Permeability properties of liposomes prepared from dipalmitoyllecithin, dimyristoyllecithin, egg lecithin, rat liver lecithin and beef brain sphingomyelin

Liposomes have been prepared from dipalmitoyllecithin, dimyristoyllecithin, egg lecithin, rat liver lecithin and beef brain sphingomyelin.Permeability properties of liposomes thus prepared were studied toward glucose. The glucose permeability of liposomes with saturated lecithins (dipalmitoyllecithin and dimyristoyllecithin) and sphingomyelin appears to be more strongly temperature dependent than that of liposomes with lecithin containing unsaturated fatty acyl chains (egg and rat liver lecithins). The permeability of glucose through vesicles of dipalmitoyllecithin or dimyristoyllecithin was enhanced drastically at their transition temperatures, while the incorporation of about 25 mole% of egg lecithin into liposomes of saturated lecithins suppressed the enhanced permeation rates of glucose above the transition temperatures.The incorporation of small amounts of cholesterol enhanced the temperature-dependent permeability of glucose through the bilayer of saturated lecithins or sphingomyelin. This tendency was best shown in the case of dipalmitoyl-lecithin, in which 20 mole% of cholesterol had the most stimulating effect on the temperature-dependent permeability. The introduction of more than 33 mole% of cholesterol showed, however, reduced effects on the temperature-dependent permeability through liposomes with saturated lecithins or sphingomyelin. It was also shown that cholesterol had a much larger effect on the regulation of the temperature-dependent permeability of liposomes prepared with saturated lecithins or sphingomyelin than on that of liposomes prepared with phospholipids containing unsaturated fatty acids.     

CHANGES IN ACYL GROUP COMPOSITION OF DIACYL-GLYCEROPHOSPHORYLETHANOLAMINE, ALKENYLACYL-GLYCEROPHOSPHORYLETHANOLAMINE AND DIACYL-GLYCEROPHOSPHORYLCHOLINE IN MYELIN AND MICROSOMAL FRACTIONS OF MOUSE BRAIN DURING DEVELOPMENT

—Age-related changes in acyl group composition of diacyl-glycerophosphorylethanolamine (GPE), alkenylacyl-GPE and diacyl-glycerophosphorylcholine (GPC) were examined in myelin and microsomal fractions of mouse brain during development. In general, the phosphoglycerides in the myelin fraction showed an increase in the proportions of 18:1 and 20:1 and a decrease in the proportions of 16:0, 20:4(n-6) and 22:6(n-3) with increasing age. These changes were especially obvious with the acyl groups of alkenylacyl-GPE. The acyl group profiles of phosphoglycerides in the microsomal fraction were different from those in the myelin fraction. During development, there was an increase in 22:6 and a decrease in 20:4 in the phosphoglycerides of microsomes. These changes were especially obvious with the diacyl-GPE. Starting from 2 weeks of age, there was also an increase in the proportions of 18:1 and 20:1 in alkenylacyl-GPE in the microsomal fraction but this change was not as dramatic as that in the myelin fraction. In general, the acyl groups of diacyl-GPC in both myelin and microsomal fractions showed only little age-related changes as compared to the ethanolamine phosphoglycerides. Results suggest an induction in the synthesis of monoenoic fatty acids in brain during development. The monoenoic fatty acids synthesized during this period are rapidly and preferentially incorporated into the ethanolamine plasmalogen for further utilization in synthesis of the myelin membranes.     

Methyl branching in short-chain lecithins: are both chains important for effective phospholipase A2 activity?

Several seven-carbon fatty acyl lecithins with varied acyl chain branching have been synthesized and characterized as potential phospholipase A2 substrates. Micellar bis(4,4-dimethylpentanoyl) phosphatidylcholine, bis(5-methylhexanoyl)phosphatidylcholine, bis(3-methylhexanoyl)phosphatidylcholine, and bis(2-methylhexanoyl)phosphatidylcholine are poor substrates for phospholipase A2 (Naja naja naja). These branched lecithins also inhibit the hydrolysis of diheptanoylphosphatidylcholine by the enzyme with Ki values comparable to or smaller than the apparent Km of the linear compound. The terminally branched lecithins are excellent substrates for another surface-active hydrolytic enzyme, phospholipase C from Bacillus cereus. When only one acyl chain bears a methyl group, the hybrid lecithins 1-heptanoyl-2-(2-methylhexanoyl)phosphatidylcholine and 1-(3-methylhexanoyl)-2-heptanoylphosphatidylcholine are substrates comparable to diheptanoylphosphatidylcholine. Analysis of micellar structure and dynamics by 1H and 13C NMR spectroscopy, quasi-elastic light scattering, and comparison of critical micellar concentrations indicates little significant difference in the conformation and dynamics of these seven-carbon fatty acyl lecithin micelles, even when the methyl groups are adjacent to the carbonyls. Phospholipase A2 UV difference spectra induced by phospholipid binding imply different enzyme conformations or aggregation states caused by linear-chain and asymmetric-chain lipids compared to bis(methylhexanoyl)phosphatidylcholines. The differences in hydrolytic activity of phospholipase A2 against the branched-chain micellar lecithins can then be attributed to an enzyme-lipid interaction at the active site. The species with both fatty acyl chains branched bind to phospholipase A2 but are not turned over rapidly. Since poor enzymatic activity only occurs for lecithins with both chains methylated, the interaction of both chains with the enzyme must be important for catalytic efficiency.     

Short-chain lecithin/long-chain phospholipid unilamellar vesicles: asymmetry, dynamics, and enzymatic hydrolysis of the short-chain component

Asymmetric unilamellar vesicles are produced when short-chain phospholipids (fatty acyl chain lengths of 6-8 carbons) are mixed with long-chain phospholipids (fatty acyl chain lengths of 14 carbons or longer) in ratios of 1:4 short-chain/long-chain component. Short-chain lecithins are preferentially distributed on the outer monolayer, while a short-chain phosphatidylethanolamine derivative appears to localize on the inner monolayer of these spontaneously forming vesicles. Lanthanide NMR shift experiments clearly show a difference in head-group/ion interactions between the short-chain and long-chain species. Two-dimensional 1H NMR studies reveal efficient spin diffusion networks for the short-chain species embedded in the long-chain bilayer matrix. The short-chain lecithin is considerably more mobile than the long-chain component but has hindered motion compared to short-chain lecithin micelles. This differentiation in physical characteristics of the two phospholipid components is critical to understanding the activity of phospholipases toward these binary systems.     

Temperature-induced changes in lecithin model membranes detected by novel covalent spin-labelled phospholipids.

Several spin-labelled phospholipids carrying covalently bound 5-doxylstearic acid (2-(3-carboxydecyl)-2-hexyl-4,4-dimethyl-3-oxazolidinoxyl) were intercalated in liposomes of saturated and unsaturated lecithins. Temperature-induced changes of these liposomes, detected by the spin-labelled phospholipids, were found to be in agreement with the previously described transitions of hydrocarbon chains of host lecithins detected by different probes and different techniques, establishing that spin-labelled phosopholipids are sensitive probes for the detection of temperature-induced changes in lecithin model membranes. In addition to the detection of already-known transitions in lecithin liposomes, the coexistence of two distinctly different enviroments was observed above the characteristic transition temperature. This phenomenon was tentatively attributed to the influence of the lecithin polar group on the fluidity of fatty acyl chains near the polar group. Combined with other results from the literature, the coexistence of two environments could be associated with the coexistence of two conformational isomers of lecithin, differing in the orientation of the polar head group with respect to the plane of bilayer. These findings have been discussed in view of the present state of knowledge regarding temperature-induced changes in model membranes.     

Regulation of phospholipase A2 activity by the lipid-water interface: a monolayer approach

Interfacial regulation of phospholipase A2 activity on lecithin monolayers was investigated by using radioactively labeled enzyme. Labeling of the protein with 125I did not produce a change of the enzyme and protein properties as compared to the 3H fully amidinated phospholipase A2. The induction time observed during pre-steady-state kinetics reflects the rate-limiting step of the penetration of the enzyme in the interface. This penetration is reversible. However, in the surface pressure range where the enzyme is able to hydrolyze the lecithin films, the desorption of the protein from the film is slow as compared to the adsorption. Below a surface pressure of 10 dyn/cm nonspecific adsorption occurs. Using lecithins with fatty acids of different chain lengths, we have shown that the kinetics of the penetration process is governed by the packing density of the substrate molecules independent of the surface pressure. However, the steady-state surface concentration of the enzyme increases with the fatty acyl chain length of the lecithin, indicating that hydrophobic interaction occurs between phospholipase A2 and the lipid molecules at the interface. From the lecithins used pancreatic phospholipase A2 preferentially splits substrate molecules with nine carbon atoms in the acyl chain.     

Molecular species of lecithins of rat heart, kidney, and plasma

The lecithins of the heart, kidney, and plasma of the rat were isolated, and the major molecular species identified and quantitatively estimated by combined thin-layer and gas-liquid chromatographic analyses and specific enzymic hydrolyses. The lecithins of the three tissues differed significantly in the composition, positional distribution, and pairing of the fatty acids. No preferential pairing of any one saturated with any other unsaturated fatty acids was observed. The three tissues contained qualitatively the same molecular species of lecithins. The lecithin profiles of the rat heart and kidney appeared to be unrelated to that of the plasma.     

Acyl chain unsaturation modulates distribution of lecithin molecular species between mixed micelles and vesicles in model bile. Implications for particle structure and metastable cholesterol solubilities.

We determined the distribution of lecithin molecular species between vesicles and mixed micelles in cholesterol super-saturated model biles (molar taurocholate-lecithin-cholesterol ratio 67:23:10, 3 g/dl, 0.15 M NaCl, pH approximately 6-7) that contained equimolar synthetic lecithin mixtures or egg yolk or soybean lecithins. After apparent equilibration (48 h), biles were fractionated by Superose 6 gel filtration chromatography at 20 degrees C, and lecithin molecular species in the vesicle and mixed micellar fractions were quantified as benzoyl diacylglycerides by high performance liquid chromatography. With binary lecithin mixtures, vesicles were enriched with lecithins containing the most saturated sn-1 or sn-2 chains by as much as 2.4-fold whereas mixed micelles were enriched in the more unsaturated lecithins. Vesicles isolated from model biles composed of egg yolk (primarily sn-1 16:0 and 18:0 acyl chains) or soy bean (mixed saturated and unsaturated sn-1 acyl chains) lecithins were selectively enriched (6.5-76%) in lecithins with saturated sn-1 acyl chains whereas mixed micelles were enriched with lecithins composed of either sn-1 18:1, 18:2, and 18:3 unsaturated or sn-2 20:4, 22:4, and 22:6 polyunsaturated chains. Gel filtration, lipid analysis, and quasielastic light scattering revealed that apparent micellar cholesterol solubilities and metastable vesicle cholesterol/lecithin molar ratios were as much as 60% and 100% higher, respectively, in biles composed of unsaturated lecithins. Acyl chain packing constraints imposed by distinctly different particle geometries most likely explain the asymmetric distribution of lecithin molecular species between vesicles and mixed micelles in model bile as well as the variations in apparent micellar cholesterol solubilities and vesicle cholesterol/lecithin molar ratios.(ABSTRACT TRUNCATED AT 250 WORDS)     

Substrate specificity of plasma lysolecithin acyltransferase and the molecular species of lecithin formed by the reaction

Human plasma lecithin-cholesterol acyltransferase also converts lysolecithin to lecithin in the presence of low density lipoproteins. To understand the physiological importance of this lysolecithin acyltransferase reaction, we investigated the molecular species of lysolecithin available for acylation in normal plasma and the lecithins which are formed by the acylation of each of these lysolecithins. Palmitate- and stearate-containing lysolecithins were formed by the lecithin-cholesterol acyltransferase reaction, whereas oleate- and linoleate-containing lysolecithins were formed by the action of post-heparin lipase(s). All the natural lysolecithins were esterified at comparable rates by the isolated enzyme. Lyso platelet-activating factor was esterified about 70% as efficiently as the lysolecithins, while lysophosphatidylethanolamine was esterified at about 30% the rate observed with lysolecithin. The 2-acyl isomers of lysolecithin were acylated to the same extent as the 1-acyl isomers, although considerable isomerization of the former took place during the incubation. There were no net changes in the concentrations of lecithin and lysolecithin after 6 h of incubation with the enzyme, although over 10% of the labeled lysolecithin was converted to lecithin, indicating that the endogenous lecithin serves as the acyl donor in the reaction. When the molecular species of lecithin formed were analyzed by high performance liquid chromatography, the same pattern of fatty acid incorporation was observed with all the lysolecithins used. The bulk of the radioactivity was incorporated into molecular species formed by the acylation with linoleic, oleic, and palmitic acids, in decreasing order. However, in each case, the lecithins formed by acylation with palmitic acid had the highest specific radioactivity, followed by those acylated with linoleic and oleic acids. From these results it is postulated that the enzyme alters the molecular species composition of lecithin in plasma without increasing the net amount of total lecithins.     

Studies on lysophospholipases, V. The action of lysolecithin-hydrolyzing enzymes on lecithins and 1-acyl lysolecithins with varying fatty acid chain-length.

The activity of two purified lysolecithin-hydrolyzing enzymes on homologous series of synthetic lecithins containing two identical fatty acyl chains and of 1-acyl-lysolecithins has been measured as a function of substrate concentration. In general, enzymatic activity toward lecithins decreased with increasing chain length. Maximal hydrolysis rates for the lysolecithin series were measured with 1-dodecanoyllysolecithin. In this series increased affinities for substrates with increasing acyl-chain length was noticed. In the substrate concentration versus enzymatic velocity curves no breaks were observed at the critical micelle concentration of the various substrates. The initial site of attack during hydrolysis of short-chain lecithins was determined using 1-octanoyl-2pentanoyl-lecithin, 1-hexanoyl-2-hexyllecithin and 1 -hexyl-2-hexanoyllecithin. Both enzymes exhibited a pronounced preference for hydrolysis of the acyl ester bond at the 1-position. Especially the enzyme from beef pancreas seems to be suitable for the enzymatic preparation of 2-acyl lysolecithins from the corresponding short-chain lecithins.     

1-Acyl-lysolecithin acyltransferase and synthesis of biliary lecithins in rat liver

The role of lysolecithin acyltransferase activities in biliary lecithin formation was investigated, using livers perfused in the presence of labeled palmitoyl-lysolecithin and albumin, overloaded or not with linoleic acid. At the end of liver perfusion, the lecithins extracted from microsomes, mitochondria and plasma membranes displayed the same specific activity. Double-labeled lysolecithin was used to prove that labeled lecithins were synthesized by lysolecithin acylation. In the absence or presence of a linoleic acid overload, the level of lysolecithin incorporation into linoleyl and arachidonyl containing lecithin was identical. Hence fatty acids did not influence phosphatidylcholine synthesis by the acylation pathway. In vitro the rate of linoleyl lecithin synthesis was the same in plasma membranes, mitochondria and microsomes provided the linoleyl-CoA concentration was lower than 30 microM. Taurocholate was essential to the excretion of lecithin synthesized from lysolecithin and stimulated its synthesis. The specific activities of the two lecithin molecular species excreted in bile (linoleyl and arachidonyl) were not significantly different. These results enabled us to evaluate the contribution of the lysolecithin pathway to the synthesis of lecithin in liver and bile: this contribution in bile was less than 2% under the perfusion conditions used.     

Conserved Fatty Acid Composition of Proteolipid Protein During Brain Development and in Myelin Subfractions

Myelin proteolipid protein (PLP) is modified after translation by the attachment of long-chain fatty acids to several cysteine residues. In this study, the amount and pattern of fatty acids covalently bound to rat PLP were determined during brain development and in myelin subfractions. For this purpose, PLP was isolated by gel-filtration chromatography in organic solvents, subjected to alkaline methanolysis, and the released fatty acid methyl esters were analyzed by gas-liquid chromatography. At all ages examined, PLP had the same amount of covalently-bound fatty acids (3–4% w/w) and palmitate, oleate and stearate were always the major acyl chains. In contrast to myelin lipids, the fatty acid composition of PLP showed only minor changes between 15-days and 90-days of age. The amount and pattern of fatty acids bound to PLP prepared from three myelin subfractions were also indistinguishable. The conservation of a characteristic PLP-fatty acid make-up during brain development and in various myelin compartments suggests that this post-translational modification is essential for the normal functioning of the protein.     

Microcalorimetric study of phospholipid binding to human apo-HDL

The binding of lysolecithin and synthetic short-chain lecithins: di-caproyl, di-lauroyl and di-myristoyl lecithins to a human apo-high density lipoprotein (apo-HDL) was followed by microcalorimetry. Complex formation was checked by ultracentrifugal flotation.The binding reaction was very rapid and strongly exothermal. The apparent binding enthalpy ΔH_B together with the complex composition were computed from the binding curves. Both quantities were of the same order of magnitude for lysolecithin and for the shorter chain lecithins while the binding of di-myristoyl lecithin was characterized by a more highly exothermal reaction.The structure of the lipid phase strongly influences the enthalpy change. In the case of lysolecithin and of the shorter chain lecithins; which form micellar structures in water, the enthalpy change is mainly due to apoprotein-phospholipid complex formation.The disrupture of the myelin figures formed by the di-myristoyl lecithin accounts for the complementary heat effect.The phospholipid composition of the complexes isolated by ultracentrifugal flotation was lower than that determined by microcalorimetry, due to the presence of high salt concentrations in the ultracentrifuge.     

EFFECT OF HYPERPHENYLALANINEMIA ON FATTY ACID COMPOSITION OF LIPIDS OF RAT BRAIN MYELIN

Abstract— We have studied the fatty acid compositions of cerebral myelin lipids in phenyl-alanine-treated and control rats. The proportion of long chain fatty acids and the ratio of unsaturated to saturated fatty acids of whole brain lipids was low in the penylalanine-treated animals. Both of these reductions were more pronounced in the myelin from phenylalanine-treated rats. The ratio of unsaturated to saturated fatty acids in ethanolamine phosphatides was markedly decreased in the hyperphenylalaninemic condition. The reduction in the proportion of long chain fatty acids was predominant in non-hydroxy fatty acids in cerebrosides and ethanolamine phosphatides. The lipid composition of the myelin expressed as mole percentages of individual phospholipid and sphingolipid components was not significantly different in the two groups of rats, nor did it change with age. Our results indicate a deficiency in the fatty acid elongation and desaturation system in the brains of phenylalanine-treated rats. We suggest that in hyperphenylalaninemic rats, a reduction in the amounts of unsaturated fatty acid and long chain fatty acid alters, respectively, the biochemical reactivity and the stability of the myelin.     

Phospholipase A2 inhibitors: monoacyl, monoacylamino-glycero-phosphocholines

In this study the relative affinities of natural lecithins and slightly modified lecithin analogues to the active site of porcine pancreatic phospholipase A2 were determined. It was found that the replacement of the phospholipase-fissile fatty acid ester bond in lecithins by an acylamino function results in the formation of potent competitive inhibitors. Substitution of the non-phospholipase-susceptible ester bond by the acylamino linkage does not result in increased affinity of the lecithin analogue to the enzyme. Most probably only the former lecithin analogues partially mimic the structure of the transition state and bind more tightly to the enzyme than the equivalent substrate molecule.     

The ionic structure of lecithin monolayers

Surface potentials of mixed monolayers of dicetyl phosphate and eicosanyl trimethylammonium bromide (1:1) were the same on subsolutions of 0.02 M NaCl or 0.01 M CaCl(2), which indicated that ionic phosphate does not interact with Ca(++) in the presence of a neighboring trimethylammonium group. Surface potential-pH plots of dicetyl phosphate, and of dipalmitoyl, egg, and dioleoyl lecithins showed that as the pH of the subsolution is decreased the phosphate groups in the monolayer are neutralized in the order: dicetyl phosphate > dipalmitoyl lecithin > egg lecithin > dioleoyl lecithin. The binding of cations (Na(+), Ca(++)) to the phosphate group of lecithin also showed the same order. The binding of Ca(++)) to egg phosphatidic acid monolayers, as measured by the increase in surface potential, is considerably greater than that to egg lecithin. These results suggest that there is an internal salt linkage between the phosphate and trimethylammonium groups on the same lecithin molecule. An increase in unsaturation of fatty acyl chains increases the intermolecular spacing, which reduces the ionic repulsion between polar groups, and hence strengthens the internal salt linkage. The results support the concept of a vertical rather than coplanar orientation of the phosphoryl choline group with respect to the interface. A position has been proposed for Ca(++) in the dipole lattice of lecithin from a consideration of the surface potential measurements.     

Fatty Acid Composition of Myelin Proteolipid Protein During Vertebrate Evolution

The hydrophobic myelin proteolipid protein (PLP) contains covalently bound long-chain fatty acids which are attached to intracellular cysteine residues via thioester linkages. To gain insight into the role of acylation in the structure and function of myelin PLP, the amount and pattern of acyl groups attached to the protein during vertebrate evolution was determined. PLP isolated from brain myelin of amphibians, reptiles, birds and several mammals was subjected to alkaline methanolysis and the released methyl esters were analyzed by gas-liquid chromatography. In all species studied, PLP contained approximately the same amount of covalently bound fatty acids (3% w/w), and palmitic, palmitoleic, oleic and stearic acids were always the major acyl groups. Although the relative proportions of these fatty acids changed during evolution, the changes did not necessarily follow the variations in the acyl chain composition of the myelin free fatty acid pool, suggesting fatty acid specificity. The phylogenetic conservation of acylation suggests that this post-translational modification is critical for PLP function.     

TURNOVER OF PHOSPHATIDYLCHOLINE IN MICROSOMES AND MYELIN IN BRAINS OF YOUNG AND ADULT RATS

We have tested the hypothesis that the turnover of phosphatidylcholine in subcellular fractions of rat brain is a function of the age at which this lipid is deposited. Rats, 60 days of age, were injected intracranially with [2-³H]glycerol and either [methyl-¹⁴C]choline (to label the base moiety) or [U-¹⁴C]glucose (to label acyl moieties). Littermates were killed up to 90 days after injection and brain microsomes and myelin isolated. Lipids were extracted and the phosphatidylcholine was isolated by 2-dimensional TLC and hydrolyzed to its constituent moieties. The ³H in the glycerol backbone and ¹⁴C in the choline or acyl residues was quantitated. The microsomal and myelin ³H/¹⁴C ratios decreased with time with either set of precursors, indicating that labeled choline and acyl moieties were reutilized more efficiently than the glycerol backbone. The various precursors exhibited first order decay curves with half-lives for the glycerol backbone of 6 and 11 days for the microsomal and myelin fractions respectively. These results contrast with those previously obtained with identical experimental procedures when 17-day-old animals were injected. In that study, although much of the phosphatidylcholine turned over rapidly as for the older animals, by 2 weeks after injection most of the remaining phosphatidylcholine was turning over more slowly with a half-life of 13 and 25 days for microsomes and myelin respectively (Miller et al., 1977). The base and acyl moieties also had a corresponding shorter half-life in older animals relative to the slow turnover phase in younger rats.     

Fatty acid composition of cholesterol esters in brains of patients with Schilder's disease, G M1 -gangliosidosis and Tay-Sachs disease, and its possible relationship to the -position fatty acids of lecithin

Abstract— Cholesterol esters were isolated from cerebral cortex and white matter of patients with Schilder's disease, G_M1-gangliosidosis and Tay-Sachs disease, and the fatty acid composition was determined by gas-liquid chromatography. The fatty acid composition was similar among the three pathological conditions, but it was entirely different from that reported for cholesterol esters of normal brain. Lecithin and ethanolamine phospholipids were isolated from the same brain specimens, treated with snake venom phospholipase A, and the fatty acids at the a’and β-positions of the glycerol moiety were determined separately. The fatty acid composition of cholesterol esters was similar to that of the β-position fatty acids of lecithin of white matter in all samples, and was quite different from those of the a'-position of lecithin, or of the a’or β-position of ethanolamine phospholipids. The results indicate that the source of fatty acids for cholesterol esterification in nonspecific sudanophilic demyelination is different from that in normal brain, and that the most likely source is the β-linked fatty acids of lecithin. There are two possible enzymic mechanisms; activation of phospholipase A and subsequent esterification of the liberated β-position fatty acids to cholesterol, or direct transacylation by lecithin-cholesterol acyl transferase.     

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.