ACYLATION OF LYSOPHOSPHATIDYLSERINE BY RAT BRAIN MICROSOMES

Abstract— The acylation of lysophosphatidylserine, prepared by snake venom digestion of phosphatidylserine, by rat brain microsomes is described. Acylation was monitored by spectrophotometric assay and by measuring the incorporation of radioactively labelled acyl CoA thioesters. Acylation was time dependent, showed an approximately linear response to enzyme concentration and had a pH optimum of 9.0. Maximum acylation was attained at a concentration of about 100 μM for lysophosphatidylserine and about 40μM for acyl CoA thioesters. Positional distribution studies with [¹⁴C]oleoyl CoA and [¹⁴C]arachidonoyl CoA showed incorporation was predominantly at position -2, but with significant labelling at position–1, particularly with oleoyl CoA, possibly as a result of isomerization of the 1–acyl isomer of lysophosphatidylserine. Both saturated and unsaturated thioesters could serve as acyl group donors. Myristoyl CoA was considerably superior to palmitoyl CoA and stearoyl CoA, which were poor acyl group donors. Some selectivity was shown among the long chain unsaturated thioesters, linoleoyl, linolenoyl and arachidonoyl CoA being the most effective acylating agents. Although docosahexaenoic acid is a major unsaturated fatty acid in brain phosphatidylserine, its CoA ester was a relatively poor acyl group donor. Relative acylation rates remained essentially constant over a wide range of lysophosphatidylserine concentrations. It is concluded that acyl transfer mechanisms are active in brain for the regulation of the fatty acid profile of phosphatidylserine.     

Metabolism of sulfoquinovosyl diglyceride in Chlorella pyrenoidosa by sulfoquinovosyl monoglyceride: fatty acyl CoA acyltransferase and sulfoquinovosyl glyceride: fatty acyl ester hydrolase pathways

Cell-free preparations of Chlorella pyrenoidosa catalyze the transfer of the fatty acyl moiety of fatty acyl CoA derivatives to sulfoquinovosyl monoglyceride to form sulfoquinovosyl diglyceride. This reaction is stimulated by Triton X-100 concentrations of up to 0.6 mg/ml and has a pH optimum of 7.7. Similar Chlorella preparations catalyze the stepwise removal of both fatty acyl groups from sulfoquinovosyl diglyceride to form sulfoquinovosyl monoglyceride and then sulfoquinovosyl glycerol. This reaction is inhibited by both calcium and magnesium. The nonionic surfactant Triton X-100 inhibits the enzymatic deacylation at concentrations of less than 0.5 mg/ml but stimulates it at higher concentrations. The pH optimum for the deacylation of sulfoquinovosyl glycerides is 8.2, with little activity observed below pH 8. The enzymatic activities for both the transacylation and deacylation reactions are associated with a 30,000 g particulate fraction of Chlorella. Sulfoquinovosyl glycerol was found not to be an acceptor of the fatty acyl moiety of fatty acyl CoA derivatives. Methods are described for the preparation of sulfoquinovosyl monoglyceride, sulfoquinovose, and 3-sulfo-1,2-propanediol.     

BIOSYNTHESIS OF PHOSPHATIDIC ACID IN RAT BRAIN VIA ACYL DIHYDROXYACETONE PHOSPHATE

Abstract— The enzymes for the biosynthesis of phosphatidic acid from acyl dihydroxyacetone phosphate were shown to be present in rat brain. These enzymes were mainly localized in the microsomal fraction of 12–14 day old rat brains. The brain microsomal acyl CoA: dihydroxyacetone phosphate acyl transferase (EC 2.3.1.42), exhibited a broad pH optimum between pH 5 and 9 with maximum activity at pH 5.4. K _m for DHAP at pH 5.4 was 0.1 m m and V _max was 0.86nmol/min/mg of microsomal protein. The corresponding microsomal enzyme for the glycerophosphate pathway (acyl CoA: sn -glycerol-3-phosphate acyl transferase EC 2.3.1.15) was shown to have a different pH optimum (pH 7.6). On the basis of the differences in pH optima, differential effects of sodium cholate in the enzymes and a common substrate competition study, these acyl transferases were postulated to be two different microsomal enzymes.
Acyl DHAP:NADPH oxidoreductase (EC 1.1.1.101) in brain microsomes was found to be quite specific for NADPH as cofactor, being able to utilize NADH only at very high concentrations. This enzyme exhibited a K _m of 8.6 μ m with NADPH and V _mx of 0.81 nmol/min/mg protein. The presence of these two enzymes and the known presence of l-acyl- sn -glycerol-3-phosphate: acyl CoA acyl transferase in brain (F leming & H ajra , 1977) demonstrated the biosynthesis of phosphatidic acid in brain via acyl dihydroxyacetone phosphate. Phosphatidic acid was shown to form when dihydroxyacetone phosphate, acyl CoA, NADPH and other cofactors were incubated together with brain microsomes. Further properties of the enzymes and the probable importance of the presence of this pathway in brain were discussed.     

Steryl glucoside and acyl glucoside biosynthesis in maturing pea seeds

Sterol glucosyltransferase activity was found in a particulate fraction of pea seeds. The activity was stimulated by Ca²⁺ and Mg²⁺ and inhibited by Zn²⁺, Cu²⁺, Hg²⁺, EDTA and EGTA. Iodoacetamide was without effect but p-chloromercuribenzoate completely inhibited the enzyme. N -Ethylmaleimide gave 60–70 % inhibition over a wide range of concentrations. The activity was stimulated by ATP in the presence of Mg²⁺. Under such conditions, steryl acyl glucoside was formed. The acyl derivative was barely detectable in the presence of Ca²⁺ either with or without ATP. Both oleyl CoA and palmityl CoA stimulated acyl glucoside synthesis. Of the four nucleoside triphosphates, ATP, GTP, UTP and CTP both ATP and CTP stimulated acylation in the presence of Mg²⁺. The observations suggest that acyl donors other than digalactosyl diglyceride and phospholipids may function in steryl acyl glucoside synthesis in plants.     

Acylation of dog heart lysophosphatidylserine by transacylase activity

Dog heart microsomes catalyze the transfer of acyl groups from the sn-2 position of phosphatidylcholine (PC) to lysophosphatidylserine (lysoPS) in the presence of coenzyme A (CoA) at pH optima of 4.5-5.0 and 7.5. Acyl transfer activity at acidic pH is about three times higher than at neutral pH. Transacylation of lysoPS by acyl transfer from PC with dog heart microsomes at neutral pH favors arachidonate over linoleate by a factor of 2.1, whereas free linoleic acid is favored by a factor of 3.7 over arachidonic acid for lysoPS acylation in the presence of acyl-CoA-generating cofactors. Considering the location and acyl composition of myocardial PS, it appears that both acyl transfer from PC and utilization of unesterified fatty acids may be involved in the acylation of lysoPS at its sn-2 position.     

MgATP may depress de novo neuronal nuclear PAF generation by promoting the formation of alkylacylglycerophosphate,an inhibitor of alkylglycerophosphate acetyltransferase

MgATP substantially inhibited 1-alkyl-sn-glycero-3-phosphate (AGP) acetyltransferase found in neuronal nuclei. Other nucleotides and the ATP analogue AMP-PNP did not show a comparable inhibition. MgATP inhibition decreased in the presence of bovine serum albumin or the fatty acyl CoA synthetase inhibitor, Triacsin C. MgATP inhibition increased when nuclei were preincubated in 50 mM Tris-HCl (pH 7.4)/1 mM MgCl(2) at 37 degrees C, and preincubations elevated levels of nuclear free fatty acid. Exogenous free fatty acid, added to the acetylation incubations, increased the inhibition seen in the presence of MgATP. Oleoyl CoA, in the absence of MgATP, also inhibited AGP acetylation. These results suggested that MgATP supported the conversion of nuclear free fatty acids to fatty acyl CoA. Fatty acyl CoA may directly inhibit nuclear AGP acetyltransferase, but inhibition brought about by MgATP was competitive for the AGP substrate, suggesting an inhibitor close in structure to AGP. 1-Hexadecyl-2-arachidonoyl-sn-glycero-3-phosphate was identified as a competitive inhibitor for AGP in the acetylation reaction. Neuronal nuclei can convert AGP to 1-alkyl-2-acyl-sn-glycero-3-phosphate (AAcylGP), a reaction dependent upon MgATP and the presence of acetyl CoA or free CoA. This nuclear acylation was increased by free fatty acid addition and was seen using oleoyl CoA in the absence of MgATP. Nuclear AAcylGP formation was inhibited by bovine serum albumin and by Triacsin C. Thus, nuclear AGP acetyltransferase may be regulated by AGP acyltransferase activity and the availability of MgATP, a nucleotide that is rapidly lost during brain ischemia.     

Selective acyl transfer in the reacylation of brain glycerophospholipids. Comparison of three acylation systems for 1-alk-1'-enylglycero-3-phosphoethanolamine, 1-acylglycero-3-phosphoethanolamine and 1-acylglycero-3-phosphocholine in rat brain microsomes

The activities of three acylation systems for 1-alkenylglycerophosphoethanolamine (1-alkenyl-GPE), 1-acyl-GPE and 1-acylglycerophosphocholine (1-acyl-GPC) were compared in rat brain microsomes and the acyl selectivity of each system was clarified. The rate of CoA-independent transacylation of 1-[3H]alkenyl-GPE (approx. 4.5 nmol/10 min per mg protein) was about twice as high as in the case of 1-[3H]acyl-GPE and 1-[14C]acyl-GPC. On the other hand, the rates of CoA-dependent transacylation and CoA + ATP-dependent acylation (acylation of free fatty acids by acyl-CoA synthetase and acyl-CoA acyltransferase) of lysophospholipids were in the order 1-acyl-GPC greater than 1-acyl-GPE much greater than 1-alkenyl-GPE. HPLC analysis of newly synthesized molecular species revealed that the CoA-independent transacylation system exclusively esterified docosahexaenoate and arachidonate, regardless of the lysophospholipid class. The CoA-dependent transacylation and CoA + ATP-dependent acylation systems were almost the same with respect to the selectivities for unsaturated fatty acids when the same acceptor lysophospholipid was used, but some distinctive acyl selectivities were observed with different acceptor lysophospholipids. 1-Alkenyl-GPE selectively acquired only oleate in these two systems. 1-Acyl-GPE and 1-acyl-GPC showed selectivities for both arachidonate and oleate. In addition, an appreciable amount of palmitate was transferred to 1-acyl-GPC, not to 1-acyl-GPE, in CoA- or CoA + ATP-dependent manner. The acylation of exogenously added acyl-CoA revealed that the acyl selectivities of the CoA-dependent transacylation and CoA + ATP-dependent acylation systems may be mainly governed through the selective action of acyl-CoA acyltransferase. The preferential utilization of oleoyl-CoA by all acceptors and the different utilization of arachidonoyl-CoA between alkenyl and acyllysophospholipids indicated that there might be two distinct acyl-CoA:lysophospholipid acyltransferases that discriminate between oleoyl-CoA and arachidonoyl-CoA, respectively. Our present results clearly show that all three microsomal acylation systems can be active in the reacylation of three major brain glycerophospholipids and that the higher contribution of the CoA-independent system in the reacylation of ethanolamine glycerophospholipids, especially alkenylacyl-GPE, may tend to enrich docosahexaenoate in these phospholipids, as compared with in the case of diacyl-GPC.     

Fatty Acid Acylation of Rat Brain Myelin Proteolipid Protein In Vitro: Identification of the Lipid Donor

The immediate acyl chain donor for fatty acid esterification of proteolipid protein (PLP) was identified in an in vitro system. Rat brain total membranes, after removal of crude nuclear and mitochondrial fractions, were incubated with radioactive acyl donors, extracted with chloroform/methanol, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of [3H]palmitic acid, CoA, ATP, and Mg2+, acylation of endogenous PLP occurred at a linear rate for at least 2 h. The radioactivity was associated with the protein via an ester linkage, mainly as palmitic acid. Omission of ATP, CoA, Mg2+, or all three reduced fatty acid incorporation into PLP to 44, 27, 8, and 4%, respectively, of the values in the complete system. Incubation of the membrane fraction with [3H]palmitoyl-CoA in the absence of CoA and ATP led to highly labeled PLP. These data demonstrate that activation of free fatty acid is required for acylation. Phospholipids and glycolipids were not able to acylate the PLP directly. Finally, when isolated myelin was incubated with [3H]palmitoyl-CoA in the absence of cofactors, only PLP was labeled, thus confirming the identity of palmitoyl-CoA as the direct acyl chain donor and suggesting that the acylating activity and the PLP pool available for acylation are both in the myelin.     

Identification of a 51-kilodalton polypeptide fatty acyl chain acceptor in soluble extracts from mouse cardiac tissue

We have identified a protein in the soluble fraction from mouse cardiac tissue extracts which is rapidly and selectively acylated by myristyl CoA. This protein was partially purified by anion-exchange chromatography and gel filtration, and the acylation reaction was measured using [3H]myristyl CoA as substrate, followed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis to resolve [3H]fatty acyl polypeptides. The [3H]acyl protein migrated as heterogeneous bands corresponding to relative masses (MrS) of 42,000-51,000 under nonreducing conditions or as a single polypeptide of Mr 51,000 in the presence of reducing agents. Fatty acyl chain incorporation into protein was very rapid and already maximum after 30 s of incubation, whereas no acylation was detected using heat-denatured samples or when the reaction was stopped immediately after initiation. Only the acyl CoA served as fatty acyl chain donor. No incorporation into protein occurred when myristyl CoA was substituted by myristic acid, ATP, and CoA. A time-dependent reduction in the level of [3H]fatty acyl polypeptide was observed upon addition of excess unlabeled myristyl CoA, indicating the ability of the labeled acyl moiety of the protein to turn over during incubation. The saturated C10:0, C14:0, and C16:0 acyl CoAs were more effective to chase the label from the [3H]acyl polypeptide than the C18:0 and C18:1 acyl CoAs. These results provide evidence for a 51-kilodalton polypeptide which serves as an acceptor for fatty acyl chains and could represent an important intermediate in fatty acyl chain transfer reactions in cardiac tissue.     

Biosynthesis of phosphatidic acid by glycerophosphate acyltransferases in rat liver mitochondria and microsomes

The acyltransferases that catalyze the synthesis of phosphatidic acid from labelled sn-[14C]glycero-3-phosphate and fatty acyl carnitine or coenzyme A derivatives have been shown to be present in both isolated mitochondria and microsomes from rat liver. The major reaction product was phosphatidic acid in both subcellular fractions. A small quantity of lysophosphatidic acid and neutral lipids were produced as by-products. Divalent cations had significant effects on both mitochondrial and microsomal fractions in stimulating acylation using palmitoyl CoA, but not when palmitoyl carnitine was used as the acyl donor. Palmitoyl CoA and palmitoyl carnitine could be used for acylation by both mitochondria and microsomes. Mitochondria were more permeable to palmitoyl carnitine and readily used it as the substrate for acylation. On the other hand, microsomes yielded a better rate with palmitoyl CoA and the rate of acylation from palmitoyl carnitine in microsomes was correlated with the degree of mitochondrial contamination. The enzymes were partially purified from Triton X-100 extracts of subcellular fractions. Based on the differences of substrate utilization, products formed, divalent cation effects, molecular weights, and polarity, the mitochondrial and microsomal acyltransferases appeared to be different enzymes.     

Coupled biosynthesis and esterification of 1,2,4‐butanetriol to simplify its separation from fermentation broth

1,2,4‐Butanetriol (BT) is a valuable chemical with versatile applications in many fields and can be produced through biosynthetic pathways. As a trihydric alcohol, BT possesses good water solubility and is very difficult to separate from fermentation broth, which does complicate the production process and increase the cost. To develop a novel method for BT separation, a biosynthetic pathway for 1,2,4‐butanetriol esters with poor water solubility was constructed. Wax ester synthase/acyl‐coenzyme A: diacylglycerol acyltransferase (Atf) from Acinetobacter baylyi, Mycobacterium smegmatis, and Escherichia coli were screened, and the acyltransferase from A. baylyi (AtfA) was found to have higher capability. The BT producing strain with AtfA overexpression produced 49.5 mg/L BT oleate in flask cultivation. Through enhancement of acyl‐CoA production by overexpression of the acyl‐CoA synthetase gene fadD and deleting the acyl coenzyme A dehydrogenase gene fadE, the production was improved to 64.4 mg/L. Under fed‐batch fermentation, the resulting strain produced up to 1.1 g/L BT oleate. This is the first time showed that engineered E. coli strains can successfully produce BT esters from xylose and free fatty acids.     

Flavonol 3-O-glycoside hydroxycinnamoyltransferases from Scots pine (Pinus sylvestris L.)

Flavonol 3-O-glucosides esterified with ferulic or p-coumaric acid at positions 3' and 6' are the major UV-B screening pigments of the epidermal layer of Scots pine (Pinus sylvestris) needles. The last steps in the biosynthesis of these compounds are catalyzed by enzymes that transfer the acyl part of hydroxycinnamic acid CoA esters to flavonol 3-O-glucosides. A newly developed enzyme assay revealed three flavonol 3-O-glucoside hydroxycinnamoyltransferases (HCTs) in Scots pine needles with specificities for positions 3', 4' or 6'. The positions of the acyl groups were identified by cochromatography with reference compounds and by NMR spectroscopy. The enzymes were characterized by molecular mass, isoelectric point, and also pH and temperature optima. Substrate specificities for flavonol glycosides and hydroxycinnamic acid CoA esters as well as kinetic properties of 3'- and 6'HCT suggested that acylation preferably occurs with glucosides and p-coumaroyl-CoA. In addition, acylation takes place in a well-defined order, beginning at position 6' followed by acylation at position 3'. These results give the first detailed characterization of flavonol 3-O-glycoside HCTs involved in the protection of plant tissues against UV-B (280-315 nm) radiation.     

Acylation of 1-alkenylglycerophosphoethanolamine and 1-acylglycerophosphoethanolamine in guinea-pig heart microsomes

Although the acylation of 1-alkenylglycerophosphocholine in mammalian heart is well documented, the acylation of 1-alkenylglycerophosphoethanolamine in the heart was not reported. In this study, the presence of acyl CoA: 1-alkenylglycerophosphoethanolamine acyltransferase in the guinea pig heart microsomes was demonstrated. 1-Alkenylglycerophosphoethanolamine acyltransferase displayed a high degree of specificity towards acyl-CoA. The order of reactivity with acyl-CoA was found to be: linoleoyl much greater than arachidonyl greater than palmitoyl greater than stearoyl = oleoyl. 1-Acylglycerophosphoethanolamine acyltransferase in the microsomes also exhibited specificity towards acyl-CoA in the following manner: linoleoyl greater than arachidonyl much greater than palmitoyl greater than oleoyl greater than stearoyl. However, such specificity appeared to be dependent on acyl-CoA concentration. The acyl-CoA specificities of both enzymes did not correlate with the C-2 acyl distribution observed in the corresponding microsomal phospholipids. Our results suggest that in addition to the acyl specificity of the acyltransferases, intracellular concentrations of acyl-CoAs may also have an important role in determining the observed acyl patterns of the phospholipids. Based on the acyl specificities, pH profiles, and their responses to heat inactivation and thiol reagents, we conclude that 1-alkenylglycerophosphoethanolamine acyltransferase and 1-acylglycerophosphoethanolamine acyltransferase in guinea-pig heart microsomes are not the same enzyme.     

Carnitine acyltransferases and their influence on CoA pools in health and disease

Cells contain limited and sequestered pools of Coenzyme A (CoA) that are essential for activating carboxylate metabolites. Some acyl-CoA esters have high metabolic and signalling impact, so control of CoA ester concentrations is important. This and transfer of the activated acyl moieties between cell compartments without wasting energy on futile cycles of hydrolysis and resynthesis is achieved through the carnitine system. The location, properties of and deficiencies in the carnitine acyltransferases are described in relation to their influence on the CoA pools in the cell and, hence, on metabolism. The protection of free CoA pools in disease states is achieved by excretion of acyl-carnitine so that carnitine supplementation is required where unwanted acyl groups build up, such as in some inherited disorders of fatty acid oxidation. Acetyl-carnitine improves cognition in the brain and propionyl-carnitine improves cardiac performance in heart disease and diabetes. The therapeutic effects of carnitine and its esters are discussed in relation to the integrative influence of the carnitine system across CoA pools. Recent evidence for sequestered pools of activated acetate for synthesis of malonyl-CoA, for the synthesis of polyunsaturated fatty acids and for the inhibition of carnitine palmitoyltransferase 1 to regulate fatty acid oxidation is reviewed.     

Solubilisation and reconstitution of acylcoenzyme A:estradiol-17 beta acyltransferase

Acylcoenzyme A:estradiol-17 beta acyltransferase in microsomes of bovine placenta cotyledons was strongly membrane bound. The enzyme was solubilised from microsomes by sodium cholate and was reconstituted into phospholipid vesicles. The apparent Km for estradiol-17 beta was 11 microM which was close to the value of 8 microM previously found with the membrane-bound enzyme. Testosterone was also a substrate for the reconstituted enzyme (apparent Km 62 microM) and was a competitive inhibitor (Ki 74 microM) of the acylation of estradiol-17 beta. Although various long-chained fatty acyl CoAs acted as acyl donors, these proved to have widely differing apparent Km values with palmitoleoyl CoA having the highest affinity (Km 24 microM) and arachidonoyl CoA the lowest affinity (Km 330 microM).     

Rapid metabolism of fatty acids covalently bound to myelin proteolipid protein

Proteolipid protein (PLP), the major protein of central nervous system myelin, contains approximately 2 mol of covalently bound fatty acids. In this study, the in vivo turnover rate of the acyl chains bound to PLP was determined in 40-day-old rats after a single intracranial injection of [3H]palmitic acid. The apparent half-life of total fatty acids bound to PLP was approximately 7 days. After correction for acyl chain interconversion, the half-life of palmitate bound to PLP was only 3 days. This turnover rate is much more rapid than that of the protein moiety calculated under the same experimental conditions (t1/2 = 1 month). Additional evidence for the dynamic metabolism of acyl groups was provided by experiments in brain tissue slices which showed that acylation of PLP occurs in adult animals as well as during active myelination. Acylation of endogenous PLP in purified myelin and its subfractions was also studied during rat brain development using either [3H]palmitoyl-CoA or [3H]palmitic acid plus ATP and CoA. Labeling of endogenous PLP with [3H]palmitoyl-CoA was observed as early as 10 days postnatal and continued at the same rate throughout development. When [3H]palmitic acid was used as precursor in the presence of both ATP and CoA, esterification of myelin PLP occurred rapidly in adult animals, indicating that both nonacylated PLP and acyl-CoA ligase are present in myelin. Finally, pulse-chase experiments in a cell-free system showed that PLP-bound fatty acids turn over with a half-life shorter than 10 min. These observations are consistent with the concept that acylation of myelin PLP is a dynamic process involved mainly in myelin maintenance and function.     

sn-Glycerol-3-phosphate acyltransferase activity in particulate preparations from anaerobic, light-grown cells of Rhodopseudomonas spheroides. Involvement of acyl thiolester derivatives of acyl carrier protein in the synthesis of complex lipids.

Crude particulate preparations obtained from anaerobic, light-grown cells of Rhodopseudomonas spheroides have been shown to possess a significant level of sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15) activity. In contrast to the enzyme from Escherichia coli, the R. spheroides glycerophosphate acyltransferase has a high specificity for acyl thiolester derivatives of acyl carrier protein (ACP) as acyl donors for the reaction. Only limited , nonlinear glycerophosphate incorporation into lipid occurs when acyl coenzyme A (CoA) derivatives are employed as acyl substrate. With oleyl-ACP as substrate, maximal enzyme activity was observed at 40 degrees, over a broad pH range (6.0 to 8.5) and did not require a divalent metal cation. The presence of dithiothreitol stimulated enzyme-activity 15 to 20%. When oleyl-ACP or palmityl-ACP was employed as sole acyl group donor, the major products recoverable from the reaction mixtures were lysophosphatidic acid, phosphatidic acid, and monoglyceride. Althouh oleyl-ACP and palmityl-ACP gave comparable maximal velocities in the initial acylation of glycerophosphate, the formation of phosphatidic acid occurred preferentially with the unsaturated acyl-ACP derivative.     

Lipolytic enzymes of Trichophyton rubrum.

Trichophyton rubrum cells contain lipase, phospholipases A and B and acyl CoA lysolecithin acyl transferase activities. This dermatophyte excretes lipase and phospholipase A into the growth medium when cultivated in Sabouraud's broth. Extracellular lipase has optimum activity at pH 8.0 whereas the intracellular lipase is maximally active at pH 8.0 whereas the intracellular lipase is maximally active at pH 7.0. The optimum pH of phospholipase A and B activities which are localized in 15000 g sedimentable cell fragments are 7.0 and 6.0 respectively. Supernatant obtained after removal of 1,005,000 g sedimentable fragments from cell extract contains acyl CoA lysolecithin acyl transferase which requires ATP, CoA, Mg2+ and an unsaturated fatty acid for its activity.     

Characterization of Lysophospholipid Metabolizing Enzymes in Human Brain

Abstract: Lysophospholipids are generated during the turnover and breakdown of membrane phospholipids. We have identified and partially characterized three enzymes involved in the metabolism of lysophospholipids in human brain, namely, lysophospholipase, lysophospholipid:acyl-CoA acyltransferase (acyltransferase), and lysophospholipid:lysophospholipid transacylase (transacylase). Each enzyme displayed comparable levels of activity in biopsied and autopsied human brain, although in all cases the activity was somewhat lower in human than that in rat brain. All three enzymes were localized predominantly in the particulate fraction, with lysophospholipase possessing the greatest activity followed by acyltransferase and transacylase. Lysophosphatidylcholine possessed a K_m in the micromolar range for lysophospholipase and transacylase, and in the millimolar range for acyltransferase, whereas arachidonyl-CoA displayed a K_m in the micromolar range for acyltransferase. The three enzymes differed in their pH optima, with lysophospholipase being most active at pH 8.0, transacylase at pH 7.5, and acyltransferase at pH 6.0. Both bromophenacyl bromide and N-ethylmaleimide inhibited lysophospholipase activity and, to a lesser extent, that of acyltransferase and transacylase. None of the enzyme activities were affected by the presence of dithiothreitol or EDTA, although particulate lysophospholipase was activated approximately two-fold by the addition of 5 mM MgCl₂ or CaCl₂ but not KCl. Transacylating activity was stimulated by CoA, the EC₅₀ of activation being 6.8 µM. Acyltransferase displayed an approximately threefold preference for arachidonyl-CoA over palmitoyl-CoA, whereas the acylation rate of different lysophospholipids was in the order lysophosphatidylinositol > 1-palmitoyl lysophosphatidylcholine > 1-oleoyl lysophosphatidylcholine ? lysophosphatidylserine > lysophosphatidylethanolamine. This, and the preference of human brain phospholipase A₂ for phosphatidylinositol, suggests that this phospholipid may possess a higher turnover rate than the other phospholipid classes examined. Human brain homogenates also possessed the ability to transfer fatty acid from lysophosphatidylcholine to lysophosphatidylethanolamine. In addition, we also present evidence that diacylglycerophospholipids can act as acyl donors for the transacylation of lysophospholipids. We have therefore demonstrated the presence of, and partially characterized, three enzymes that are involved in the metabolism of lysophospholipids in human brain. Our results suggest that lysophospholipase may be the major route by which lysophospholipids are removed from the cell membrane in human brain. However, all three enzymes likely play an important role in the remodeling of membrane composition and thereby contribute to the overall functioning of membrane-associated processes.     

Biosynthesis of Long-Chain Alcohols by Developing and Regenerating Rat Sciatic Nerve

Abstract: Cell-free preparations of rat sciatic nerve were found to catalyze the reduction of fatty acid to alcohol in the presence of NADPH as reducing cofactor. The reductase was membrane-bound and associated primarily with the microsomal fraction. When fatty acid was the substrate, ATP, coenzyme A (CoA), and Mg²⁺ were required, indicating the formation of acyl CoA prior to reduction. When acyl CoA was used as substrate, the presence of albumin was required to inhibit acyl CoA hydro-lase activity. Fatty acid reductase activity was highest with palmitic and stearic acids, and somewhat lower with lauric and myristic acids. It was inhibited by sulfhydryl reagents, indicating the participation of thiol groups in the reduction. Only traces of long-chain aldehyde could be detected or trapped as semicarbazone. Fatty acid reductase activity in rat sciatic nerve was highest between the second and tenth days after birth and decreased substantially thereafter. Microsomal preparations of sciatic nerve from 10-day-old rats exhibited about four times higher fatty acid reductase activity than brain or spinal cord microsomes from the same animals. Wallerian degeneration and regeneration of adult rat sciatic nerve resulted in enhanced fatty acid reductase activity, which reached a maximum at about 12 days after crush injury.