Understanding in vivo carbon precursor supply for fatty acid synthesis in leaf tissue

The principal supply of carbon precursors for fatty acid synthesis in leaf tissue has been a much debated topic, with some experiments suggesting a direct supply from the C3 products of photosynthetic carbon fixation and colleagues suggesting the utilization of free acetate (for which concentrations in leaves in the range of 0.05-1.4 mM have been reported). To address this issue we first reassessed the in vivo rate of fatty acid synthesis using a new method, that of [13C]carbon dioxide labeling of intact Arabidopsis plants with the subsequent analysis of fatty acids by gas chromatography-mass spectrometry (GC-MS). This method gave an average value of 2.3 mmoles carbon atoms h-1 mg chlorophyll-1 for photosynthetic tissues. The method was extended by isotopic dilution analysis to measure the rate of fatty acid synthesis in the dark. There was negligible fatty acid synthesis (< 5% of the rate in the light) in the dark. In addition, the method allowed an estimate of the absolute rate of fatty acid degradation of about 4% of the total fatty acid content per day. With the in vivo rate of fatty acid synthesis in the light defined, if the bulk tissue acetate concentration available for fatty acid synthesis is 1 mM, this acetate pool can sustain fatty acid synthesis for approximately 60 min. When the leaves of Arabidopsis, barley and pea were given a 5 min pulse of [14C]carbon dioxide, the label rapidly appeared in fatty acids with a lag phase of less than 2-3 min. Continuous labeling with [14C]carbon dioxide, for up to 1 h, showed a similar result. Furthermore, 14C-label in free acetate was less than 5% of that in fatty acids. In conclusion, these data suggest that either the bulk pool of acetate is not involved in fatty acid synthesis or the concentration of acetate must be less than 0.05 mM under strong illumination.     

On the control of long-chain-fatty acid synthesis in isolated intact spinach (Spinacia oleracea) chloroplasts

1. Chloroplasts isolated from spinach leaves by using the low-ionic-strength buffers of Nakatani & Barber [(1977) Biochim. Biophys. Acta.461, 510-512] had higher rates of HCO(3) (-)-dependent oxygen evolution (up to 369mumol/h per mg of chlorophyll) and higher rates of [1-(14)C]acetate incorporation into long-chain fatty acids (up to 1500nmol/h per mg of chlorophyll) than chloroplasts isolated by using alternative procedures. 2. Acetate appeared to be the preferred substrate for fatty acid synthesis by isolated chloroplasts, although high rates of synthesis were also measured from H(14)CO(3) (-) in assays permitting high rats of photosynthesis. Incorporation of H(14)CO(3) (-) into fatty acids was decreased by relatively low concentrations of unlabelled acetate. Acetyl-CoA synthetase activity was present 3-4 times in excess of that required to account for rates of [1-(14)C]acetate incorporation into fatty acids, but pyruvate dehydrogenase was either absent or present in very low activity in spinach chloroplasts. 3. Rates of long-chain-fatty acid synthesis from [1-(14)C]acetate in the highly active chloroplast preparations, compared with those used previously, were less dependent on added cofactors, but showed a greater response to light. The effects of added CoA plus ATP, Triton X-100 and sn-glycerol 3-phosphate on the products of [1-(14)C]acetate incorporation were similar to those reported for less active chloroplast preparations. 4. Endogenous [(14)C]acetyl-CoA plus [(14)C]malonyl-CoA was maintained at a constant low level even when fatty acid synthesis was limited by low HCO(3) (-) concentrations. Endogenous [(14)C]acyl-(acyl-carrier protein) concentrations increased with increasing HCO(3) (-) concentration and higher rates of fatty acid synthesis, but were slightly lower in the presence of Triton X-100. It is proposed that rates of long-chain-fatty acid synthesis in isolated chloroplasts at saturating [1-(14)C]acetate concentrations and optimal HCO(3) (-) concentrations may be primarily controlled by rates of removal of the products of the fatty acid synthetase.     

The effect of red and far red light on the desaturation of Fatty acids in barley leaves

Barley (Hordeum vulgare) leaf tissue was either (a) exposed to continuous red light or (b) exposed to red, far red, or red followed by far red light. The fatty acid composition and incorporation of acetate-2-¹⁴C into linolenate were determined. Changes occurred in the fatty acid composition of dark-grown barley leaves regardless of whether the plants were subsequently exposed to red light or whether the tissue remained in the dark. Measurements were also made of the fatty acids of the coleoptile. Red light treatment did not reduce the lag period for the synthesis of linolenate when chlorophyll synthesis was inhibited. It appears that the desaturation process per se in the synthesis of linolenate is not phytochrome-mediated but may appear to be phytochrome mediated if, possibly, galactolipid and chlorophyll syntheses occur concomitantly.     

In vivo pools of free and acylated acyl carrier proteins in spinach. Evidence for sites of regulation of fatty acid biosynthesis

In order to examine potential regulatory steps in plant fatty acid biosynthesis, we have developed procedures for the analysis of the major acyl-acyl carrier protein (ACP) intermediates of this pathway. These techniques have been used to separate and identify acyl-ACPs with chain configurations ranging from 2:0 to 18:1 and to determine the relative in vivo concentrations of acyl-ACPs in spinach leaf and developing seed. In both leaf and seed as much as 60% of the total ACPs were nonesterified (free), with the remaining proportion consisting of acyl-ACP intermediates leading to the formation of palmitate, stearate, and oleate. In spinach leaf the proportions of the various acyl groups esterified to each ACP isoform were indistinguishable, indicating that these isoforms are utilized similarly in de novo fatty acid biosynthesis in vivo. However, the acyl group distribution pattern of seed ACP-II differed significantly from that of leaf ACP-II. The malonyl-ACP levels were less than the 4:0-ACP and 6:0-ACP levels in leaf, and in contrast, the malonyl-ACP-II levels in seed were approximately 3-fold higher than the 4:0-ACP-II and 6:0-ACP-II levels. In addition, the ratio of oleoyl-ACP-II (18:1) to stearoyl-ACP-II (18:0) was higher in seed than in leaf. These data suggest that the differences in acyl-ACP patterns reflect a tissue/organ-specific difference rather than an isoform-specific difference. In extracts prepared from leaf samples collected in the dark, the levels of acetyl-ACPs were approximately 5-fold higher compared to samples collected in the light. The levels of free ACPs showed an inverse response, increasing in the light and decreasing in the dark. Notably there was no concomitant increase in the malonyl-ACP levels. The most likely explanation for the major increase in acetyl-ACP levels in the dark is that light/dark control over the rate of fatty acid biosynthesis occurs at the reaction catalyzed by acetyl-CoA carboxylase.     

Fatty acid synthesis by spinach chloroplasts I. Property of fatty acid synthesis from acetate

Intact chloroplasts (about 70% Class I chloroplasts) isolatedfrom spinach leaves incorporated 150 nmoles of [1-¹⁴C] acetateinto fatty acids per mg chlorophyll in 1 hr at pH 8.3, 25°Cand 25,000 lux. On electron and phase-contrast microscopiescombined with hypotonic treatment of chloroplasts, this syntheticactivity was shown to be proportional to the percentage of ClassI chloroplasts in the preparation. Light was necessary for thesynthesis, the activity in the complete reaction mixture inthe dark being only 2% of that in the light. The synthetic activityincreased with increasing intensities of light to reach saturationat 6,000 lux. CoA and ATP were most effective as cofactors,HCO₃^–, HPO₄^2–, Mg^2$ and Mn^2$ were less effective.ATP could be replaced by ADP in the presence of Pi, suggestingpossible supply of ATP by photophosphorylation. Omission ofthe NADPH-generation system and NADH did not affect the synthesis,indicating sufficient provision of endogenous NADPH and NADHin intact chloroplasts under light. Addition of DTE did notcause recovery of the synthetic activity of intact chloroplastsin the dark. ¹ Present address: Radioisotope Centre, University of Tokyo,Yayoi, Bunkyo, Tokyo 113, Japan. (Received August 26, 1974; )     

Photosynthesis of Lipids from CO(2) in Spinacia oleracea

Young expanding spinach leaves exposed to ¹⁴CO₂ under physiological conditions for up to 20 minutes assimilated CO₂ into lipids at a mean rate of 7.6 micromoles per milligram chlorophyll per hour following a lag period of 5 minutes. Label entered into all parts of the lipid molecule and only 28% of the ¹⁴C fixed into lipids was found in the fatty acid moieties, i.e. fatty acids were synthesized from CO₂in vivo at a mean rate of 2.1 micromoles per milligram chlorophyll per hour. Intact spinach chloroplasts isolated from these leaves incorporated H¹⁴CO₃ into fatty acids at a maximal rate of 0.6 micromole per milligram chlorophyll per hour, but were unable to synthesize either the polar moieties of their lipids or polyunsaturated fatty acids. Since isolated chloroplasts will only synthesize fatty acids at rates similar to the one obtained with intact leaves in vivo if acetate is used as a precursor, it is suggested that acetate derived from leaf mitochondria is the physiological fatty acid precursor.     

Effect of one night with "low light'on uptake, reduction and storage of nitrate in spinach

During the night, shoot nitrate concentration in spinach (Spinacia oleracea L. cv. Vroeg Reuzenblad) increased due to increased uptake of nitrate by the roots. When the plants were subjected to a one night “low light’period at 35 μmol m^?2 s^?1, the shoot nitrate concentration did not increase and was reduced by 25% compared to control plants in the dark. The major contribution to this decrease was located in the leaf blades, where the nitrate concentration was decreased by 60%, while the petiole nitrate concentration decreased by only 9%. Nitrate accumulated in the leaf blade vacuoles during a dark night, but this was not the case during the “low light’period. This decrease in vacuolar nitrate concentration, compared to control plants in the dark, was not caused by increased amounts of leaf blade nitrate reductase (NR; EC 1.6.6.1). During a “low light’night period, the cytoplasmic soluble carbohydrate concentration was increased compared to the control plants in the dark. Calculations showed in situ NR activity to be higher than in the control plants in the dark. This increase in NR activity, however, was not large enough to account for the total difference found in the shoot nitrate concentration. Net uptake of nitrate by the roots was increased during the initial hours of the dark night, while vacuolar nitrate concentration in the leaf blades increased at the same time. During the “low light’night period, however, net uptake of nitrate by the roots did not increase, and vacuolar nitrate concentration did not change. We conclude that nitrate uptake by the roots and vacuolar nitrate concentration in the leaf blades are tightly coupled. The decreased shoot nitrate concentration is mainly caused by a reduction in net uptake of nitrate by the roots. During the “low light’night period, carbohydrates and malic acid partly replaced vacuolar nitrate. A “low light’period one night prior to harvest provides a valuable tool to reduce shoot nitrate concentrations in spinach grown in greenhouses in the winter months.     

Thermodynamic bases for fatty acid ethyl ester synthase catalyzed esterification of free fatty acid with ethanol and accumulation of fatty acid ethyl esters

Myocardial homogenates rapidly synthesize fatty acyl ethyl esters from nonesterified fatty acid and ethanol in the absence of coenzyme A or ATP, and the enzyme catalyzing this reaction, fatty acid ethyl ester synthase, has been purified 5400-fold to homogeneity [Mogelson, S., & Lange, L. G. (1984) Biochemistry (preceding paper in this issue)]. To define the factors permitting this de novo synthesis of ester bonds and the consequent accumulation of fatty acyl ethyl esters in myocardium, we determined thermodynamic parameters relevant to the kinetics and equilibria of this reaction and specifically characterized (1) the rates of synthesis of ethyl oleate, in both the presence and absence of purified enzyme catalyst, and (2) the physical properties of the product, ethyl oleate, in an aqueous milieu. Compared to the reaction of ethanol and oleate in the absence of catalyst, fatty acid ethyl ester synthase enhanced the rate of ethyl oleate synthesis by reducing the free energy of activation (delta G) from 32.5 to 19.9 kcal/mol, effected in large part by a positive entropy shift, delta Senz - delta S uncat = 23.9 cal/(mol.deg). Rate constants in the presence and absence of enzyme at 37 degrees C were 6 X 10(-2) s-1 and 7.8 X 10(-11) M-1 s-1, respectively, indicating a catalytic power of at least 10(8)M for this enzyme. Kinetic data indicated an enzymatic Vmax of 1.25 nmol/(mg.s) (37 degrees C). The equilibrium constant was calculated for the reaction oleate + ethanol in equilibrium ethyl oleate and was 0.095 M-1 at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of adenylate levels in intact spinach chloroplasts

Starch phosphorylase activity in extracts of spinach or pea leaves and of isolated chloroplasts was determined and separated by electrophoresis in polyacrylamide gels. In spinach leaf extracts, a specific activity of 16 nmol glucose 1-phosphate formed per min per mg protein was found, whereas a lower value (6 nmol per min per mg protein) was observed in preparations of isolated chloroplasts which were about 75% intact. In the spinach leaf extracts two forms of phosphorylase were found; chloroplast preparations almost exclusively contained one of these. In pea leaf extracts the specific activity was 10 nmol glucose 1-phosphate formed per min per mg protein. Three forms of phosphorylase contributed to this activity. Preparations of isolated chloroplasts with an intactness of about 85% exhibited a lower specific activity (5nmol per min per mg protein) and contained two of these three phosphorylase forms.Abbreviations G1P Glucose 1-phosphate - P_i orthophosphate - Tris Tris (hydroxymethyl)aminomethane - MES 2(N-morpholino)ethane sulphonic acid - EDTA ethylenediamine tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid     

The Effect of Light and Hormones on Leaf Senescence

With wheat leaves as material, the changes of superoxide dismutase (SOD), lipid peroxi-dation and membrane permeability during leaf senescence in light or dark, and treated withphytohormones (KT or ABA) have been studied. The changes of chlorophyll content, lipidperoxidation and fine structure of spinach chloroplasts senescing in light or dark have alsobeen studied. When leaves senesce in light, the activity of SOD increased at first then decreased. The increase of SOD activity was able to result from the synthesis of new protein. Lightwas found to delay the leaf senescence obviously but also accelerate leaf senescence by causinglipid peroxidation when prolonged the illumination time. The delay or acceleration of leafsenescence by exogenous hormones were observed, it may be due to the control of lipid peroxi-dation by adjusting the activity of SOD. O2-participated the chlorophyll decomposition andlipid peroxidation during chloroplasts senesce in light. A favourable role of light in mainta-lng the fine structure of isolated chloroplasts was clear.     

Maltose is the major form of carbon exported from the chloroplast at night

Transitory starch is formed in chloroplasts during the day and broken down at night. We investigated carbon export from chloroplasts resulting from transitory-starch breakdown. Starch-filled chloroplasts from spinach (Spinacia oleracea L. cv. Nordic IV) were isolated 1 h after the beginning of the dark period and incubated for 2.5 h, followed by centrifugation through silicone oil. Exported products were measured in the incubation medium to avoid measuring compounds retained inside the chloroplasts. Maltose and glucose made up 85% of the total exported products and were exported at rates of 626 and 309 nmol C mg^–1 chlorophyll h^–1, respectively. Net export of phosphorylated products was less than 5% and higher maltodextrins were not detected. Maltose levels in leaves of bean (Phaseolus vulgaris L. cv. Linden), spinach, and Arabidopsis thaliana (L.) Heynh. were low in the light and high in the dark. Maltose levels remained low and unchanged during the light/dark cycle in two starch-deficient Arabidopsis mutants, stf1, deficient in plastid phosphoglucomutase, and pgi, deficient in plastid phosphoglucoisomerase. Through the use of nonaqueous fractionation, we determined that maltose was distributed equally between the chloroplast and cytosolic fractions during darkness. In the light there was approximately 24% more maltose in the cytosol than the chloroplast. Taken together these data indicate that maltose is the major form of carbon exported from the chloroplast at night as a result of starch breakdown. We hypothesize that the hydrolytic pathway for transitory-starch degradation is the primary pathway used when starch is being converted to sucrose and that the phosphorolytic pathway provides carbon for other purposes.Abbreviations CAM crassulacean acid metabolism - Chl chlorophyll - DHAP dihydroxyacetone phosphate - FBPase fructose bisphosphatase - GAP glyceraldehyde-3-phosphate - G6P glucose 6-phosphate - PGA 3-phosphoglycerate - TPT triose phosphate translocator - WT wild type     

Lipid biosynthesis by chloroplasts isolated from developing Zea mays

Fatty acid biosynthesis by isolated plastids has been examined in relation to chloroplast development and differentiation in leaves of maize plants grown in light for 7 days. Biosynthesis of fatty acids from acetate by proplastids prepared from the basal regions of the leaf was low and mainly palmitate was synthesized. The greatly increased utilization of acetate for fatty acid biosynthesis as the plastids increased in size was due to an increased synthesis of oleate. The maximum synthesis of total fatty acids and monoenoic fatty acids was obtained in chloroplasts prepared from leaf tissue 6–8 cm from the base of the plant where granal formation was most active. Fully-developed chloroplasts prepared from distal regions of the leaf were less active in fatty acid biosynthesis. Maize chloroplasts failed to synthesize fatty acids when isolated by methods commonly used to prepare active spinach chloroplasts. The method of isolation which included a density gradient gave a high proportion of Class I chloroplasts from maize leaves and incorporated up to about 10% of the acetate used. Biosynthesis of unsaturated fatty acids, especially with chloroplasts prepared from the most mature tissue, was increased by the addition of both mitochondrial and microsomal fractions. Increases in polyunsaturated fatty acids were also obtained but the proportions in the newly-synthesized fatty acids were well below the endogenous levels. Monoenoic synthesis was greatly stimulated by increasing the pH in the range 7·0–8·0 and also the highest proportions of unsaturated fatty acids were obtained at short incubation times.     

Studies on Greening of Etiolated Seedlings

Evidence is given that a selective light-pretreatment of the embryonic axis exerts a deep influence on the greening in primary leaves of 8-day-old etiolated bean seedlings (Phaseolus vulgaris cv. Limburg). After a subsequent dark incubation of sufficient length and a final exposure of the entire plants to continuous illumination the lag phase of chlorophyll synthesis is completely removed. In particular the highly meristematic hook tissue seems to be responsible for this light effect. Lengthening of the dark period following pre-irradiation increased the capability of chlorophyll production in the main white light period, reaching its maximum after about 12 hours of darkness. The period of dark incubation for elimination of the lag phase is considerably longer in plants with shielded leaves than the length of the lag phase in etiolated seedlings of the same age, exposed entirely to continuous light. This difference may be explained by the synergistic effect between leaves and embryonic axis. Evidence for this interorgan cooperation is given by experiments with a selective light-pretreatment of leaves and embryonic axis. After a 5 min pre-exposure to white light of whole plants the leaves of some of the plants were shielded and these plants received a further pre-illumination of 2 hours on their embryonic axis. In all the pre-irradiated, etiolated plants the lag phase of chlorophyll synthesis was eliminated during the main white light period, following a dark incubation of 2 hours. Additional and preferential light activation of the embryonic axis during the pretreatment had no significant effect on chlorophyll production during the white light illumination after a 2 hours dark incubation, but resulted in a lower yield of chlorophylls after 18 hours dark incubation compared to the white light controls, receiving no selective light-pretreatment on the embryonic axis. From our results we can decisively conclude that a simultaneous light-pretreatment of both, leaves and embryonic axis, is more effective and beneficial for building up a capacity of chlorophyll synthesis in the leaves than either a selective light-pretreatment of the embryonic axis alone or a simultaneous pre-illumination of leaves and embryonic axis, immediately followed by an additional preirradiation of the embryonic axis. Therefore, we think that several photoactive sites are involved in de-etiolation processes of intact, etiolated seedings. Light activation of the embryonic axis stimulates the development of this organ and contributes to the greening processes in the leaf. At the same time, by irradiating the leaf, light activates the photo-sensitive site in the leaf itself, which also develops a capacity for chlorophyll synthesis. Both photo-acts are cooperative, explaining the enhanced chlorophyll production. Additional pre-irradiation of the embryonic axis after a short illumination of whole plants favours its own development and reduces the synthetic capacity of the leaf. A prolonged far-red pretreatment induces qualitatively the same response as white light. We assume that these effects on lag phase removal and chlorophyll production, induced in etiolated, primary bean leaves by selective irradiation of the embryonic axis, is a phytochrome-mediated process. Our results indicate a transmission of light-induced stimuli from one organ to another.     

Fatty-acid-induced release of manganes from chloroplasts

The effect of both endogenous and exogenous unsaturated free fatty acids on manganese release from chloroplasts of chill-resistant (spinach) and chill-sensitive (tomato, bean) plants was studied. The level of endogenous free fatty acids increased 2–3-fold during cold and dark storage of leaves of chill-sensitive plants and was accompanied by depletion of about 60% of total chloroplast manganese content. Similar effects were observed when accumulation of free fatty acids in chloroplasts was achieved by storage of growing tomato plants for a few days in the dark at room temperature. In contrast, the cold and dark treatment of leaves of chill-resistant plant (spinach) affected neither free fatty acid, manganese levels nor Hill-reaction activity in chloroplasts. Incubation of chloroplasts of both chill-sensitive and chill-resistant plants with bean leaf galactolipase resulted in an accumulation of free fatty acids and a release of approx. 60% of total manganese content. The same amount of total manganese content was released following 3 h incubation of chloroplasts with linolenic acid at fatty acid/chlorophyll ratio (w/w, 2:1–10:1). The efficiency of C₁₈ unsaturated fatty acids/linolenic, linoleic, oleic on manganese release from chloroplasts was established in decreasing order C_18:3 > C_18:2 > C_18:1. The results indicate that the inhibitory effect of both endogenous and exogenous fatty acids on Hill reaction depends on the release from chloroplasts of functionally active, loosely bound manganese. Thus, similarly to both Tris and hydroxylamine treatments of chloroplasts, the incubation of chloroplast preparations with unsaturated fatty acids may be a useful tool for manganese depletion of chloroplasts.     

Effects of ethephon on aging and photosynthetic activity in isolated chloroplasts

Chloroplasts, isolated from the primary leaves of 7-day-old seedlings, were incubated in vitro at 25°C with 2-chloroethylphosphonic acid (ethephon) under light (0.16 milliwatts per square centimeter) and dark conditions. Ethephon at 1 micromolar (0.1445 ppm), 0.1 and 1 millimolar, or 5 microliters ethylene promoted the deterioration of chloroplasts, increased proteolysis, and reduced the chlorophyll content and PSI and PSII during 72 hours under both light and dark conditions. The decline in PSI and PSII occurred prior to a measurable loss of chlorophyll. The loss of photosynthetic activity affected by ethephon was initiated prior to 12 hours of incubation. After 24 hours in light, 0.1 millimolar (1.445 ppm) epthephon significantly reduced PSI and PSII and promoted the total free amino acid liberation in isolated chloroplasts. In darkness the rate of loss of PSI activity was about 50% of that in light. After 24 hours, in light at 1 millimolar epthephon, PSII activity was 55% of the control, yet nearly 90% of the chlorophyll remained, which indicates that the loss of thylakoid integrity was promoted by ethephon. Ethylene injected in the chloroplast medium at 5 microliters (0.22 micromolar per milliliter) reduced PSI by nearly 50% of the initial in 12 hours. In leaf sections floated in 5 microliters per milliliter suspension medium, a 36% loss of chlorophyll of the control in 36 hours was observed. Cycloheximide at 0.5 millimolar masked the effect of 1 millimolar ethephon and maintained the initial chlorophyll content during the 72 hour period.     

Fatty acid uptake by human hepatoma cell lines represents a carrier-mediated uptake process

Cellular influx kinetics of a representative long chain fatty acid, [3H]oleate, were examined in monolayer cultures of three different human hepatoma cell lines (Hep G2; PLC/PRF 5; Mz-Hep-1). The cultures were incubated with 173 microM [3H]oleate in the presence of various concentrations of albumin which served to modulate the unbound oleate concentration in the medium. For all [3H]oleate-albumin complexes incubated, it was shown that cellular uptake of [3H]oleate over the initial 30 s incubation period was maximal, linear and independent of intracellular fatty acid metabolism, representing cellular influx. With increasing unbound oleate concentrations in the medium cellular influx by all three cell lines revealed similar saturation kinetics with Km values of 112.6 +/- 14.5 nM and Vmax values of 7.19 +/- 0.32 nmol.min-1 per mg cell protein. When these hepatoma cell lines were pretreated with the IgG fraction of a monospecific antibody to the rat liver membrane fatty acid binding protein (MFABP), initial uptake of [3H]oleate was selectively inhibited compared to controls pretreated with the IgG fraction of the preimmune serum. Furthermore, immunoblot analysis with the monospecific antibody to the rat MFABP revealed reactivity with a single 40 kDa protein in the homogenates of all three cell lines. These data suggest that uptake of fatty acids by human hepatoma cells may be mediated by a specific membrane fatty acid binding protein.     

A chloroplast-associated fatty acid synthetase system in Euglena

Fatty acid synthetase activity in etiolated Euglena gracilis strain Z is independent of added ACP and associated with a high-molecular-weight complex of the type found in yeast. Cells grown in the dark and then greened by illumination in a resting medium develop a second enzyme system which is dependent on added ACP and generally resembles the corresponding E. coli and plant enzymes. Cycloheximide has no effect on the appearance of the ACP-dependent fatty acid synthetase in greening cells whereas chloramphenicol causes complete inhibition at concentrations which decrease chlorophyll synthesis by 66%. An induction of the ACP-dependent fatty acid synthetase in the absence of chloroplast development occurs on exposure of dark-grown cells to doses of ultraviolet light which selectively affect proplastid nucleoprotein. This enzyme induction by ultraviolet light is inhibited by chloramphenicol. The protein synthesis machinery of the chloroplast appears to be responsible, either directly or indirectly, for the appearance of the ACP-dependent fatty acid synthetase of Euglena.     

Prenyl lipid formation in spinach chloroplasts and in a cell-free system of Synechococcus (Cyanobacteria): polyprenols,chlorophylls, and fatty acid prenyl esters

Isolated chloroplasts from spinach leaf cells, chloroplast subfractions, and a cell-free system of the cyanobacterium Synechococcus CCAP 6312 incorporated [1-¹⁴C]isopentenyl pyrophosphate in high yields into prenyl lipids. Products were polyprenols (C₂₀, C₄₅) chlorophylls, quinoid compounds, and fatty acid prenyl esters; prenyl pyrophosphates occurred in trace amounts, and carotenes were only formed to a limited extent in the Synechococcus system. The formation of fatty acid prenyl esters, which is described here for the first time, was found to occur in two different ways in the chloroplast system; by an acyl-CoA: polyprenol acyltransferase reaction associated with the envelope membranes and by a transesterification reaction from chlorophyll associated with the thylakoids. Endogenous fatty acid prenyl esters made up about 3% by weight of total lipids in spinach chloroplasts and were also found to be natural constituents of the cyanobacterial cells.Abbreviations Chl chlorophyll - Chl_GG chlorophyll a containing a geranylgeranyl side chain - IPP isopentenyl pyrophosphate     

Purification to homogeneity and characterization of major fatty acid ethyl ester synthase from human myocardium

Non-oxidative metabolism of ethanol via fatty acid ethyl ester synthase is present in those extrahepatic organs most commonly damaged by alcohol abuse. DEAE-cellulose chromatography of human myocardial cytosol at pH 8.0 separated synthase I, minor and major activities, eluting at conductivities of 5, 7 and 11 mS, respectively. The major synthase was purified 8900-fold to homogeneity by sequential gel permeation, hydrophobic interaction, and anti-human albumin affinity-chromatographies with an overall yield of 25%. SDS-PAGE showed a single polypeptide with a molecular mass of 26 kDa and gel permeation chromatography under nondenaturing conditions indicated a molecular mass of 54 kDa for the active enzyme. The purified enzyme catalyzed ethyl ester synthesis at the highest rates with unsaturated octadecanoic fatty acid substrates (Vmax = 100 and 65 nmol/mg/h for oleate and linoleate, respectively). Km values for oleate, linoleate, arachidonate, palmitate and stearate were 0.22 mM, 0.20 mM, 0.13 mM, 0.18 mM and 0.12 mM, respectively. Thus, human heart fatty acid ethyl ester synthase (major form) is a soluble dimeric enzyme comprised or two identical, or nearly identical, subunits (Mr = 26000).     

Relationship between glycerol-3-phosphate dehydrogenase,fatty acid synthase and fatty acid binding proteins in developing human placenta

The activities of the enzymes glycerol-3-phosphate dehydrogenase and fatty acid synthase are inhibited by palmitoyl-coenzyme A and oleate. The two isoforms of fatty acid binding proteins (PI 6.9 and PI 5.4) enhance the activities of glycerol-3-phosphate dehydrogenase and fatty acid synthase in the absence of palmitoyl-coenzyme A or oleate and also protect them against palmitoyl-coenzyme A or oleate inhibition. Levels of fatty acid binding proteins, the activities of the enzymes fatty acid synthase and glycerol-3-phosphate dehydrogenase increase with gestation showing a peak at term. However, the activity of fatty acid synthase showed the same trend up to the 30th week of gestation and then declined slightly at term. With the advancement of pregnancy when more lipids are required for the developing placenta, fatty acid binding proteins supply more fatty acids and glycerol-3-phosphate for the synthesis of lipids. Thus a correlation exists between glycerol-3-phosphate dehydrogenase, fatty acid synthase and fatty acid binding proteins in developing human placenta.