Arachidoyl- and arachidonoyl-CoA elongation mechanism in swine cerebral microsomes

Long-chain saturated and polyunsaturated fatty acyl-CoA elongations were studied in swine cerebral microsomes. The elongation of endogenous palmitoyl-CoA to stearate was highly active in both cerebral and liver microsomes, whereas those of arachidoyl-CoA (20:0-CoA) and endogenous arachidonoyl-CoA (20:4-CoA) were high in cerebral microsomes, but negligible in liver microsomes. The elongation of 22:4 to 24:4 was also observed in cerebral microsomes. Both NADPH and NADH at 500 microM were effective in elongation of 16:0-, 20:0- and 20:4-CoA, whereas NADPH was more effective in elongation of 22:4 to 24:4 than NADH. The incorporation of deuterium atoms to the elongated product was detected by the technique of mass fragmentography when the NADPH-dependent elongations of 20:0-CoA and 20:4-CoA were performed in 2H2O medium upon cerebral microsomes. The number of incorporated deuterium atoms into 22:0 elongated from 20:0-CoA was mainly two, and that into 22:4 elongated from 20:4-CoA was mainly three. These results indicated that part of hydrogens in elongated arachidoyl- and arachidonoyl-CoA were transferred from NADPH.     

Characteristics of Synthesis of Very-Long-Chain Saturated and Tetraenoic Fatty Acids in Swine Cerebral Microsomes

The elongation of arachidoyl-CoA (20:0-CoA) yielded 22:0 and 24:0 concomitantly, whereas the elongation of behenoyl-CoA (22:0-CoA) yielded only a negligible amount of 24:0 in adult swine cerebral microsomes. The dependence on time, pH, and the substrate concentrations were examined for the synthesis of 22:0 and 24:0 from 20:0-CoA. A microcomputer-aided simulation study suggested that there were two parallel pathways in the elongation of 20:0-CoA to 22:0 and 24:0. The elongation of 22:0-CoA could not be observed in adult swine cerebral microsomes; however, it was observed clearly in newborn swine and rat brain microsomes. A dilution experiment with the addition of cold 22:0-CoA in the reaction of elongation of 20:0-CoA confirmed the above suggestion that no intermediate 22:0 appeared during the synthesis of 24:0 from 20:0-CoA. The elongation of endogenous 20:4-CoA to 22:4 and 24:4 was examined in newborn swine cerebral microsomes, and the presence of two parallel pathways in the elongation of 20:4-CoA to 22:4 and 24:4 similar to those involved in the elongation of 20:0-CoA to 22:0 and 24:0 was suggested.     

Activation of synthesis of hexacosenoic acid by sulfhydryl reagents in swine cerebral microsomes

The elongation of icosenoyl-CoA (20:1-CoA) in swine cerebral microsomes resulted in the synthesis of docosenoic acid (22:1) and tetracosenoic acid (24:1), but the synthesis of hexacosenoic acid (26:1) was negligible. In contrast, in the presence of sulfhydryl reagents (0.6 mM N-ethylmaleimide [NEM] or 0.3 mM p-chloromercuriphenylsulfonic acid [PCMPS]) the synthesis of 26:1 was remarkably enhanced. We suggest that the synthesis of 26:1 from 20:1-CoA was more enhanced by NEM or PCMPS as a result of activation of the condensation step in the elongation of 24:1 (intermediate) to 26:1.     

Comparison between condensation and overall chain elongation of arachidoyl-CoA and arachidonoyl-CoA in swine cerebral microsomes

The condensation and overall elongation products of exogenous arachidoyl-CoA (20:0-CoA) and endogenous fatty acids in swine cerebral microsomes were detected by radio gas chromatography. In addition, the condensation products with malonyl-CoA as substrate were analyzed by radio high-performance liquid chromatography. Three main condensation products were detected; the overall elongation products of exogenous 20:0-CoA were 22:0 and 24:0, and those of endogenous substrates were 18:0, 22:4, and 24:4. The yield was estimated for the conversion of 3-ketoacyl-CoAs to the corresponding saponification products (methyl ketones or R-2-one; e.g., 2-heptadecanone = 17:0-2-one); these products were identified in the preceding paper (S. Yoshida and M. Takeshita (1987) Arch. Biochem. Biophys. 254, 170-179). The extraction of R-2-one by hexane depended on the acyl chain length. The yield of 2-heneicosanone (21:0-2-one) detected by radio gas chromatography was 80% whereas the yields of 17:0-2-one and 2-heneicosatetraenone (21:4-2-one) from the corresponding 3-ketoacyl-CoAs were 56 and 48%, respectively. A quantitative comparison was performed for the condensation and overall elongation activity; it was noticed that the condensation activity for the system which simultaneously produced two elongation products was nearly the same as that of the corresponding overall elongation activity. This result suggests that the condensation step may be at least one of the rate-limiting steps in the overall elongation of very-long-chain fatty acyl-CoA.     

Fatty acyl-CoA elongation in Blatella germanica integumental microsomes

Insect cuticular hydrocarbons are synthesized de novo in integumental tissue through the concerted action of fatty acid synthases (FASs), fatty acyl-CoA elongases, a reductase, and a decarboxylase to produce hydrocarbons and CO2. Elongation of fatty acyl-CoAs to very long chain fatty acids was studied in the integumental microsomes of the German cockroach, Blatella germanica. Incubation of [1-14C]palmitoyl-CoA, malonyl-CoA, and NADPH resulted in the production of 18-CoA with minor amounts of C20, C22, C24, C30, and C32 labeled acyl-CoA moieties. Similar experiments with [1-14C]stearoyl-CoA rendered C20-CoA as the major product, and lesser amounts of C22 and C24-CoAs were also detected. After solubilization of the microsomal FAS, kinetic parameters were determined radiochemically or by measuring NADPH consumption. The reaction velocity was linear for up to 3 min incubation time, and with a protein concentration up to 0.025 microg/microl. The effect of the chain length on the reaction velocity was compared for palmitoyl-CoA, stearoyl-CoA, and eicosanoyl-CoA. The optimal substrate concentration was 10 microM for C16-CoA, between 8 and 12 microM for C18-CoA, and close to 3 microM for C20-CoA. In vivo hydrocarbon biosynthesis was inhibited from 55.5 to 72.5% in the presence of 1 mM trichloroacetic acid, a known inhibitor of elongation reactions.     

Inhibitory effect of very-long-chain monounsaturated fatty-acyl-CoAs on the elongation of long-chain fatty acid in swine cerebral microsomes

Characteristics of condensation and overall elongation of very-long-chain fatty-acyl-CoAs in swine cerebral microsomes were studied using radio high-performance liquid chromatography (RHPLC) and gas chromatography-mass spectrometry (GC-MS). The monounsaturated fatty-acyl-CoA depressed both the condensation and overall elongation activities of endogenous substrates and also of exogenous saturated fatty-acyl-CoA. The extent of the decrease of the elongation activity was dependent on the concentration and the chain length of the exogenous fatty-acyl-CoAs. The dependence of the condensation activity of monounsaturated fatty-acyl-CoA on the concentration of malonyl-CoA suggested that the non-Michaelis-Menten type kinetics was dominant for oleoyl-CoA, however, a normal kinetic pattern was obtained for endogenous palmitoyl-CoA and arachidonoyl-CoA with Km = 37 microM to malonyl-CoA. The condensation activity for icosanoyl-CoA (20:0-CoA) was inhibited by icosenoyl-CoA (20:1-CoA) in a non-competitive manner, which suggested that the condensation enzyme, or at least the active center of the enzyme for icosenoyl-CoA, was different from that for icosanoyl-CoA.     

Analysis of the condensation step in elongation of very-long-chain saturated and tetraenoic fatty acyl-CoAs in swine cerebral microsomes

The condensation products in the elongation of exogenous arachidoyl-CoA (20:0-CoA) and endogenous fatty acids in adult swine cerebral microsomes were isolated and purified by using HPLC and a radioanalyzer. A saponification product of the condensation reaction of 20:0-CoA with malonyl-CoA was identified by gas chromatography-mass spectrometry as 2-heneicosanone (21:0-2-one). The endogenous substrates (16:0-CoA and 20:4-CoA) were likewise identified as 2-heptadecanone (17:0-2-one) and 2-heneicosatetraenone (21:4-2-one). Quantitative analysis of condensation activity was performed using electron-impact mass fragmentography. A characteristic fragment ion (m/z 59) of 21:0-2-one was used to estimate the condensation activity for 20:0-CoA, and fragment ions at m/z 58 and 80 were monitored for the endogenous substrates (16:0-CoA and 20:4-CoA, respectively). The molecular ion for each product was detected using chemical ionization. A comparative study of the condensation of 20:0-CoA and endogenous substrates was carried out for microsomes obtained from white matter, gray matter, and isolated neuronal cells; the activity for 20:0-CoA was significantly lower in gray matter and neuronal cells than in white matter, whereas the activity for endogenous substrates was almost the same for microsomes obtained from gray and white matter. This result suggests that the condensation enzyme for 20:0-CoA may be different from that for endogenous 16:0-CoA or 20:4-CoA in swine cerebral microsomes.     

Concomitant decrease in the elongation and condensation activities of very-long chain fatty acyl-CoA in jimpy mouse

Overall elongation and condensation of long-chain and very-long-chain fatty acids have been studied in the brain microsomes of jimpy mice. Both the elongation and condensation activities with stearoyl (18:0)-, oleoyl (18:1)- and arachidoyl (20:0)-CoA were severely diminished in jimpy brain, but the decrease in the activity with the exogenous palmitoyl (16:0)-CoA was less pronounced. The decrease in the elongation and condensation reactions with endogenous palmitic and arachidonic (20:4) acids was not distinct in the mutant. The decrease in the activity of condensation reaction may be responsible for the reduced rate of overall fatty acid elongation.     

Effect of fatty-acyl-CoAs on the elongation of saturated fatty acid in porcine aorta microsomes

The microsomal elongation system from porcine aorta for longchain fatty-acyl-CoAs was investigated. Palmitoleoyl-CoA (16:1-CoA), oleoyl-CoA (18:1-CoA), and eicosenoyl-CoA (20:1-CoA) remarkably depressed the elongation activity for 16:0-CoA in aorta microsomes by 44.8, 52.4, and 43.7% of the control activity, respectively. Saturated and polyunsaturated fatty-acyl-CoAs had little effect on the 16:0-CoA elongation activity. These results indicate that monounsaturated long-chain fatty acyl-CoAs can regulate the synthesis of saturated fatty acids in the vessel walls.     

Solubilization of C18-Co A and C20-CoA elongases from Allium porrum L. epidermal cell microsomes

The effects of n-octyl-β-D-glucopyranoside, Triton X-100 and deoxycholate on acyl-CoA elongation by Allium porrum L. epidermal cell microsomes showed that the Triton X-100 specifically stimulated the synthesis of C22–C26 acids using C18-CoA as primer, whereas the fatty acid elongation products of C20-CoA remained essentially unchanged. n-Octyl-β-D-glucopyranoside increased the C20 and C22 fatty acid syntheses to the same extent and deoxycholate inhibited C18-CoA and C20-CoA elongation. The presence of two different elongation systems, as suggested by these results, has been demonstrated. After solubilization by Triton X-100, the C18-CoA and C20-CoA elongases were separated by sucrose density centrifugation. The fractions corresponding to sucrose concentrations of 0.51 and 0.62 M presented the maximal activities for C18-CoA and C20-CoA elongases, respectively. In addition, by gel filtration on a Sephacryl S-300 column, the C20-CoA and the C18-CoA elongases have estimated apparent molecular masses under detergent conditions of 600 and 350 kDa, respectively.     

Stereospecific incorporation of hydrogens from NADPH in elongation of very long fatty acyl-CoA by swine cerebral microsomes

The elongation of arachidoyl-CoA by swine cerebral microsomes resulted in the production of behenic acid (22:0) and lignoceric acid (24:0) concomitantly. When 4S-[4-²H₁]NADPH was used for the elongation of arachidoyl-CoA, the incorporation of two deuterium atoms into 22:0 was observed by the technique of mass fragmentography. Furthermore, the incorporation of four deuterium atoms into 24:0 was also detected. On the other hand, when 4R-[4-²H₁]NADPH was used, no deuterium was incorporated into the elongated products.     

Tissue and chain length specificity of the fatty acyl-CoA elongation system in the American cockroach

The elongation of fatty acyl-CoAs, reactions involved in hydrocarbon biosynthesis, was examined in the cockroach, Periplaneta americana. Products were analyzed by radio-HPLC and radio-GLC. The majority of the elongation activity was observed in microsomes prepared from abdominal epidermal tissue. Linoleoyl-CoA (18:2-CoA) was elongated most efficiently followed by stearoyl-CoA (18:0-CoA), linolenoyl-CoA (18:3-CoA; n-3) and oleoyl-CoA (18:1-CoA). The products of 18:2-CoA elongation included all even numbered acyl groups up to 28 carbons, and the products of 18:0-CoA included all even numbered acyl groups to 26 carbons. The 18:3-CoA was elongated only to 20 and 22 carbons. Radioactivity from both 18:2-CoA (5.4%) and 18:0-CoA (1.2%) was recovered in the hydrocarbon fraction. Analysis of this hydrocarbon fraction showed that the radio-activity from 18:2-CoA was present in (Z,Z)-6,9-heptacosadiene and that the radioactivity from 18:0-CoA was present in n-pentacosane. These data demonstrate for the first time in an in vitro insect system that the fatty acid elongation reactions are coupled with the conversion of the elongated product to hydrocarbon. Thus, each of the expected intermediates in the conversion of 18:0 and 18:2 to 25 and 27 carbon hydrocarbons, respectively, was observed, and the results demonstrate high tissue, substrate, and product specificity.     

Study of the 3-hydroxy eicosanoyl-coenzyme A dehydratase and (E)-2,3 enoyl-coenzyme A reductase involved in acyl-coenzyme A elongation in etiolated leek seedlings

(R,S)-[1-¹⁴C]3-Hydroxy eicosanoyl-coenzyme A (CoA) has been chemically synthesized to study the 3-hydroxy acyl-CoA dehydratase involved in the acyl-CoA elongase of etiolated leek (Allium porrum L.) seedling microsomes. 3-Hydroxy eicosanoyl-CoA (3-OH C20:0-CoA) dehydration led to the formation of (E)-2,3 eicosanoyl-CoA, which has been characterized. Our kinetic studies have determined the optimal conditions of the dehydration and also resolved the stereospecificity requirement of the dehydratase for (R)-3-OH C20:0-CoA. Isotopic dilution experiments showed that 3-hydroxy acyl-CoA dehydratase had a marked preference for (R)-3-OH C20:0-CoA. Moreover, the very-long-chain synthesis using (R)-3-OH C20:0-CoA isomer and [2-¹⁴C]malonyl-CoA was higher than that using the (S) isomer, whatever the malonyl-CoA and the 3-OH C20:0-CoA concentrations. We have also used [1-¹⁴C]3-OH C20:0-CoA to investigate the reductant requirement of the enoyl-CoA reductase of the acyl-CoA elongase complex. In the presence of NADPH, [1-¹⁴C]3-OH C20:0-CoA conversion was stimulated. Aside from the product of dehydration, i.e. (E)-2,3 eicosanoyl-CoA, we detected eicosanoyl-CoA resulting from the reduction of (E)-2,3 eicosanoyl-CoA. When we replaced NADPH with NADH, the eicosanoyl-CoA was 8- to 10-fold less abundant. Finally, in the presence of malonyl-CoA and NADPH or NADH, [1-¹⁴C]3-OH C20:0-CoA led to the synthesis of very-long-chain fatty acids. This synthesis was measured using [1-¹⁴C]3-OH C20:0-CoA and malonyl-CoA or (E)-2,3 eicosanoyl-CoA and [2-¹⁴C]malonyl-CoA. In both conditions and in the presence of NADPH, the acyl-CoA elongation activity was about 60 nmol mg⁻¹ h⁻¹, which is the highest ever reported for a plant system.     

Acyltransferase activities in adult rat type II pneumocyte-derived subcellular fractions

Acyl-CoA: lysophosphatidylcholine, acyl-CoA: lysophosphatidylethanolamine, and lysophosphatidylcholine:lysophosphatidylcholine acyltransferases were investigated using subcellular fractions derived from adult rat type II pneumocytes in primary culture. Acyl-CoA:lysophospholipid acyltransferase activities were determined to be microsomal, while lysophosphatidylcholine:lysophosphatidylcholine acyltransferase activity was found to be cytosolic. Total palmitoyl CoA:lysophosphatidylcholine acyltransferase activity was 30-fold greater than lysophosphatidylcholine:lysophosphatidylcholine acyltransferase activity, indicating that the former enzyme is more important in the synthesis of dipalmitoyl phosphatidylcholine. Palmitoyl-CoA and oleoyl-CoA lysophosphatidylcholine acyltransferase activities were approximately equal under optimal substrate conditions. Specific activities of the enzyme using arachidoyl-CoA and arachidonoyl-CoA were 46% and 18%, respectively, of those with palmitoyl-CoA. Acyl-CoA:lysophosphatidylethanolamine acyltransferase showed a preference for palmitoyl-CoA as opposed to oleoyl-CoA under optimal conditions. However, when equimolar concentrations of either palmitoyl-CoA and oleoyl-CoA or palmitoyl-CoA and arachidoyl-CoA were assayed together, the relative utilization of the two substrates was found to be dependent on total acyl-CoA concentration. At higher concentrations, the incorporation of palmitoyl-CoA into phosphatidylcholine was less than other acyl-CoAs. However, at lower concentrations palmitoyl-CoA was utilized quite selectively. Whole lung microsomes did not show as marked a preference for palmitoyl-CoA as did type II pneumocyte microsomes under these same conditions. In similar experiments, low total acyl-CoA concentrations produced greater incorporation of oleoyl-CoA into phosphatidylethanolamine. For both enzymes total activity at the lowest concentrations used was at least 45% that at optimal conditions. This demonstrates that the type II pneumocyte acyltransferase system(s) can selectively utilize palmitoyl-CoA. No evidence for direct exchange of palmitoyl-CoA with 1-saturated-2-unsaturated phosphatidylcholine in subcellular fractions from type II pneumocytes was found.     

Effect of phospholipase A2 and free fatty acids on lipid-protein interactions in long- and very-long-chain fatty acyl-CoA elongation enzyme systems of brain microsomes

The elucidation of the mechanism of phospholipase A2-induced inactivation of the condensation enzyme provided evidence concerning the important role of lipid-enzyme interactions in maintaining the condensation activity in swine cerebral microsomes. A quantitative analysis of fatty acid release by phospholipase A2 from the microsomal membrane revealed that only 5 nmol of free fatty acid per mg microsomal protein was released, including oleic acid and arachidonic acid, by treatment with 0.4 unit of phospholipase A2 per mg microsomal protein for 15 s at 23 degrees C. Under these conditions, the condensation activity for endogenous 16:0-CoA and 20:4-CoA decreased to half and that for exogenous 20:0-CoA decreased to 75%. However, the addition of free fatty acids and lysophospholipids or a mixture of them at 5-10 nmol/mg protein did not change the condensation activity for endogenous 16:0-CoA and 20:4-CoA, or for exogenous 20:0-CoA. These results indicated that phospholipase A2 inhibited the condensation activity by acting directly on phospholipids that are indispensable to maintaining the function of the condensation enzyme. The Arrhenius plot for the condensation of endogenous 16:0-CoA showed a break at around 16 degrees C, whereas no break of the plot was observed for the condensation of 20:0-CoA and 20:4-CoA. The activation energy for the condensation of 16:0-CoA and 20:4-CoA was decreased by the addition of free fatty acids such as oleic acid and stearic acid, with disappearance of the Arrhenius break for 16:0-CoA condensation, whereas the activation energy for the condensation of 20:0-CoA was not changed. These results suggest that the type of lipid-protein interaction in the condensation enzyme for 20:0-CoA is different from that for 16:0-CoA and 20:4-CoA.     

Long-chain and very long-chain fatty acyl-coa synthetase activity in microsomes of jimpy mouse brain

The objective of this study was to determine whether the conversion of free, very long chain fatty acids (C₂₂–C₂₆) to their CoA-esters are involved in cerebroside synthesis, since cerebrosides are uniquely rich in very long chain fatty acids including lignoceric acid (C_24:0). We have studied lignoceroyl-CoA synthetase activity in the microsomes isolated from normal and jimpy mouse brain. The jimpy mouse lacks the ability to make myelin and is deficient in enzyme activities involved in the synthesis of myelin components, including cerebrosides. Unexpectedly, the lignoceroyl-CoA synthetase activity in jimpy brain microsomes was slightly higher than that in control microsomes. The palmitoyl (C_16:0)-CoA synthetase activity in jimpy brain was not different from the control. The level of cerebrosides in microsomes was grossly lower in jimpy brain. The implication of these findings and the involvement of lignoceric acid activation in cerebroside synthesis is discussed.     

Palmitoyl-CoA Elongation in Brain Microsomes: Dependence on Cytochrome b5 and NADH-Cytochrome b5 Reductase

Experiments were performed to demonstrate the involvement of electron transport system in fatty acid elongation in rat brain microsomes. Mercuric chloride and p-chloromercuriphenylsulfonate, inhibitors on NADH-cytochrome b5 reductase, at 32 microM inhibited NADH-supported palmitoyl-CoA elongation to 30 and 60% of control activity, respectively, whereas NADPH-supported palmitoyl-CoA elongation was unaffected by these mercurials. An antibody to rat liver NADH-cytochrome b5 reductase inhibited brain microsomal NADH-cytochrome b5 reductase activity and NADH-dependent palmitoyl-CoA elongation. Treatment of brain microsomes with trypsin diminished the cytochrome b5 content; NADH- and NADPH-cytochrome c reductase activities were significantly decreased, but the decrease in NADH-cytochrome b5 reductase activity was relatively small. Whereas essentially no incorporation of malonyl-CoA into palmitoyl-CoA was observed with trypsin-treated microsomes, addition of detergent-solubilized cytochrome b5 resulted in a recovery of fatty acid elongation. These results indicate the presence of an electron transport system, NADH-NADH-cytochrome b5 reductase-cytochrome b5-fatty acid elongation, in brain microsomes.     

Inhibition of very-long-chain fatty acid formation by indanofan, 2-[2-(3-chlorophenyl)oxiran-2-ylmethyl]-2-ethylindan-1,3-dione, and its relatives

Indanofan and its analogs inhibited the elongation of stearoyl- or arachidoyl-CoA by [2-14C]-malonyl-CoA in leek microsomes from Allium porrum. Although the precise mode of interaction of indanofan at the molecular level is not completely clarified by the present study, it is concluded that indanofan and analogs act as inhibitor of the elongase enzyme involved in de novo biosynthesis of fatty acids with an alkyl chain longer than C18, called very-long-chain fatty acids (VLCFAs). For a strong inhibition of VLCFA formation chloro substituents at the benzene ring and the oxirane group were necessary. Furthermore, the greenhouse test showed strong activity for indanofan and its analogs, and the scores coincided with cell-free elongation inhibition. The cell-free assay, however, failed to indicate any activity for an analog having a methylene instead of the oxirane group, while both Digitaria ciliaris and Echinochloa oryzicola were killed with 1 kg a.i./ha. This finding cannot be discussed because the applied use rate of 1 kg a.i./ha is too high to allow for a score differentiation. For high concentrations of this compound additional unknown inhibitory effects may be involved besides fatty acid elongation.     

Regulation of sex pheromone biosynthesis in the housefly, Musca domestica: relative contribution of the elongation and reductive steps.

The regulation of production of the sex pheromone (Z)-9-tricosene (Z9-23:Hy) in the housefly, Musca domestica, was studied by examining the chain length specificity of the fatty acyl-CoA elongation reactions and the reductive conversion of fatty acyl-CoAs to alkenes in 1- and 4-day-old male and female houseflies. Microsomal preparations from 4-day-old female insects produced as the predominant alkene Z9-23:Hy when incubated with malonyl-CoA, NADPH, and [9,10-3H2]oleoyl-CoA (18:1-CoA), whereas microsomal preparations from 4-day-old male insects produced predominantly (Z)-9-heptacosene (Z9-27:Hy). These are the major alkenes produced in vivo by Day 4 females and males, respectively. Microsomes prepared from both Day 1 males and Day 1 females produced Z9-27:Hy as the major alkene from labeled 18:1-CoA. This is the major alkene produced in vivo by both sexes at Day 1. An examination of the chain length specificity of the elongation reactions showed that microsomes prepared from Day 4 male insects readily elongated both 18:1-CoA and 15-[15,16-3H2]tetracosenoyl-CoA (24:1-CoA) to 28-carbon moieties, whereas microsomes from Day 4 female insects did not efficiently elongate either substrate beyond 24 carbons. With high substrate concentrations, microsomes prepared from male insects converted 24:1-CoA to Z9-23:Hy more efficiently than did those from females, whereas under lower and presumably more physiological substrate concentrations, microsomes from females had slightly higher activity than did those from males. Taken together, these data show that the regulation of the chain length of the alkenes, and thus sex pheromone production, in the housefly resides predominantly in the elongation reactions and not in the step which converts the fatty acyl-CoA to hydrocarbon.     

Elongation of fatty acids by microsomal fractions from the brain of the developing rat.

Elongation of fatty acids by microsomal fractions obtained from rat brain was measured by the incorporation of [2-14C]malonyl-CoA into fatty in the presence of palmitoyl-CoA or stearoyl-CoA. 2. Soluble and microsomal fractions were prepared from 21-day-old rats; density gradient centrifugation demonstrated that the stearoyl-CoA elongation system was localized in the microsomal fraction whereas fatty acid biosynthesis de novo from acetyl-CoA occurred in the soluble fraction. The residual activity de novo in the microsomal fraction was attributed to minor contamination by the soluble fraction. 3. The optimum concentration of [2-14C]malonyl-CoA for elongation of fatty acids was 25 mum for palmitoyl-CoA or stearoyl-CoA, and the corresponding optimum concentrations for the two primer acyl-CoA esters were 8.0 and 7.2 muM respectively. 4. Nadph was the preferred cofactor for fatty acid formation from palmitoyl-CoA or stearoyl-CoA, although NADH could partially replace it. 5. The stearoyl-CoA elongation system required a potassium phosphate buffer concentration of 0.075M for maximum activity; CoA (1 MUM) inhibited this elongation system by approx. 30%. 6. The fatty acids formed from malonyl-CoA and palmitoyl-CoA had a predominant chain length of C18 whereas stearoyl-CoA elongation resulted in an even distribution of fatty acids with chain lengths of C20, C22 and C24. 7. The products of stearoyl-CoA elongation were identified as primarily unesterified fatty acids. 8. The developmental pattern of fatty acid biosynthesis by rat brain microsomal preparations was studied and both the palmitoyl-CoA and stearoyl-CoA elongation systems showed large increases in activity between days 10 and 18 after birth.