concurrent studies on long-chain fatty acid biosynthesis using developing seeds from nasturtium (17), jojoba (15, 16), and rapeseed.The biosynthesis of 20:1(1 Ic) and 22:1(13c) in oilseeds by chain elongation of preformed oleate rather than by a complete de novo biosynthesis is now a well-documented phenomenon (1,5,(15)(16)(17). Our recent studies with cell-free extracts from developing jojoba cotyledons have shown that long-chain acyl-CoA thioesters, in-

Oleate A'2-hydroxylase activty was measured in extracts of developing castor bean seeds. Most of the hydroxylase activity is associated with microsomes. However, when microsomes are washed, the activity is completely lost. Some (50%) of the activity can be restored by addition of the 100,000g supernatant to the washed microsomes. Supernatant extracts (100,000g) of developing safflower seeds are able to restore all (100%) of the hydroxylase activity to the washed castor bean microsomes. 2 Abbreviations: PC, phosphatidylcholine; ACP, acyl carrier protein.as described below. The TLC spot containing PC was sequentially eluted with 2 ml 50 mm acetic acid, 3.3 ml ethanol, and 6.6 ml chloroform. ACP was isolated from Escherichia coli by the method of Majerus et al. (9). All other reagents were obtained from Sigma. Developing castor bean seeds (Ricinus communis var Baker 290)were harvested from the field from August to December, 1979. Preparation of Cell Fractions. As mentioned previously (6), maximum hydroxylase activity was obtained during a narrow period of seed development. For the study reported here, seeds were chosen when their endosperm filled all but the outer 1 mm of the volume of the seed. A previous study showed that, at this stage, 80 to 90%7o of the storage lipid had been synthesized (11).After removal of the seed coat, the endosperm and embryonic axes were washed in cold water and homogenized with a mortar and pestle in 2 volumes grinding buffer (0.6 M sucrose, 0.15 M Tricine, 10 mm KCI, I mM MgCl2, I mm EDTA, and the pH was adjusted to 7.5 with KOH). The homogenate was filtered through 44-,um nylon cloth and centrifuged at 500g for 10 min. The 5OOg supernatant and fat pad were combined and centrifuged at 12,000g for 30 min. To 2 ml 12,000g supernatant was added 0.02 volume 100 mm a-tocopherol (or other antioxidants when noted) in ethanol, and the preparation was centrifuged at 105,000g for I h. The 100,000g pellet (microsomes) was resuspended in 2 ml grinding buffer and 0.02 volumes 100 mm a-tocopherol with a Teflon homogenizer. The resuspended microsomes then were washed by recentrifugation at 105,000g for I h. This washed microsomal pellet was finally resuspended in 2 ml grinding buffer and 0.02 volume 100 mmi a-tocopherol.Enzyme Assays. Oleate hydroxylase was routinely assayed for 30 min at 30 C in screw-cap vials by a radioisotopic method. The reaction mixture contained 0.3 M sucrose, 0.1 M Tris-HCl buffer (pH 7.0), 0.2 ms NADH, 0.4 nmol [1-14C]oleoyl-CoA (about 40,000 cpm), and 25 pl enzyme in a total volume of 1 ml. The reaction was stopped by adding 1 ml 15% methanolic KOH. The lipids were saponified by capping the vials and heating at 80 C for 30 min. The samples then were cooled and neutralized with HCI, and the lipids were extracted with 7 ml hexane:isopropanol (3:2) and 5 ml 6.7% Na2SO4. The fatty acids were methylated with diazomethane and separated on a stainless steel column (155 cm x 7 mm) packed with GP88-SE30 (14.3% on Chromosorb W, AW 60/80 mesh; Analabs). A Varian-Aerograph 920...