Long chain fatty acid synthesis in developing castor bean seeds I. The operation of the path from acetate to long chain fatty acids in a subcellular particulate system

The path of LFA synthesis from acetate in developing castorbean seeds was associated with subcellular 10,000 g particles.Further fractionation of these particles by a stepwise densitygradient method showed the high possibility that the site ofLFA synthesis is the proplastid. A study on cofactor requirementswhen [1-¹⁴C]acetate predominantly incorporated into LFAs indicatedthat synthesis would be achieved by acetyl-CoA carboxylation,malonyl-ACP condensation. ATP, CoA, HCO₃^– and Mg⁺⁺ orMn⁺⁺ were essential for synthesis from acetate by the 10,000gparticulate system. Results of inhibhitor experiment suggestedthat the supply of ATP to the LFA synthesizing system is broughtabout by mitochondrial oxidative phosphorylation, when acetateis the sole precursor for LFA synthesis in this system. TheNADPH generating system was contained in the paticles, althoughthe addition of NADP⁺ and G-6-P increased synthesis. NADH markedlystimulated LFA synthesis from acetate. The primary role of NADHseems to be as a direct reductant in both steps involving thereduction and oxidative desaturation of fatty acid chains; particularly,in the former step, although NADH partially contributes to thesupply of ATP as a respiratory substrate. It is unlikely thatNADH works as a hydrogen donor to NADP⁺. LFA synthesis by thecastor bean particulate system was not stimulated by light,thus differing from that by leaf chloroplasts. (Received July 23, 1973; )     

Cellular uptake and intracellular trafficking of long chain fatty acids.

While aspects of cellular fatty acid uptake have been studied as early as 50 years ago, recent developments in this rapidly evolving field have yielded new functional insights on the individual mechanistic steps in this process. The extremely low aqueous solubility of long chain fatty acids (LCFA) together with the very high affinity of serum albumin and cytoplasmic fatty acid binding proteins for LCFA have challenged the limits of technology in resolving the individual steps of this process. To date no single mechanism alone accounts for regulation of cellular LCFA uptake. Key regulatory points in cellular uptake of LCFA include: the aqueous solubility of the LCFA; the driving force(s) for LCFA entry into the cell membrane; the relative roles of diffusional and protein mediated LCFA translocation across the plasma membrane; cytoplasmic LCFA binding protein-mediated uptake and/or intracellular diffusion; the activity of LCFA-CoA synthetase; and cytoplasmic protein mediated targeting of LCFA or LCFA-CoAs toward specific metabolic pathways. The emerging picture is that the cell has multiple, overlapping mechanisms that assure adequate uptake and directed intracellular movement of LCFA required for maintenance of physiological functions. The upcoming challenge is to take advantage of new advances in this field to elucidate the differential interactions between these pathways in intact cells and in tissues.     

Inhibition of chymase activity by long chain fatty acids

The activity of chymase was markedly inhibited by fatty acids with carbon chain lengths of 14-22 at doses greater than 0.02 microM, irrespective of the number of double bonds. Cis acids with a carbon chain length of 18, such as stearic acid, oleic acid, linoleic acid, and linolenic acid were potent inhibitors, whereas the trans isomer of oleic acid, elaidic acid, showed less inhibitory activity. The extent of inhibition by oleyl alcohol was almost the same as that by oleic acid, suggesting that the acid moiety itself was not necessary for the inhibition; but a fatty acid with a terminal functional amide, oleamide, showed little inhibitory activity. The inhibition was noncompetitive and was reversible, and the Ki value of oleic acid was 2.7 microM. Stearic acid and oleic acid inhibited all chymotrypsin-type serine endopeptidases tested. The ID50 values of these fatty acids for atypical mast cell protease were higher than those for the other chymotrypsin-type serine endopeptidases tested. Other proteases, such as papain, trypsin, collagenase, and carboxypeptidase A, except cathespin D, were not affected by stearic or oleic acid.     

The cellular localization of long chain fatty acids in sponges

Examination of fractionated sponge tissue shows that long chain fatty acids (LCFAs) occur in high proportions in cell membranes. This conclusion refutes a recent suggestion made by other workers that sponge membranes would contain conventional fatty acids similar to those found in membranes from other organisms.     

Desaturation of fatty acids in seeds of higher plants

Photosynthesizing flax, soybean, and safflower plants were exposed to (14)CO(2) at seed-setting stage for a 1 hr period. Seed was sampled periodically thereafter and the lipids were extracted. A triglyceride-rich fraction was methanolyzed; the resultant methyl esters were analyzed by gas-liquid chromatography and assayed for radioactivity. Of the C(18) unsaturated acids, oleic was the first to acquire radioactivity, which subsequently and successively appeared in linoleic and linolenic acids. The shapes of the radioactivity-time curves provide evidence that consecutive desaturation reactions occur in the seeds of these higher plants.     

Mechanisms of cellular uptake of long chain free fatty acids

Cells take up long chain free fatty acids (FFA) in vivo from the non-protein bound ligand pools in extracellular fluid and plasma, which contain ~100 and 600 M albumin, respectively. The physiologic range of unbound FFA concentrations in such fluids has traditionally been calculated at < 1 M. Studies of [3H]-oleate uptake by hepatocytes, adipocytes, cardiac myocytes and other cell types demonstrate that FFA uptake within this range is saturable, and exhibits many other kinetic properties indicative of facilitated transport. Within this range, the uptake kinetics of the acidic (pKa = 0.5) FFA analog 2,2,3- heptafluorostearate are similar to those of stearate. Thus, uptake of physiologic concentrations of FFA involves facilitated transport of the FFA anion (FA-). Over a much wider range of unbound FFA concentrations hepatocellular [3H]-oleate uptake exhibits both saturable and non-saturable components. Oleate binding to liver plasma membranes (LPM) also demonstrates such components. Comparing the two components of FFA uptake to the corresponding components of binding permits estimates of trans-membrane transport rates. T1/2 for saturable uptake (~ 1 sec) is less than for non-saturable uptake (~ 14 sec). Others have determined the flip-flop rates of protonated FFA (FAH) across small and large unilamellar vesicles (SUV, LUV) and across cellular plasma membranes. These reported flip-flop rates, measured by the decrease in pH resulting from the accompanying proton flux, exhibit a highly significant inverse correlation with cell and vesicle diameter (r = 0.99). Although T1/2's in vesicles are in the msec range, those in cells are > 10 sec, and thus comparable to the rates of non-saturable uptake we determined. Thus, under physiologic conditions, the predominant mechanism of cellular FFA uptake is facilitated transport of FA-; at much higher, non- physiologic FFA concentrations, passive flip-flop of FAH predominates. Several plasma membrane proteins have been identified as potential mediators of facilitated FFA transport. Studies in animal models of obesity and non-insulin dependent diabetes mellitus demonstrate that tissue-specific regulation of facilitated FFA transport has important pathophysiologic consequences.     

Desaturation,chain scission,and register-shift of oxygen-substituted fatty acids during reaction with stearoyl-ACP desaturase

Stearoyl acyl carrier protein Delta(9) desaturase catalyzes the NADPH- and O(2)-dependent insertion of a cis double bond between the C-9 and C-10 positions of the acyl chain in the kinetically preferred natural substrate 18:0-ACP. In this work, substrate analogues with an oxygen atom singly replacing the methylene groups at the 8, 9, 10, and 11 positions of the stearoyl chain were synthesized, converted to acyloxy-ACPs, and used as probes of desaturase reactivity. Evidence for desaturation, acyloxy chain scission, and register-shift in binding prior to chain scission was obtained. Reactions with acyloxy-ACPs having either O-8 or O-11 substitutions gave a single desaturation product consistent with the insertion of a cis double bond between C-9 and C-10. The k(cat)/K(M) values for the O-8- and O-11-substituted acyloxy-ACPs were comparable to that of the natural substrate, indicating that the presence of an ether group adjacent to the site of reactivity did not significantly interfere either with the desaturation reaction or with the binding of substrate in the proper register for desaturation between C-9 and C-10. For reactions with the O-9 and O-10 acyloxy-ACPs, the k(cat) values were decreased to approximately 3% of that observed for 18:0-ACP, and upon reaction, the acyloxy chain was broken to yield an omega-hydroxy fatty alkanoyl-ACP and a volatile long-chain aldehyde. For the O-9 substitution, 8-hydroxyoctanoate and 1-nonanal were obtained, corresponding to the anticipated binding register and subsequent reaction between the O-9 and C-10 positions. In contrast, the O-10 substitution yielded 9-hydroxynonanoyl-ACP and 1-octanal, corresponding to an obligate "register-shift" of acyloxy chain binding prior to reaction between the O-10 and C-11 positions. Register-shift is thus defined as a mechanistically relevant misalignment of acyl chain binding that results in reaction at positions other than between C-9 and C-10. The inability of the O-10 acyloxy probe to undergo reaction between the C-9 and O-10 positions provides evidence that the Delta9D-catalyzed desaturation of stearoyl-ACP may initiate at C-10. Possible mechanisms of the acyl chain scission and implications of these results for the desaturation mechanism are considered.     

Exposure to long chain polyunsaturated fatty acids triggers rapid multimerization of synucleins.

Detergent-stable multimers of alpha-synuclein have been found specifically in the brains of patients with Parkinson's disease and other neurodegenerative diseases. Here we show that recombinant alpha-synuclein forms multimers in vitro upon exposure to vesicles containing certain polyunsaturated fatty acid (PUFA) acyl groups, including arachidonoyl and docosahexaenoyl. This process occurs at physiological concentrations and much faster than in aqueous solution. PUFA-induced aggregation involves physical association with the vesicle surface via the large apolipoprotein-like lipid-binding domain that constitutes the majority of the protein. beta- and gamma-synucleins, as well as the Parkinson's disease-associated alpha-synuclein variants A30P and A53T, show similar tendencies to multimerize in the presence of PUFAs. Multimerization does not require the presence of any tyrosine residues in the sequence. The membrane-based interaction of the synucleins with specific long chain polyunsaturated phospholipids may be relevant to the protein family's physiological functions and may also contribute to the aggregation of alpha-synuclein observed in neurodegenerative disease.     

Long chain fatty acid synthesis in developing castor bean seeds II. Operation of the path from sucrose to long chain fatty acids in a subcellular particulate system

Particles spun down at 10,000 ? g from developing castor beanseeds were capable of synthesizing LFAs from sucrose, a physiologicalprecursor transferred from leaves as a photosynthetic product.Tracer experiments, in combination with inhibitor effects, intermediatedilutions and cofactor requirements, indicated the operationof the following path: sucroseUDPGG-1-PG-6-PGAPpyruvateacetyl-CoAmalonyl-CoALFA.The whole path appears to be associated with 10,000 ? g particles,because repeated washings were unsuccessful in dissociatinga partial path from the particles, depsite of disorganizingthe structure of the particles. Based on the occurrence of freehexose(s) and the utilization of UDPG similar to that of sucroseor G-1-P in this reaction, it is probable that hexose phosphateis formed from sucrose via UDPG and fructose, although the conversionof sucrose to hexose phosphates via glucose and furctose isnot ruled out. Inhibitor experiments showed that ATP is self-supportingover the whole path, the ATP formed in the glycolytic path beingconsumed in a acetyl-CoA carboxylation step. Since oxidizedpyridine nucleotides are as available as reduced ones for LFAsynthesis, they seem to shuttle between the reduction in theconversion of sucrose to acetyl-CoA and the oxidation in LFAsynthesis from acetyl-CoA. From the pattern of the LFAs synthesized,NAD⁺ is available for the synthesis of saturated LFAs (18: 0,16: 0). whereas NADP⁺ is available for that of unsaturated LFAs(18: 1, 16: 1). (Received July 23, 1973; )