Coexistence of phosphatidylcholine-specific phospholipase C and phospholipase D activities in rat cerebral cortex synaptosomes

DAG derived from phosphatidylcholine (PtdCho) acts as a lipid second messenger. It can be generated by the activation of phospholipase D (PLD) and the phosphatidic acid phosphohydrolase type 2 (PAP2) pathway or by a PtdCho-specific phospholipase C (PtdCho-PLC). Our purpose was to study PtdCho-PLC activity in rat cerebral cortex synaptosomes (CC Syn). DAG production was highly stimulated by detergents such as Triton X-100 and sodium deoxycholate. Ethanol and tricyclodecan-9-yl-xanthate potassium salt decreased DAG generation by 42 and 61%, respectively, at 20 min of incubation. These data demonstrate that both the PLD/PAP2 pathway and PtdCho-PLC contribute to DAG generation in CC Syn. PtdCho-PLC activity remained located mainly in the synaptosomal plasma membrane fraction. Kinetic studies showed K _m and V _max values of 350 μM and 3.7 nmol DAG × (mg protein × h)⁻¹, respectively. Western blot analysis with anti-PtdCho-PLC antibody showed a band of 66 KDa in CC Syn. Our results indicate the presence of a novel DAG-generating pathway in CC Syn in addition to the known PLD/PAP2 pathway.     

Characterization of the diacylglycerol acyltransferase activity in the membrane fraction from a fungus

In an attempt to clarify the mechanism of lipid accumulation inMortierella ramanniana var.angulispora, diacylglycerol acyltransferase (DGAT) in the membrane fraction from this fungus was characterized. The enzyme had an optimum pH of 7.0–7.5, and enzyme activity was blocked by SH-reagents. Metal ions were not essential for maintaining DGAT activity.n-Octyl-β-d-glucoside, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and Tween 80 were found to preserve activity, while Triton X-100 and sucrose monolaurate inhibited it. As the inhibition of DGAT activity by Triton X-100 was overcome by the addition of diacylglycerol (DG), the dependency of DGAT activity on exogenous DG was determined in the presence of 0.1% Triton X-100. DGAT activity in the membrane fraction was traced in fungi cultured for different time periods or in media at different carbon to nitrogen (C/N) ratios. Although the increase in total lipid content with culture time was accompanied by an increase in DGAT activity, total lipid changes related to changes in C/N ratio did not correlate with DGAT activity. Factors other than DGAT activity in the membrane fraction would appear to be involved in the regulation of total lipid content in this fungus.     

Difficulties in the assay of phosphatidate phosphohydrolase activity. Influence of ionic strength,detergent, and selection of substrate

In the present paper, problems in connection with assay of the activity of magnesium-dependent rat liver phosphatidate phosphohydrolase (PAP) are discussed. PAP activity is usually measured by following the production of diacylglycerol or inorganic phosphate from the substrate phosphatidate. These two methods may give widely different results due to a number of factors that may affect the assay. One such factor is the composition of the substrate. Higher apparent enzyme activity was observed with dioleoyl-phosphatidate than with dipalmitoyl-phosphatidate. This substrate-dependent difference in apparent PAP activity was 2-2.5-fold in the absence and 10-fold in the presence of Triton X-100, respectively. Triton X-100 reduced the activity as measured with the dipalmitoyl-phosphatidate substrate. In contrast, the activity of PAP as measured with dioleoyl-phosphatidate was stimulated by Triton X-100 The stimulatory effect of Triton was reduced or abolished when the ionic strength in the assay mixture was increased. Assays based on³²P-labeled substrate are rapid and sensitive. It is shown here that³³P can be used as an alternative. This radionuclide has a longer half-life and also emits particles with lower energy, thus posing less potential health hazards for the user.     

Characterization of phospholipase A2 from rabbit lung microsomes

A phospholipase A₂ activity associated with the microsomal fraction of rabbit lung homogenates was studied. The enzyme showed specificity for thesn ⁻² ester bond of phosphatidylcholine, had an alkaline pH optimum and required Ca²⁺ for activity. Other divalent cations were unable to support hydrolysis. In the absence of detergents, exogenous phosphatidylethanolamine was deacylated at a rate sevenfold higher than phosphatidylcholine. The activity toward both substrates could be enhanced by sodium deoxycholate or, more effectively, by sodium taurodeoxycholate. Phosphatidylethanolamine required higher detergent/phospholipid molar ratios than phosphatidylcholine. Under these conditions, the preference for the former substrate over the latter was nearly abolished. The zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) and the nonionic detergent Triton X-100 were either ineffective (phosphatidylcholine) or inhibitory (phosphatidylethanolamine). Addition of KCl produced opposite effects on the activity depending on the bile salt used to disperse the substrate. The phospholipase A₂ activity was inhibited byp-bromophenacyl bromide but remained unaffected after treatment with diisopropylfluorophosphate or NaF. N-Ethylmaleimide, but not other thiol reagents, partially inhibited the activity. Presented in part at the 29th CFBS meeting, Guelph, Canada, June 1986, and at the symposium “25 Years Lipids and Biomembranes,” Utrecht, The Netherlands, June 1986.     

Characterization of phospholipase D activity in bovine photoreceptor membranes

Phospholipase D (E.C. 3.1.4.4.) was detected in isolated bovine rod outer segments (ROS) and its properties determined. The enzyme activity was assayed using either a sonicated microdispersion of 1,2-diacyl-sn-[2³H]glycerol-3-phosphocholine (PC), or [¹⁴C]ethanol. Using [³H]PC and ethanol as a substrate, we were able to detect the hydrolytic properties as well as the transphosphatidylation reaction catalyzed by phospholipase D (PLD): formation of [³H]phosphatidic acid and phosphatidylethanol [³H]PtdEt; whereas with [¹⁴C]ethanol or [³H]glycerol in the absence of exogenous PC, only transphosphatidylation reactions were detected (formation of [¹⁴C]PtdEt or [³H]phosphatidylglycerol, respectively). The use of varying concentrations of [³H]PC and 400 mM of ethanol gave an apparent K _m value for PC of 0.51 mM and a V _max value of 111 nmol × h⁻¹ × (mg protein)⁻¹. The activity was linear up to 60 min of incubation and up to 0.2 mg of protein. The optimal ethanol concentration was determined to be 400 mM, with an apparent K _m of 202 mM and a V _max value for ethanol of 125 nmol × h⁻¹ × (mg protein)⁻¹. A clear pH optimum was observed around 7. PLD activity was increased in the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate or sodium deoxycholate and inhibited with Triton X-100. The enzyme activity was also activated in the presence of Ca²⁺ or Mg²⁺ (1 mM) although these ions were not required for measuring PLD activity. The high specific activity of PLD found in purified ROS compared to the activity found in other subcellular fractions of the bovine retina suggests that this enzymatic activity is native to ROS. The present report is the first evidence of PLD activity associated with photoreceptor ROS.     

CDPcholine:1,2-diacylglycerol cholinephosphotransferase from rat liver microsomes. I. Solubilization and characterization of the partially purified enzyme and the possible existence of an endogenous inhibitor

The solubilization and partial purification of cholinephosphotransferase (CDPcholine:1,2-diacylglycerol cholinephosphotransferase, EC 2.7.8.2) from rat liver microsomes were examined in the presence of ionic (sodium deoxycholate), nonionic (Triton X-100,n-octylglycoside), or zwitter ionic (CHAPS) detergents. Among the four detergents tested, only sodium deoxycholate was found to be an efficient solubilizer of cholinephosphotransferase activity from microsomal membranes, whereas the other three detergents caused irreversible inactivation of the enzyme at the solubilization step. Addition of phospholipids at the solubilization step, or after solubilization of the membrane proteins, could not preserve or reconstitute activity to any extent. The sodium deoxycholate-solubilized activity was partially purified by gel permeation chromatography (Superose 12HR). The partially purified preparation appeared to consist of a large aggregate containing phospholipids; further dissociation of the protein-phospholipid complex caused complete inactivation of the enzyme. The partially purified cholinephosphotransferase showed a specific activity of 100–130 nmol/min/mg protein, which is the highest activity reported to date from any tissue source; this amounts to a 4-fold enrichment of cholinephosphotransferase activity from the original KCl-washed rat liver microsomes. Ethanolaminephosphotransferase (CDPethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase, EC 2.7.8.1) activity was copurified and 6-fold enriched with a total recovery of 60%. During the purification of cholinephosphotransferase activity, a putative endogenous inhibitor of cholinephosphotransferase was also solubilized and was isolated from the microsomal membranes. This heat-labile, nondialyzable inhibitor was shown to act specifically on cholinephosphotransferase and not on ethanolaminephosphotransferase. Further characterization of the inhibitory activity revealed that it may act at the binding step of the cholinephosphotransferase to its lipid substrate, diacylglycerol.     

A highly active soluble diacylglycerol synthesizing system from developing rapeseed,Brassica napus L.

The subcellular distribution of the enzymes of triacylglycerol biosynthesis has been studied in developing oilseed rape. All in vitro enzymatic activities from oleoyl-CoA to triacylglycerol were sufficient to account for the known rate of oleate deposition in triacylglycerol in vivo. The enzymatic activities from oleoyl-CoA to diacylglycerol preferentially were localized in a 150,000 g supernatant fraction, while the diacylglycerol acyl-transferase mostly was associated with the microsomal (20,000 g pellet and 150,000 g pellet) and oil-body fractions. The soluble (150,000 g supernantant) fraction rapidly incorporated oleate from [1-¹⁴C]oleoyl-CoA into diacylglycerol with rates of 40 nm min⁻¹ g⁻¹ FW at 20 μM oleoyl-CoA. The pH optimum was 7.5–9.0, and normal saturation kinetics were seen with oleoyl-CoA; the S_0.5 was about 32 μM. Exogenous acyl acceptors, such as glycerol 3-phosphate, lysophosphatidic acid and lysophosphatidylcholine stimulated oleate incorporation into diacylglycerol. The detergents Triton X-100 and sodium cholate inhibited diacylglycerol formation at concentrations in the region of their critical micellar concentration, while n-octyl-β,D-glyco-pyranoside had no effect, even at high concentration. The significance of these findings for the mechanism of oil-body formation in developing oilseeds is discussed.     

Properties of monoacylglycerol lipase in rabbit aorta

Monoacylglycerol lipase activity was characterized in a soluble preparation from rabbit aorta (intima-media) obtained by combining a 100,000×g supernatant fraction with activity solubilized from the 100,000×g precipitate fraction by treatment with Triton X-100. Rates of hydrolysis with 1-monoolein and 2-monoolein substrates were nearly identical. 1-Monoolein was a competitive inhibitor (Ki 65 μM) of 2-monoolein hydrolysis. 2-Monoolein and 2-monopalmitin were both hydrolyzed more rapidly than 2-monoarachidonin. Lipase activity measured with a 2-monoolein substrate was inhibited by the addition of oleate, NaF and CaCl₂ to the assay. Preincubation of the lipase preparation with p-bromophenacyl bromide resulted in a potent inhibition of lipase activity; this inhibition could be prevented by dithiothreitol.     

Stimulation of a neutral triacylglycerol hydrolase from rat heart by phosphatidylethanolamine and lysophosphatidylethanolamine

Triacylglycerol hydrolase activity measured at pH 7.5 in a pH 5.2 precipitate fraction from rat heart was increased two-to three-fold by the presence of phosphatidylethanolamine (PE) or lysophosphatidylethanolamine (LPE). This stimulatory effect also could be obtained in assays with particulate and soluble subcellular fractions and was observed with two different methods of preparing triolein substrate emulsions. Ethanolamine and glycerophosphorylethanolamine had no effect on hydrolase activity, whereas phosphatidylcholine (PC) and acidic phospholipids such as cardiolipin were inhibitory. Palmitic acid, palmityl CoA and palmityl carnitine inhibited PE-stimulated hydrolase activity, but ethyl esters of palmitate had no effect. The preparation of acetone-ether powders resulted in a marked reduction of triacylglycerol hydrolase activity, but PE and LPE now stimulated hydrolase activity by ten-fold or greater, suggesting that these phospholipids may have an obligatory role in modulating triacylglycerol hydrolase activity. Triton X-100 also stimulated hydrolase activity in acetone-ether powders.     

Biosynthesis and localization of phosphatidyl-scyllo-inositol in barley aleurone cells

A novel isomer of phosphatidylinositol (PI), phosphatidyl-scyllo-inositol, was characterized in the aleurone cells of barley seeds. In this investigation, the subcellular localization of scyllo-PI and the relative rates of biosynthesis and accumulation of [³²P]phosphoric acid ([³²Pi])-labeled scyllo- and myo-phosphoinositides in the plasma membrane and intracellular membrane pools were investigated. About 25% of the [³²Pi]-labeled phospholipids were present in plasma membrane and 75% in intracellular membranes. Incorporation of [³²Pi] into scyllo-PI was greater than into myo-PI in both the plasma membranes and intracellular membranes at all time points investigated, thus suggesting a higher rate of biosynthesis; however, the data do not preclude reduced breakdown of labeled scyllo-PI as a contributing factor. In vitro studies were conducted to investigate the presence of cytidinediphosphate diacylglycerol (CDP-DG):scyllo-inositol 3-phosphatidyltransferase (scyllo-PI synthase) and to optimize enzymatic activity. The inclusion of nonionic detergents (Brij 58 and Triton X-100) effected significant enhancement in the biosynthesis of scyllo-PI, whereas anionic, cationic, and zwitterionic detergents had little or no effect. This is the first evidence for CDP-DG:scyllo-inositol 3-phosphatidyltransferase activity.     

Sulfatide biosynthesis by intact microsomes and triton extracts of normal and “Quaking” mouse brain

Microsomes from “Quaking” mice were unable to catalyze the transfer of³⁵S from PAP³⁵S to ceramide galactoside to form sulfatide. However Triton X-100 extracts were found to possess this activity.     

Fast-swelling porous starch-g-poly(acrylic acid) superabsorbents

The fast-swelling porous starch-g-poly(acrylic acid) (St-g-PAA) superabsorbents were prepared simply by free-radical graft polymerization between acrylic acid and potato starch in the aqueous phase by using Triton X-100 as the pore-forming agent. The porous superabsorbents were characterized by FTIR and scanning electron microscopy. The effects of weight ratio of acrylic acid to starch and the Triton X-100 content on water absorbency were investigated. In addition, the effects of Triton X-100 on swelling rate, water absorbency in 0.9 % NaCl and in the artificial urine solution, and reswelling ability were studied in detail. The superabsorbent prepared with an acrylic acid/starch ratio of 6:1 and a Triton X-100 content of 0.4 % has a water absorbency of 683 g g^?1 in distilled water and 72.8 g g^?1 in 0.9 % NaCl. The content of Triton X-100 has great influence on the water absorbency and the swelling rate. The porous sample prepared with 0.4 % Triton X-100 only needs about 5 min to reach 80 % of its equilibrium water absorbency which is obviously shorter in comparison with the nonporous sample (about 90 min). The porous samples also show nice urine absorbency and excellent reswelling ability. We believe the reported method may find some applications in preparing novel porous hydrogels.     

Solubilization of Acyl-CoA elongases from developing rapeseed (Brassica napus L.)

Acyl-CoA elongases are important in producing high-erucic acid rapeseed. The effects of Triton X-100,N-octyl-β-D-glucopyranoside and deoxycholate on the C18:1-CoA and C20:1-CoA elongase(s) have been studied by using a 15,000 ×g pellet from developing rapeseed. The synthesis of very long chain monounsaturated fatty acids (VLCMFA) and, in particular, that of erucic acid were stimulated by Triton X-100, whatever the substrate used. In the presence ofN-octyl-β-D-glucopyranoside, the elongase activity was practically unchanged, whereas deoxycholate strongly inhibited VLCMFA synthesis. Triton X-100 was chosen for the solubilization, at an optimal Triton X-100/protein (w/w) ratio of 2.5. Acyl-CoAs were the major products synthesized by the solubilized acyl-CoA elongase(s). The analysis of the reaction intermediates showed that the entire elongation complex has been solubilized and was still functional.     

A Spectrophotometric Microtiterplate Assay to Determine the Transphosphatidylation Potential of Phospholipase D

In addition to the hydrolysis of the terminal phosphate ester bond in glycerophospholipids, phospholipases D (PLDs) are able to catalyze the exchange of the polar head group. The biocatalytic potential of PLDs strongly depends on the ratio of the transphosphatidylation to hydrolysis rate which, therefore, is an important criterion in the screening for efficient PLDs from natural sources or combinatorial DNA libraries. Here, we present a fast spectrophotometric assay that allows the determination of the rate of both hydrolysis and transphosphatidylation of PLD-containing solutions including cell extracts in one microtiterplate. The assay is based on the reaction of phosphatidylcholine solubilized in Triton X-100 micelles with ethanolamine and the determination of phosphatidic acid (PA) and choline. PA is determined via Fe(III) complexation and represents hydrolysis, while choline is determined by the conventional choline oxidase/peroxidase assay and yields the total conversion. The difference between both values corresponds to the transphosphatidylation product. The method is suitable for measuring reactions rates as well as product yields after defined time periods. As shown for E. coli cells expressing PLD from cabbage, the assay can be applied to extracts of cells grown and lysed in microtiterplates.     

Phospholipid Hydrolysis and Transphosphatidylation by Phospholipase D from Indian Mustard Seeds in Two-Phase Systems

Phospholipase D (PLD), isolated from Indian mustard seeds and purified to homogeneity, has recently been identified as typical α‐type PLD with a high activity toward phosphatidylcholine (PC) in an aqueous mixed micellar system (Khatoon et al. 2014, Phytochemistry 117, 65–75). In light of biocatalytical application, we have now studied the enzyme in aqueous‐organic two‐phase systems and compared the results with the properties of the enzyme in aqueous mixed micellar systems. n‐Hexane containing 5–10 mol% of 1‐ or 2‐octanol proved to be optimal as an organic solvent in the two‐phase system, whereas anionic detergents such as sodium dodecyl sulfate or sodium deoxycholate were favorable components in the mixed micellar system. The optimum pH value (5.5–5.6) and the optimum Ca²⁺ ion concentration (70 mM) were independent of the reaction system. The head group selectivity in terms of activity toward different phospholipids (PC > phosphatidylethanolamine > phosphatidylglycerol) was similar in both types of reaction systems. Also, the K_M values toward PC were on the same order of magnitude. In contrast, the V_max value in the two‐phase system was 20‐fold lower than in the mixed micellar system. The enzyme was able to catalyze the substitution of choline in PC by ethanolamine, glycerol, and ethylene glycol with high efficiency. l ‐Serine was exchanged for choline with low activity. Myo‐inositol was not an alcohol acceptor, but promoted the hydrolysis of PC.     

Electrodeposition of Pt-Ru nanoparticles on fibrous carbon substrates in the presence of nonionic surfactant: Application for methanol oxidation

Liquid crystalline and micellar aqueous solutions of the nonionic surfactant Triton X-100 were used to direct the electrodeposition of Pt-Ru nanoparticles onto graphite felts, which were investigated as novel anodes for the direct methanol fuel cell. The effects of surfactant concentration, current density and deposition time in the preparation of these three-dimensional electrodes were studied in a factorial experiment and the electrodes were characterized by SEM and ICP-AES. Cyclic voltammetry, chronoamperometry and chronopotentiometry were carried out to assess the activity of the catalyzed felts for methanol oxidation. The presence of Triton X-100 (40-60 wt.%) coupled with an acidic plating solution were essential for the efficient co-electrodeposition of Ru in the presence of Pt to yield approximately a 1:1 Pt:Ru atomic ratio in the deposit. The highest mass specific activity, 24 A g⁻¹ at 298 K (determined by chronoamperometry after 180 s at 0 V versus Hg/Hg₂SO₄, K₂SO_4std), was obtained for the Pt-Ru electrodeposited in the presence of 40 wt.% Triton X-100 at 60 A m⁻², 298 K for 90 min. Surfactant mediated electrodeposition is a promising method for meso-scale (ca. 10-60 nm diameter) catalyst particle preparation on three-dimensional electrodes.     

The formation of lysophosphatidylinositol phosphate in human platelet microsomes

The formation of lysophosphatidylinositol phosphate (lysoPIP) from lysophosphatidylinositol (lysoPI) via kinase activity was studied in microsomal preparations from human platelets. For this purpose, [³H]lysoPI or [³H]phosphatidylinositol ([³H]PI) was prepared and incubated in the presence or absence of ATP, MgCl₂ and Triton X-100, and the appearances of radioactivity in [³H]lysoPIP and [³H]phosphatidylinositol phosphate ([³H]PIP), respectively, were monitored using thin layer chromatography. Both lysoPI and PI phosphorylations were completely dependent upon the presence of ATP and MgCl₂ in the incubation medium; Triton X-100 addition stimulated both reactions, with the stimulation of PI conversion being considerably greater than that for lysoPI conversion. The present results demonstrate that lysoPI can be converted to lysoPIP by phosphorylation in human platelet microsomes. The potential significance of this enzymatic reaction in stimulated cells is discussed in relation to the generation of inositol-1,4,5-trisphosphate, an important intracellular second messenger.     

Purification of the acyl-CoA elongase complex from developing rapeseed and characterization of the 3-ketoacyl-CoA synthase and the 3-hydroxyacyl-CoA dehydratase

Oleoyl-CoA elongase catalyzes four successive reactions: condensation of malonyl-CoA to oleoyl-CoA, reduction, dehydration, and another reduction. Evidence supporting this mechanism and the multienzymatic nature of the elongation complex are reported. A particulate membrane fraction from rapeseed is able to elongate intermediates (R,S) 3-hydroxy-20∶0-CoA and (E) 2,3–20∶1-CoA to very long chain fatty acids in the presence of malonyl-CoA. Studies of the 3-ketoacyl-CoA synthase activities showed that maximal activity could be measured by using 15 to 30 μM 18∶1-CoA and 30 μM malonyl-CoA, and that 18∶0-CoA and 18∶1-CoA were the best substrates. Comparison of the condensation and the overall elongation activities indicated that condensation is the rate-limiting step of the elongation process. The 3-hydroxyacyl-CoA dehydratase activity was maximal in the presence of 75 μM Triton X-100 and 25 μg of proteins. Finally, the acyl-CoA elongase complex was solubilized and purified. During the purification process, the 3-hydroxyacyl-CoA dehydratase copurified with the elongase complex, strongly suggesting that this enzyme belongs to the elongase complex. The apparent molecular mass of 700 kDa determined for the elongase complex, and the fact that four different polypeptide bands were detected after sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the purified fraction, further suggest that the acyl-CoA elongase is a multienzymatic complex.     

Characteristics of solubilization and encapsulation of fullerene C60 in non-ionic Triton X-100 micelles

The solubilization and encapsulation of monomeric C₆₀ in Triton X-100 micelles were investigated. Characteristic hydrophobic interactions of the type π-π and CH-π between the Triton X-100 micelle and C₆₀ resulted in stable aqueous dispersions of C₆₀ in the micellar medium, as evidenced from UV-vis, fluorescence emission and micro-Raman spectroscopy. Cyclic voltammetry of C₆₀ encapsulated Triton X-100 in aqueous 5 mM LiClO₄ solution revealed a quasi-reversible one-electron reduction peak with E_1/2 = −0.61 V and a reversible reduction peak at E_1/2 = −1.11 V vs. Ag/AgCl reference electrode at a scan rate of 10 mV s⁻¹, a redox behaviour drifting substantially from that of pure C₆₀. An onset concentration of ∼0.025 mM for C₆₀ aggregation in the micellar core was substantiated from the characteristic absorption spectral broadening and quenching of pyrene fluorescence. The molar solubilization capacity of C₆₀ in aqueous Triton X-100 micellar solution was estimated spectrophotometrically to be 0.22.     

Fatty acid and sterol specificity of cholesterol esterifying enzyme in developing rat brain

The properties and fatty acid and sterol specificity of cholesterol-esterifying enzyme (EC 3.1.1.13) in rat brain were studied. The enzyme utilized free fatty acid for esterification, and activity was maximal at pH 5.6. Exogenous ATP and CoA did not stimulate the incorporation of free fatty acids into sterol esters. Substrates dispersed in Tween 20 or Triton X-100 were just as effective as the substrates dissolved in acetone solution, while dispersion in propylene glycol or sodium taurocholate was not as effective. Snake venom phospholipase A₂ (EC 3.1.1.4) increased the esterification of cholesterol in the absence of added fatty acid. The fatty acid specificity data indicated that oleic and palmitic acids were the preferred fatty acids. Little or no esterification occurred in the presence of long chain fatty acids (C₂₀–C₂₄). Esterification of cholesterol with palmitate or stearate was not affected by the presence of oleic acid in the mixture. Thus, the nonrequirement of the brain-esterifying enzyme for a bile acid or for an amphiphile such as an unsaturated fatty acid suggests that micellar solubilization of the substrate is not essential for activity. Although the brain enzyme catalyzed the esterification of desmosterol, cholesterol was the preferred substrate. Neither lanosterol (C₂₉ sterols) nor Δ7-dehydrocholesterol was esterified to any significant extent. The presence of low concentrations of desmosterol increased cholesterol esterification slightly, while there was a concentration-dependent inhibition of demosterol esterification by cholesterol. These data on fatty acid and sterol specificity of the esterifying enzyme correlate well with the composition of sterol esters present in developing rat brain.