Characterization of fatty acid elongation system in porcine neutrophil microsomes

Microsomes purified from porcine neutrophils containing the fatty acid chain-elongation system for long- and very-long-chain fatty acyl-CoAs, and several enzymatic characters for the elongation of palmitoyl-CoA (16:0-CoA) and arachidoyl-CoA (20:0-CoA) were examined. The heat-inactivation profile for the elongation of 16:0-CoA was different from that of 20:0-CoA, suggesting the presence of different enzyme systems for palmitoyl-CoA and arachidoyl-CoA. Contrary to the elongation system of brain microsomes, the successive synthesis of lignoceric acid (24:0) from 20:0-CoA at 60 microM was not prominent under normal conditions in the neutrophil microsomes. The synthesis of behenic acid (22:0) was slightly inhibited by 0.5 mM N-ethylmaleimide (NEM) present in the assay mixture, whereas the pre-treatment of microsomes with 0.5 mM NEM largely inhibited the synthesis of 22:0 from 20:0-CoA. The synthesis of 24:0, however, was enhanced by 0.5 mM NEM in the elongation of 20:0-CoA and the rate of 24:0 synthesis became dominant over the synthesis of 22:0. These results suggested that the elongation enzyme for very-long-chain fatty acyl-CoA, especially for 20:0-CoA elongation to 22:0 in the neutrophil microsomes contained NEM-sensitive sulfhydryl groups in the active center and the mechanism for the synthesis of 24:0 through successive elongation from 20:0-CoA was different from that of 22:0, as the former was enhanced by NEM whereas the latter was strongly inhibited.     

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.     

Oleoyl-CoA is not an immediate substrate for fatty acid elongation in developing seeds of Brassica napus

The substrate specificity of fatty acid elongase was studied using an oil body fraction from developing seeds of Brassica napus. ATP was essential for high rates of elongase activity, but there was no apparent requirement for oleoyl-CoA, oleic acid (18:1) or CoA. Furthermore, ¹⁴C from 18:1-CoA was incorporated into eicosenoic (20:1) and erucic (22:1) acids at a much slower rate than ¹⁴C from malonyl-CoA. Incubation of [¹⁴C]18:1-CoA with the oil body fraction resulted in a rapid loss of [¹⁴C]18:1-CoA into several lipid fractions whether in the absence or presence of ATP, but the loss of 18:1-CoA had a comparatively small effect on the overall rate of elongation. Acyl-CoAs were derivatized to their respective acylbutylamide and analyzed by gas chromatography-mass spectrometry. This analysis of acyl-CoAs demonstrated that there was no detectable 20:1-CoA or 22:1-CoA at 0 min incubation, while newly synthesized 20:1-CoA and 22:1-CoA were present at 10 min. Analysis of the %¹⁴C of the substrates and products of the elongation reaction revealed that the endogenous pool of 18:1-CoA is quite small in elongase preparations. In addition, [¹⁴C]18:1-CoA added to the incubation, although incorporated into lipids, was not significantly diluted by turnover or new synthesis. In contrast, the %¹⁴C of the 20:1-CoA was two- to threefold less than that of the 18:1-CoA. Taken together, these results indicate that the [¹⁴C]18:1 from the [¹⁴C]18:1-CoA was diluted in an intermediate 18:1 pool and that the 18:1-CoA was not the major donor of the acyl group to the elongase reaction.     

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.     

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.     

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.     

Chain elongation in the formation of polyunsaturated fatty acids by brain: Some properties of the microsomal system

Chain elongation of polyunsaturated acids has been investigated using microsomes from developing rat brain. With 18:3(n ? 6) in 0.05% detergent as an acceptor and [2-¹⁴C]malonyl-coenzyme A (CoA) as a two-carbon donor, incorporation of radioactivity into 20:3 was optimal (and incorporation into other acyl chains was minimal) in the presence of 100 μm substrate, 200 μmp-bromophenacylbromide and 10 mm KCN. Up to 30% of the labeled products were incorporated into phospholipids and triacylglycerol. Maximal microsomal elongation activity was observed at 3–4 weeks of age. Several other fatty acid or acyl-CoA acceptors tested in this system were elongated at slower rates compared to 18:3(n ? 6) [e.g., 16:0-CoA, 75%; 20:4(n ? 6), 57%; 18:3(n ? 3), 13%; 18:2(n ?6), 10%; 20:3(n ? 6), 6%]. The rate of elongation of chemically synthesized 18:3-CoA was only 50% of the detergent-suspended acid and was optimal at 6 μm substrate; inhibition above 6 μm 18:3-CoA was reduced by bovine serum albumin, but incorporation of label into palmitate was greatly stimulated. CoA markedly inhibited elongation of 18:3(n ? 6) or 18:3-CoA; N-ethylmaleimide at equimolar amounts reversed this CoA inhibition but did not alter the inhibition caused by concentrations of 18:3-CoA above 6 μm. ATP was absolutely required for elongation of either the free acid or the acyl-CoA derivative, whereas exogenous MgCl₂ had little effect.     

Studies on the metabolic fate of n-3 polyunsaturated fatty acids

Several different processes involved in the metabolic fate of docosahexaenoic acid (DHA, C22:6n-3) and its precursor in the biosynthesis route, C24:6n-3, were studied. In cultured skin fibroblasts, the oxidation rate of [1-14C] 24:6n-3 was 2.7 times higher than for [1-14C]22:6n-3, whereas [1-14C]22:6n-3 was incorporated 7 times faster into different lipid classes than was [1-14C]24:6n-3. When determining the peroxisomal acyl-CoA oxidase activity, similar specific activities for C22:6(n-3)-CoA and C24:6(n-3)-CoA were found in mouse kidney peroxisomes. Thioesterase activity was measured for both substrates in mouse kidney peroxisomes as well as mitochondria, and C22:6(n-3)-CoA was hydrolyzed 1.7 times faster than C24:6(n-3)-CoA. These results imply that the preferred metabolic fate of C24:6(n-3)-CoA, after its synthesis in the endoplasmic reticulum (ER), is to move to the peroxisome, where it is beta-oxidized, producing C22:6(n-3)-CoA. This DHA-CoA then preferentially moves back, probably as free fatty acid, to the ER, where it is incorporated into membrane lipids.     

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.     

Adrenoleukodystrophy. The chain shortening of erucic acid (22:1(n-9)) and adrenic acid (22:4(n-6)) is deficient in neonatal adrenoleukodystrophy and normal in X-linked adrenoleukodistrophy skin fibroblasts

The metabolism of long chain unsaturated fatty acids was studied in cultured fibroblasts from patients with X-linked adrenoleukodystrophy (ALD) and with neonatal ALD. By using [14-14C] erucic acid (22:1(n-9)) as substrate it was shown that the peroxisomal beta-oxidation, measured as chain shortening, was impaired in cells from patients with neonatal ALD. The beta-oxidation of adrenic acid (22:4(n-6)), measured as acid-soluble products, was also reduced in the neonatal ALD cells. The peroxisomal beta-oxidation of [14-14C]erucic acid (22:1(n-9)) and [2-14C]adrenic acid (22:4(n-6)) was normal in cells from X-ALD patients. The beta-oxidation, esterification and chain elongation of [1-14C]arachidonic acid (20:4(n-6)) and [1-14C]eicosapentaenoic acid (20:5(n-3)) was normal in both X-linked ALD and in neonatal ALD. Previous studies suggest that the activation of very long chain fatty acids by a lignoceryl (24:0)-CoA ligase is deficient in X-linked ALD, while the peroxisomal beta-oxidation enzymes are deficient in neonatal ALD. The present results suggest that the peroxisomal very long-chain acyl-CoA ligase is not required for activation of unsaturated C20 and C22 fatty acids and that these fatty acids can be efficiently activated by the long chain acyl-(palmityl)-CoA ligase.     

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.     

Metabolism of hexacosatetraenoic acid (C26:4,n-6) in immature rat brain.

Rat brain was recently found to contain polyenoic very-long-chain fatty acids (VLCFA) belonging to the n-3 and n-6 series with four, five and six double bonds and even-carbon chain lengths from 24 to 38 [Robinson, Johnson & Poulos (1990) Biochem. J. 265, 763-767]. In the present paper, the metabolism in vivo of hexacosatetraenoic acid (C26:4,n-6) was studied in neonatal rat brain. Rats were injected intracerebrally with [1-14C]C26:4,n-6 and the labelled metabolites were examined after 4 h. Radioactivity was detected mainly in non-esterified fatty acids, with smaller amounts in other neutral lipids and phospholipids. Radiolabelled fatty acid products included C28-36 tetraenoic and C26-28 pentaenoic VLCFA formed by elongation and desaturation of the substrate, and C14-24 saturated, C16-24 monoenoic, C18-24 dienoic, C18-22 trienoic and C20-24 tetraenoic fatty acids formed from released [1-14C]acetate either by synthesis de novo or by elongation of endogenous fatty acids. The data suggest that polyenoic VLCFA are synthesized in brain from shorter-chain precursor fatty acids and undergo beta-oxidation.     

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.     

Site of inhibition of rat liver microsomal fatty acid chain elongation system by dec-2-ynoyl coenzyme A. Possible mechanism of inhibition

The present study examines the effect of the acetylenic thioester dec-2-ynoyl-CoA (delta 2 10 identical to 1-CoA) on the microsomal fatty acid chain elongation pathway in rat liver. When the individual reactions of the elongation system were measured in the presence of delta 2 10 identical to 1-CoA, the trans-2-enoyl-CoA reductase activity was markedly inhibited (Ki = 2.5 microM), whereas the activities of the condensing enzyme, the beta-ketoacyl-CoA reductase, and the beta-hydroxyacyl-CoA dehydrase were not affected. The absence of inhibition of total microsomal fatty acid elongation was attributed to the significant accumulation of the intermediates, beta-hydroxyacyl-CoA and trans-2-enoyl-CoA, without formation of the saturated elongated product, indicating that the trans-2-enoyl-CoA reductase-catalyzed reaction was the only site affected by the inhibitor. The nature of the inhibition was noncompetitive. In contrast to the delta 2 10 identical to 1-CoA, delta 3 10 identical to 1-CoA did not inhibit trans-2-enoyl-CoA reductase activity, suggesting that the mode of inhibition was not via formation of the 2,3-allene derivative. Based on the observation (a) that p-chloromercuribenzoate markedly inhibits reductase activity, (b) that dithiothreitol protects the enzyme against inactivation by delta 2 10 identical to 1-CoA, (c) of the spectral manifestation of the interaction between thiol reagents and delta 2 10 identical to 1-CoA depicting an absorbance peak similar to that of the beta-ketoacyl thioester-Mg2+ enolate complex, (d) of a similar absorbance spectrum formed by the interaction between delta 2 10 identical to 1-CoA and liver microsomes, and (e) of the absence of formation of a similar spectrum by delta 3 10 identical to 1-CoA, trans-2-10:1-CoA, or delta 2 10 identical to 1 free acid with liver microsomes, we propose that delta 2 10 identical to 1-CoA inactivates trans-2-enoyl-CoA reductase by covalently binding to a critical sulfhydryl group at or in close proximity to the active site of the enzyme.     

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.     

N-ethylmaleimide inhibits Ncd motor function by modification of a cysteine in the stalk domain.

N-Ethylmaleimide (NEM), which reacts readily with exposed sulfhydryl groups, has been shown to inhibit the activity of the microtubule (MT) motors kinesin, Ncd, and dynein. Currently, the mechanism of inhibition is not known for any of these proteins. To investigate the mechanism by which NEM inhibits Ncd, the recombinant Ncd motor-stalk protein MC1 (modified claret 1) was treated with varying concentrations of NEM (0-10 mM) and cosedimentation and ATPase assays were used to assess the effects of modification on MC1 interactions with MTs. In the cosedimentation assay, treatment with /=0.5 mM NEM induced aggregation of MC1 and resulted in sedimentation of the motor in the absence of MTs. NEM modification had no effect on the basal ATPase rate but produced a decrease in the MT-stimulated ATPase rate. Labeling of MC1 with [3H]NEM indicated that enhanced MT binding was associated with an average labeling of 1 Cys residue per MC1 polypeptide, while aggregation was associated with an average labeling of 2 Cys residues per MC1 polypeptide. Protein digestion, structural analysis, and mass spectrometry indicate that modification of Cys313 or Cys324 in the stalk domain is correlated with enhanced binding of MC1 to MTs. These results suggest that NEM enhances Ncd binding to MTs by disruption of neck and/or stalk function and demonstrate the importance of this region in motor function.     

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.     

Determination of the primary structure of intermolecular cross-linking sites on the Ca2(+)-ATPase of sarcoplasmic reticulum using 14C-labeled N,N'-(1,4-phenylene)bismaleimide or N-ethylmaleimide

To determine the intermolecular cross-linking site on the primary structure sarcoplasmic reticulum (SR) Ca-ATPase, the conditions for the specific binding of 14C-labeled 1,4-phenylene bis maleimide (PBM) or 14C-labeled N-ethylmaleimide (NEM) to the ATPase were explored. SR vesicles were preincubated with nonradioactive PBM in the presence of 1 mM vanadate for 1 h, then washed by centrifugation to remove free PBM and vanadate. When the pretreated SR vesicles were allowed to react with 1 mM [14C]PBM in the presence of 1 mM AMPPNP, the amount of [14C]PBM incorporated into the ATPase increased with time in parallel with the formation of dimeric ATPase and reached the maximum labeling density of 1 mol of [14C]PBM per mol of dimeric ATPase at 40 min after the start of the reaction. When the pretreated SR vesicles were allowed to react with 2 mM [14C]NEM in the absence of AMPPNP, a maximum of about 2 mol of NEM was bound per mol of the ATPase monomer. The labeling density of [14C]NEM decreased from 2 to 1 mol per mol of the ATPase when the SR vesicles were allowed to react with [14C]NEM in the presence of AMPPNP. From the analysis of the amino acid composition of the two major [14C]NEM-labeled peptides isolated from the thermolytic digest of the enzyme after the reaction of SR with [14C]NEM in the absence of AMPPNP, we deduced that [14C]NEM was incorporated into Cys377 and Cys614. On the other hand, the labeling of SR in the presence of AMPPNP resulted in inhibition of the [14C]NEM binding to Cys614, leaving Cys377 unaltered.(ABSTRACT TRUNCATED AT 250 WORDS)     

Seed-specific heterologous expression of a nasturtium FAE gene in Arabidopsis results in a dramatic increase in the proportion of erucic acid

The fatty acid elongase [often designated FAE or beta-(or 3-) ketoacyl-CoA synthase] is a condensing enzyme and is the first component of the elongation complex involved in synthesis of erucic acid (22:1) in seeds of garden nasturtium (Tropaeolum majus). Using a degenerate primers approach, a cDNA of a putative embryo FAE was obtained showing high homology to known plant elongases. This cDNA contains a 1,512-bp open reading frame that encodes a protein of 504 amino acids. A genomic clone of the nasturtium FAE was isolated and sequence analyses indicated the absence of introns. Northern hybridization showed the expression of this nasturtium FAE gene to be restricted to the embryo. Southern hybridization revealed the nasturtium beta-ketoacyl-CoA synthase to be encoded by a small multigene family. To establish the function of the elongase homolog, the cDNA was introduced into two different heterologous chromosomal backgrounds (Arabidopsis and tobacco [Nicotiana tabacum]) under the control of a seed-specific (napin) promoter and the tandem 35S promoter, respectively. Seed-specific expression resulted in up to an 8-fold increase in erucic acid proportions in Arabidopsis seed oil, while constitutive expression in transgenic tobacco tissue resulted in increased proportions of very long chain saturated fatty acids. These results indicate that the nasturtium FAE gene encodes a condensing enzyme involved in the biosynthesis of very long chain fatty acids, utilizing monounsaturated and saturated acyl substrates. Given its strong and unique preference for elongating 20:1-CoA, the utility of the FAE gene product for directing or engineering increased synthesis of erucic acid is discussed.     

Metabolism of saturated and polyunsaturated fatty acids by normal and Zellweger syndrome skin fibroblasts.

The metabolism of 1-11C-labelled derivatives of palmitic (C16:0), arachidonic (C20:4,n-6) lignoceric (C21:0) and tetracosatetraenoic (C24:4,n-6) acids was studied in normal skin fibroblast cultures and in cultures of fibroblasts from peroxisome-deficient (Zellweger's syndrome) patients. Radiolabelled products of the fatty acids included carbon dioxide. C14-24 saturated and mono-unsaturated fatty acids formed from released acetate either by synthesis de novo or by elongation of endogenous fatty acids, fatty acids formed by 2-6-carbon elongation of added substrates, and a number of water-soluble compounds, some of which were tentatively identified as the amino acids glutamine, glutamic acid and asparagine. The labelled amino acids were found predominantly in the culture medium. Zellweger's syndrome fibroblasts showed a marked decrease in radiolabelled carbon dioxide and water-soluble-product formation from (I-14C)-labelled arachidonic, tetracosatetraenoic and lignoceric acids but not from [I-14C]palmitic acid, and the production of radiolabelled C14-18 fatty acids was also diminished. However, the elongation of individual fatty acids was either normal or above normal. Our data support the view that the oxidation of 20:4, 24:4 and 24:0 fatty acids in cultured skin fibroblasts takes place largely in peroxisomes, and further that the acetyl-CoA released by the beta-oxidation process is available for the synthesis of fatty acids and amino acids. We speculate that the generation of C2 units used for synthesis is a major peroxisomal function and that this function is absent or greatly impaired in Zellweger's syndrome cells.