INCORPORATION OF [1-14C]OLEIC ACID AND [1-14C]ARACHIDONIC ACID INTO LIPIDS IN THE SUBCELLULAR FRACTIONS OF MOUSE BRAIN

Abstract— The distribution of radioactivity among lipids of subcellular membrane fractions was examined after intracerebral injections of [1-¹⁴C]oleic and [1-¹⁴C]arachidonic acids. Labelled free fatty acids were distributed among the synaptosomal-rich, microsomal, myelin and cytosol fractions at 1 min after injection. However, incorporation of the fatty acids into phospholipids and trïacylglycerols after pulse labelling occurred mainly in the microsomal and synaptosomal-rich fractions. With both types of labelled precursors, there was a higher percentage of radioactivity of diacyl-glycerophosphoryl-inositols in the synaptosomal-rich fraction as compared to the microsomal fraction. Radioactivity of [1-¹⁴C]oleic acid was effectively incorporated into the triacylglycerols in the microsomal fraction whereas radioactivity of the [1-¹⁴C]arachidonic acid was preferentially incorporated into the diacyl-glycerophosphorylinositols in the synaptosomal-rich fraction. Result of the study indicates that synaptosomal-rich fraction in brain is able to metabolize long chain free fatty acids in vivo and to incorporate these precursors into the membrane phosphoglycerides.     

The biosynthesis of cytochrome c. Sequence of incorporation in vivo of [14C]lysine into cytochrome c and total proteins of rat-liver subcellular fractions

1. In order to determine the initial intracellular site of synthesis of cytochrome c in the liver cell, groups of rats were injected with [(14)C]lysine and killed 7.5, 15, 30 and 60min. later. The livers were homogenized in 0.3m-sucrose and subcellular fractions obtained. The mitochondrial fraction was further subfractionated. Pure cytochrome c was isolated from extracts of each fraction, obtained first with water at pH4.0 and then with 0.15m-sodium chloride. 2. A comparison of the kinetics of incorporation of [(14)C]lysine into total protein for each particulate fraction showed the usual two different kinds of kinetics. Incorporation into all the mitochondrial subfractions and the nuclear fraction rose gradually to a plateau value at about 20min., in contrast with that into the two microsomal fractions which rose rapidly to a peak value about seven times that for the mitochondrial fractions. The kinetics for the incorporation into mitochondrial cytochrome c showed a plateau value at 30min. about three times that for the total mitochondrial protein. There was no difference in the specific radioactivity of the mitochondrial cytochrome c extracted with water or 0.15m-sodium chloride or between the different mitochondrial subfractions. In contrast, the cytochrome c isolated from water extracts of the microsomal fractions had a lower specific radioactivity than that obtained from the 0.15m-sodium chloride extract. The specific radioactivity of the latter showed a rapid rise to a peak value about four times that for the mitochondrial cytochrome c, and the shape of the curve was similar to that for the total protein of the microsomal fraction. The results suggest that cytochrome c is synthesized in toto by the morphological components of the microsomal fraction. It seems first to be bound tightly to a microsomal particle, passing then to a looser microsomal binding and being finally transferred to the mitochondria. The newly synthesized cytochrome c in the mitochondrion could not be differentiated from the old by its degree of extractability at pH 4.0.     

Products of fatty acid synthesis by a particulate fraction from germinating pea (Pisum sativum L.).

The synthesis of lipids and acyl thioesters was studied in microsomal preparations from germinating pea (Pisum sativum cv. Feltham First) seeds. Under conditions of maximal synthesis (in the presence of exogenous acyl-carrier protein) acyl-acyl-carrier proteins accounted for about half the total incorporation from [14C]malonyl-CoA. Decreasing the concentrations of exogenous acyl-carrier protein lowered the overall synthesis of fatty acids by decreasing, almost exclusively, the radioactivity associated with acyl-acyl-carrier proteins. A time-course experiment showed that acyl-acyl-carrier proteins accumulated most of the radioactive label at the beginning of the incubation but, eventually, the amount of radioactivity in that fraction decreased, while a simultaneous increase in the acyl-CoA and lipid fractions was noticed. Addition of exogenous CoA (1 mM) produced a decrease of total incorporation, but an increase in the radioactivity incorporated into acyl-CoA. The microsomal preparations synthesized saturated fatty acids up to C20, including significant proportions of pentadecanoic acid and heptadecanoic acid. Synthesis of these 'odd-chain' fatty acids only took place in the microsomal fraction. In contrast, when the 18,000g supernatant (containing the microsomal and soluble fractions) was incubated with [14C]malonyl-CoA, the radioactive fatty acid and acyl classes closely resembled the patterns produced by germinating in the presence of [14C]acetate in vivo. The results are discussed in relation to the role of acyl thioesters in the biosynthesis of plant lipids.     

Nature of the reaction product of [1-14C]stearoyl-CoA elongation by etiolated leek seeding microsomes

The elongation of [1-14C]stearoyl-CoA by microsomes from etiolated leek seedlings, in the presence of malonyl-CoA and NADPH, has been studied at different substrate and enzyme concentrations. The HPTLC analysis of the whole reaction mixture, followed by the analysis of the label in the fatty acid methyl esters of long-chain acyl-CoAs, phosphatidylcholine (PC), and neutral lipids, showed that the acyl-CoA fraction contained most of the labeled very-long-chain fatty acids. The very-long-chain fatty acids were rapidly formed and released from the elongase(s) as acyl-CoAs. The label of long-chain acyl-CoAs increased for 20 min and then decreased, whereas it increased in PC. Labeled very-long-chain fatty acids appeared in the neutral lipid + free fatty acid fraction after a 20-min lag.     

Effect of evisceration on the disposal of [14C] palmitate in the rat.

The utilization in vivo of [1-(14)C] palmitate was studied in hepatectomized-nephrectomized rats and their sham-operated controls. After i.v. injection of the tracer, the [14C] lipids in plasma disappeared more slowly in eviscerated animals than in their controls. More label reappeared in plasma as esterified fatty acids in the latter group. At 30 min after the tracer, the amount of label found in the lipidic fraction of carcass and heart was much greater in eviscerated animals than in their controls although the percentile distribution of labelled lipidic fractions remained stable, a considerable proportion being present in the esterified fatty acid form. On the basis of these findings, the rapid increase in the plasma levels of FFA in eviscerated animals must be the result of augmented lipolytic activity more than reduced utilization of these metabolites.     

Intracellular transport and processing of lysosomal cathepsin B

Intracellular transport and processing of lysosomal cathepsin B was investigated in the subcellular fractions of rat liver by pulse-labeling experiments with [35S]methionine in vivo. A newly synthesized procathepsin B with a molecular weight of 39 kDa firstly appeared in the rough microsomal fraction at 10 min postinjection of label. This procathepsin B moved from the microsomal fractions to the Golgi subfractions at 30 min postinjection, and then a processed mature enzyme appeared in the lysosomal fraction at 60 min. These results suggest that the propeptide-processing of procathepsin B takes place in lysosomes in the course of intracellular transport from endoplasmic reticulum through Golgi complex to lysosomes.     

Distribution of arachidonic acid in choline- and ethanolamine-containing phosphoglycerides in subfractionated human neutrophils

Human neutrophils were fractionated on Percoll gradients and the various subcellular fractions were analyzed for phospholipid and fatty acid composition. The results showed that plasma membranes and azurophilic granules were enriched with ethanolamine-(PE) relative to choline-(PC) containing phosphoglycerides. A remarkable degree of uniformity existed throughout the gradient with respect to the subclass composition of the subcellular PC and PE components. In each fraction 50-60% of the PC was diacyl, 40-45% was 1-O-alkyl-2-acyl (ether linked), and 2-5% was 1-O-alk-1'-enyl-2-acyl (plasmalogenic). For PE, 20-25% was diacyl, 7-12% ether linked, and 64-76% plasmalogenic. When neutrophils were incubated for 15 min with [1-14C]arachidonic acid and subfractionated most of the PC-associated label was intracellularly localized. A similar result was observed in PE, however, when the cells were allowed to stand for 2 h in fatty acid-free buffer following the 15 min of labeling and then subfractionated there was a sizable migration of [14C]arachidonate into plasma membrane PE. In all cases the diacyl subclass was labeled most heavily after 15 min but after an additional 2 h of incubation in fatty acid-free buffer there was a direct transfer of label to the ether- and plasmalogenic-linked PC and PE subclasses. It was also found that arachidonoyl-coenzyme A 1-acyl-lysophosphatide acyltransferase activity was inherent in all three major membrane types but was enriched in the endoplasmic reticulum/secondary granule fraction. Arachidonate consistently accounted for roughly 5% of the PC and 17% of the PE fatty chain composition in each subcellular fraction. These findings demonstrate that, despite the uniform arachidonate and PC and PE subclass composition within the various neutrophil subcellular fractions, the bulk of the PC- and PE-associated arachidonate is localized in intracellular membranes.     

Synthesis of fatty acids from [1-14C]acetylcoenzyme A in subcellular particles of rat epididymal adipose tissue

1. Mitochondrial and microsomal fractions of rat epididymal adipose tissue incorporated [1-(14)C]acetyl-CoA equally well into various fatty acids by a chain-elongation mechanism. C(18) and C(20) fatty acids were the two major products, and comprised about 80% of the total fatty acids synthesized in both particles. 2. When incubated in air, mitochondria synthesized stearic acid, octadecenoic acid and eicosamonoenoic acid in almost equal amounts (about 20% each), whereas in microsomal fractions, the synthesis of octadecenoic acid was more than fivefold the stearic acid formation. In both fractions, major components of synthesized monoenoic fatty acids were the Delta(11:12) isomers. Hexadecenoic acid and octadecenoic acid from whole adipose tissue contained approx. 11 and 14% of the Delta(11:12) isomer respectively. 3. When mitochondria or microsomal fractions were incubated in nitrogen, there was increased synthesis of stearic acid and palmitic acid and less of C(16) and C(18) monoenoic acids; synthesis of C(20) acids remained predominantly of the monoenoic acids. Determination of the position of the double bond in the monoenoic acids supported the view that the synthesis of hexadecenoic acid and octadecenoic acid involves a desaturase activity, whereas eicosamonoenoic acid and eicosadienoic acid are formed only by elongation of endogenous fatty acids. 4. Most of the radioactivity was found in free fatty acids (63%) and the phospholipid (26%) fraction. In phospholipids, phosphatidylcholine and phosphatidylethanolamine were the two major components. 5. Most of the fatty acids synthesized, including those not normally found in particle lipids (arachidic acid, eicosamonoenoic acid and eicosadienoic acid) were distributed fairly evenly in the phospholipid and free fatty acid fractions. However, stearic acid was found predominantly in the phospholipid fraction.     

Formation of mevalonic acid, sterols and bile acids from [1-14C]acetyl-CoA and [2-14C]malonyl-CoA in the liver of rabbits with experimental hypercholesterolemia

The effect of cholesterol diet on the rate of mevalonic acid biosynthesis from 1-14C acetyl-CoA, 2-14C malonyl-CoA and the incorporation of these substrates into sterols and bile acids in rabbit liver were studied. Simultaneously, the activities of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) and acetyl-CoA carboxylase and the biosynthesis of fatty acids from acetyl-CoA and malonyl-CoA were measured. Hypercholesterolemia was found to be concomitant with the inhibition of acetyl-CoA carboxylase activity only in cell-free (700 g) and mitochondrial fractions and slightly decreased the incorporation of acetyl-CoA and malonyl-CoA into fatty acids in the postmitochondrial fraction. The HMG-CoA reductase activity in all subcellular fractions except for the postmicrosomal one was inhibited under these conditions. A significant decrease of acetyl-CoA incorporation and an increase in malonyl-CoA incorporation into mevalonic acid in all liver fractions except for microsomal one were observed in rabbits with hypercholesterolemia. These data provide evidence for the existence of two pathways of mevalonic acid synthesis from the above-said substrates that are differently sensitive to cholesterol. Cholesterol feeding resulted in a decreased synthesis of the total unsaponified fraction including cholesterol from acetyl-CoA, malonyl-CoA and mevalonic acid. The rate of incorporation of these substrates into lanosterol was unchanged. All the indicated substrates (acetyl-CoA, malonyl-CoA, mevalonic acid) are precursors of bile acid synthesis in rabbit liver. Cholesterol feeding and the subsequent development of hypercholesterolemia resulted in bile acid synthesis stimulation, preferentially in the formation of the cholic + deoxycholic acids from these precursors.     

The lipid composition of ovine placental tissue homogenate and its subcellular fractions

Homogenates of the placental tissue of near term sheep were separated by differential centrifugation into mitochondrial, microsomal and cytosolic fractions. The relative proportions of the major neutral lipids and phospholipids, together with their fatty acid compositions, were determined in the homogenates and in each subcellular fraction. The cytosolic fraction contained the highest proportion of cholesteryl esters (CEs) and these possessed a fatty acid composition markedly different from the total CEs extracted from the homogenate. Both the mitochondrial and microsomal fractions contained significant proportions of solvent front phospholipid (SFP) and whereas the mitochondrial SFP displayed the relatively unsaturated fatty acid composition characteristic of diphosphatidylglycerol (cardiolipin), the fatty acids of the microsomal SFP were distinctly more saturated. These results are compared with those obtained from other mammalian tissues, both ruminant and non-ruminant, and discussed in terms of the function of the components of the subcellular fractions.     

Preferential In Vivo Incorporation of [3H]Arachidonic Acid from Blood into Rat Brain Synaptosomal Fractions Before and After Cholinergic Stimulation

Abstract: Awake adult male rats were infused intravenously with [³H]arachidonic acid for 5 min, with or without prior administration of an M1 cholinergic agonist, arecoline (15 mg/kg i.p.). Methylatropine was also administered (4 mg/kg s.c.) to control and arecoline-treated animals. At 15 min postinfusion, the animals were killed, brains were removed and frozen, and subcellular fractions were obtained from homogenates of whole brain. Total radioactivity and radioactivity in various lipid classes were determined for each fraction following normalization for exposure by use of a unidirectional incorporation coefficient, k ^⋆_brain. In control animals, incorporation was greatest in synaptosomal and microsomal fractions, accounting for 50 and 30% of total label incorporated into membrane lipids, respectively. Arecoline increased incorporation in these two fractions by up to 400% but did not increase incorporation into the myelin, mitochondrial, or cytosolic fractions. Of the incorporated radioactivity, 50–80% was in phospholipid in microsomal and synaptosomal fractions, indicating that phospholipid is the major lipid affected by cholinergic stimulation. These results demonstrate that plasma [³H]arachidonic acid is preferentially incorporated into phospholipids of synaptosomal and microsomal fractions of rat brain. Cholinergic stimulation increases incorporation into these fractions, likely by activation of phospholipase A₂ and/or C in association with acyltransferase activity. Thus, intravenously infused radiolabeled arachidonic acid can be used to examine synapse-mediated changes in brain phospholipid metabolism in vivo.     

Polyunsaturated fatty acid incorporation into plasmalogens in plasma membrane of glioma cells is preceded temporally by acylation in microsomes.

Plasmalogens (1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine) are major phospholipids in many tissues and cells, particularly of neural origin. Using cultured C6 glioma cells and subcellular fractions isolated on Percoll gradients we investigated selectivity for esterification of several polyunsaturated fatty acids (PUFA) in the sn-2 position of plasmalogens compared to [1-14C]hexadecanol, representative of de novo synthesis of the ether-linked sn-1 position. In whole cells at a final concentration of 105 microM PUFA, 2-4 nmol plasmalogen/mg protein was labeled in 4 h and 10-14 nmol in 24 h, representing 8-15% and 35-50%, respectively, of initial plasmalogen mass. Incorporation of label from hexadecanol was lower than PUFA incorporation (20:5(n-3) greater than 20:4(n-6) greater than 18:3(n-3) much greater than 18:2(n-6)) suggesting deacylation-reacylation at the sn-2 position. Plasmalogens accounted for 50% of total cell ethanolamine phospholipids and 75% in plasma membrane. Using a novel, improved method for extraction of subcellular fractions containing Percoll, plasma membrane also was enriched in plasmalogen relative to microsomes (107.4 +/- 5.2 vs. 40.0 +/- 2.9 nmol/mg protein). Selectivity for esterification at the sn-2 position of plasmalogens with respect to chain length and unsaturation of the fatty acyl chain was similar in both subcellular fractions and reflected that of whole cells. Labeling of plasma membrane with PUFA and fatty alcohol lagged behind that of microsomes. Chase experiments in cells prelabeled with [1-14C]18:3(n-3) for 2 h showed no significant reduction of label in plasmalogen of any subcellular fraction although accumulation of label in the microsomal fraction was slowed initially. Reduction of plasmalogen label (40-50%) did occur in microsomes and plasma membrane when cells prelabeled for 24 h were switched to chase medium with or without chase fatty acid. Our data suggest that esterification of PUFA to plasmalogen may occur at the endoplasmic reticulum with subsequent translocation to plasma membrane resulting in accumulation of relatively stable pools of plasmalogen that are not readily accessible for deacylation-reacylation exchange with newly appearing PUFA. Alternatively, deacylation-reacylation may occur in a more stable phospholipid pool within the plasma membrane but would involve a slower process than at the endoplasmic reticulum.     

Biosynthesis of prostaglandin E 2 in human skin: subcellular localization and inhibition by unsaturated fatty acids and anti-inflammatory drugs

The biosynthesis of prostaglandin E(2) (PGE(2)) from [1-(14)C]arachidonic acid has been demonstrated in homogenates and subcellular fractions of human epidermis. This biosynthetic capacity is localized in the microsomal fraction, indicating the presence of an active prostaglandin synthetase system associated with membranes of the skin. The incorporation of (14)C from [1-(14)C]arachidonic acid into PGE(2) by the microsomal fraction was enhanced by EDTA. This apparent increase in (14)C incorporation into PGE(2) in the presence of EDTA could be due at least in part to its chelating properties of removing the divalent cations in the homogenate that enhance the selective formation of PGF(2alpha) and the suppression of the activity of epidermal phospholipase A, which causes the release of nonradioactive fatty acid precursors from endogenous phospholipids. This study has also demonstrated that the formation of PGE(2) from arachidonic acid by the microsomal fraction from human skin could be inhibited by polyunsaturated fatty acids, suggesting a possible regulatory role of fatty acids released from endogenous phospholipids on prostaglandin synthesis in this tissue. The inhibitory effects of some anti-inflammatory drugs on skin microsomal prostaglandin synthetase were also demonstrated in these studies. Results from these studies indicate that the skin is therefore a useful tissue for the study of mechanisms of prostaglandin biosynthesis and the mode of action of various anti-inflammatory drugs.     

Effects of postdecapitation ischemia on the metabolism of [14C]arachidonic acid and [14C]palmitic acid in the mouse brain

The effect of postdecapitation ischemia on the labeling of the free fatty acid pool and their incorporation in lipids was examined during the first 10 min after decapitation in mouse brain that had been injected intracerebrally with either [1-14C]arachidonic acid or [1-14C]palmitic acid. One min after decapitation, animals injected with labeled arachidonic acid exhibited a greatly reduced incorporation of label in brain phospholipids, diglycerides, and triglycerides. When radioactive palmitic acid was used, brain lipids exhibited considerably less inhibition of label. However, a similar degree of inhibition was observed 10 min after decapitation with both fatty acids. At this time, free arachidonic acid had decreased 84% as compared to the 24% decrease observed in the controls, and about 77% of the free palmitic acid remained in the free fatty acid fraction as compared with 30% in the controls. This decreased labeling may reflect ATP shortage that affects the fatty acid activation-reacylation reactions or the enzymes involved. Alternatively, the enhanced endogenous free arachidonic acid may compete with the radiolabeled arachidonic acid resulting in an inhibition of lipid labeling. Inhibition of label may have been greater in radiolabeled arachidonic acid than palmitic because of the larger accumulation of the former endogenous fatty acid during early ischemia.     

Localization of [14C]DDT in the ventral nerve cord of the cockroach,Periplaneta americana (L.)

[¹⁴C]DDT was used as a probe to determine the subcellular localization of DDT in the ventral nerve cord (VNC) of the cockroach, Periplaneta americana (L.). Male cockroaches were injected intra-abdominally with [¹⁴C]DDT and their VNCs removed at 1 h post-injection. The VNCs were then subjected to homogenization and differential centrifugation to isolate plasma membrane, mitochondrial, and microsomal fractions. Results indicate that the plasma membrane fraction contained the greatest amount of [¹⁴C]DDT, with the mitochondrial and microsomal fractions containing significantly less. Calculations and a comparison with I₅₀ values for oligomycin-sensitive (OS)Mg-ATPase from the literature support the prediction that an insufficient amount of DDT reaches the ventral nerve cord mitochondria of a cockroach to effect an I₅₀ level of inhibition of the (OS)Mg-ATPase.     

Methylmalonic and propionic acidemias: lipid profiles of normal and affected human skin fibroblasts incubated with [1-14C]propionate

Normal human skin fibroblasts and those from methylmalonic acidemia and propionic acidemia patients were grown in culture. Following incubation with [1-14C]propionate, the major lipid classes in the cells were separated by thin layer chromatography and isolated fractions analyzed by radio gas chromatography for the presence of odd-numbered long-chain fatty acids; the pattern of even-numbered long-chain fatty acids was obtained also. Normal fibroblasts incorporated a small percentage of propionate into odd-numbered fatty acids which were present in all lipids studied. The abnormal cells incorporated a larger amount while maintaining the characteristic ratios of odd-numbered fatty acids found in the normal line. Most of the radioactivity was associated with phospholipids which are the predominant constituents of cell membranes. A characteristic C15/C17 ratio was found for different phospholipids and the triglyceride fraction; pentadecanoic acid was the principal odd-numbered fatty acid utilized in the assembly of complex lipids. Compared to even-numbered long-chain fatty acids the absolute amount of odd-numbered fatty acids was low (1-2%), even in affected cells. An unusual polar lipid fraction was isolated in the course of the study. In the normal cell it contained several unlabeled eicosanoids which were missing from the same fraction of both affected cell lines.     

The metabolism of (1-14C) stearic acid in rat testicular tissue.

(1) The metabolism of stearic acid was studied in vivo following intratesticular injection of [1-14C] stearate. Soon after injection 14C activity was found mainly in the free fatty acid pool. This was followed at later time periods by transfer of label primarily to the phosphatide pool. During each time period significant amounts of label were recovered at 14CO2. (2) Analysis of 14C-labeled fatty acids from the injected testes demonstrated an initial rapid rate of oxidation and desaturation of [1-14C] stearate followed by a slower steady state rate. It was concluded that the initial rate was due to the rapid turnover of the highly labeled free fatty acid pool followed by a much slower rate as [14C] stearate was esterified to the more metabolically stable phospholipids. Elongation of the labeled stearic or its desaturated derivative was not observed. (3) The rate of desaturation in vitro of stearic acid was measured in microsomal preparations from rat testes and found to be 12.0 +/- 0.5 pmol/min/mg compared to the estimated in vivo value of 22 pmol/min/mg and the value of 390 pmol/min/mg for hepatic microsomal desaturase.     

Decarboxylation of malonyl-CoA and biosynthesis of mevalonic acid in rat liver]

Biosynthesis of mevalonic acid (MVA), total formation of 14CO2 from [1,3-14C]malonyl-CoA and the activity of malonyl-CoA decarboxylase in subcellular fractions of rat liver were studied. The dependence of the rate of MVA biosynthesis on malonyl-CoA concentration was found to be linear both in 140,000 g supernatant and solubilized microsomal fractions. It was shown that in a composite system (140,000 g supernatant fraction added to washed microsomes, 10 : 1) the optimal concentration ratio for the substrates of MVA biosynthesis (malonyl-CoA and acetyl-CoA) is 1 to 2. In the absence of acetyl-CoA decarboxylation of [1,3-14C]malonyl-CoA was prevalent. In all subcellular fractions studied decarboxylation of [1,3-14C]malonyl-CoA prevailed over its incorporation into MVA, total non-saponified lipid fraction and fatty acids. The degree of malonyl-CoA, decarboxylation was not correlated with the rate of its incorporation into MVA, i. e. the increase in the 14CO2 formation was not accompanied by stimulation of [1,3-14C]malonyl-CoA incorporation either into MVA or into total non-saponified lipid fractions. The incorporation of [1-14C]acetyl-CoA into MVA under the same conditions was considerably lower than that of [1,3-14C]malonyl-CoA. In all subcellular fractions under study the activity of malonyl-CoA decarboxylase was found. The experimental data suggest that a remarkable part of malonyl-CoA is incorporated into MVA without preliminary decarboxylation. A possible role of malonyl-CoA decarboxylase as an enzyme which protects the cell against accumulation of malonyl-CoA and its immediate metabolites -- malonate and methylmalonyl-CoA is disucssed.     

Cerebral metabolism of plasma [14C]palmitate in awake,adult rat: Subcellular localization

Following intravenous injection of [U-¹⁴C]palmitate in awake adult rats, whole brain radioactivity reached a broad maximum between 15–60 min, then declined rapidly to reach a relatively stable level between 4 hr and 20 hr. At 44 hr total radioactivity was 57% of the 4 hr value (p<0.05). About 50% of palmitate which entered the brain from the blood was oxidized rapidly, producing¹⁴C-labeled water-soluble components which later left the cytosol. Radioactivity in the cytosolic fraction peaked at 45 min and then declined, coincident with the decline in total brain radioactivity. Membrane fractions were rapidly labeled to levels which remained relatively stable from 1 to 44 hr. Increases in the relative distributions of radioactivity were seen between 1 and 4 hr for the microsomal and mitochondrial fractions, and beyond 4 hr for the synaptic and myelin membrane fractions (p<0.05). Radioactivity in membrane fractions was 80–90% lipid, 5–13% water-soluble components and 3–17% protein. The proportion of label in membrane-associated protein increased with time. Proportions of radioactivity in the combined membrane fractions increased from 65% to 76% to 80% at 4, 20 and 44 hr, respectively. The results show that plasma-derived palmitate enters oxidative and synthetic pathways to an equal extent, immediately after entry into the brain. At and after 4 hr, the radiolabel resides predominantly in stable membrane lipids and protein. Brain radioactivity at 4 hr can be used therefore, to examine incorporation of palmitate into lipids in vivo, in different experimental conditions.     

The kinetics of incorporation in vivo of [14C]acetate and [14C]carbon dioxide into the fatty acids of glycerolipids in developing leaves

1. The patterns of incorporation of (14)C into glycerolipid fatty acids of developing maize leaf lamina from supplied [1-(14)C]acetate and from (14)CO(2) during steady-state photosynthesis were similar. Oleate of phosphatidylcholine and palmitate of phosphatidylglycerol attained linear rates of labelling more rapidly than did other fatty acids, particularly the linoleate and linolenate of monogalactosyl diacylglycerol. 2. After the transfer of lamina from labelled to unlabelled acetate, there was a decrease in labelled oleate and linoleate of phosphatidylcholine and a concomitant increase in the amount of radioactivity in the linoleate and linolenate of monogalactosyl diacylglycerol. 3. The rapidly labelled phospholipids, phosphatidylcholine and phosphatidylglycerol, were shown by differential and sucrose-density-gradient centrifugation to be associated with different organelles, the former being mainly in a low-density membrane fraction, probably microsomal, and the latter mainly in chloroplasts. 4. During a 48h period after supplying spinach leaves with [(14)C]acetate, radioactivity was lost from the oleate of phosphatidylcholine present in fractions sedimented at 12000g and 105000g, and accumulated in the linolenate of monogalactosyl diacylglycerol of the chloroplast. 5. It is proposed that the phosphatidylcholine of some non-plastid membranes is intimately involved in the process of oleate desaturation and that this lipid serves as a donor of unsaturated C(18) fatty acids to other lipids, principally monogalactosyl diacylglycerol, of the chloroplasts.