Myelin Galactolipid Synthesis in Different Strains of Mice

Previous studies have indicated that the brains of DBA/2J (D2) mice have a more heavily myelinated CNS than those of C57BL/6J (B6) at postnatal days 17-21. However, the amount of myelin in the brains of F1 (B6 X D2) hybrids is even higher than in their parental strains. To investigate further factors involved in regulating myelinogenesis in these mice, we have focused on the synthesis of cerebrosides and sulfatides, galactolipids enriched in myelin. Brain slices from 14-, 17-, and 21-day-old D2, B6, and F1 mice were incubated with [3H]galactose and [35S]sulfate. After incubation, microsomes, myelin, and oligodendroglial cells were isolated, and the galactolipids were analyzed. At 21 days of age, the labeling of cerebrosides in F1 mice was higher than in D2 and B6 mice when the results were expressed as microsomal or myelin radioactivity per gram wet weight. At 14 and 17 days of age, the labeling of cerebrosides in F1 animals was similar to that in D2 mice and was considerably higher than that in B6 mice. The labeling of sulfatides in F1 animals was significantly higher than in the B6 parent at all ages studied, whereas it remained higher than that in the D2 parent only at 17 days of age. A similar relationship among the strains was observed when the synthesis of myelin galactolipids was estimated by measuring the in vitro activity of UDP-galactose:ceramide galactosyltransferase and 3'-phosphoadenylyl sulfate:galactosylceramide 3'-sulfotransferase. The results indicate that the increased accumulation of myelin galactolipids previously reported in the F1 mice is partially due to enhanced synthetic activity.     

The turnover of phosphoglycerides in the subcellular fractions of mouse brain: a study using (1-14C)oleic acid as precursor

Abstract— The turnover of phosphoglycerides in subcellular fractions of adult mouse brain was examined after intracerebral injection of [1-¹⁴C]oleic acid. Radioactivity of the total brain homogenate decreased rapidly thereafter, with only 4 per cent of the radioactivity remaining at the end of 3 months. The rate of decrease of radioactivity in the subcellular fractions was in the order: cytosol, microsomes, synaptosomes and myelin. Increasing amounts of radioactivity were detected in the alkenyl groups and cerebrosides, but metabolic conversions were not as extensive as found previously with the palmitoyl group. The specific radioactivities for diacyl sn-glycero-3-phosphorylcholine and diacyl sn-glycero-3-phosphorylethanolamine were highest in the microsomal fraction and decreased with time. The apparent half-lives for the diacyl sn-glycero-3-phosphorylcholine and the diacyl sn-glycero-3-phosphorylethanolamine in the microsome and synaptosome-rich fractions were 1-3.5 days when estimated between 1 and 7 days after injection. The rate of decay for the brain membrane phosphoglycerides was not linear with time, probably because of the extensive amount of recycling occurring within the system. Radioactivity was incorporated into the phosphoglycerides of the myelin but equilibration of radioactivity between microsomes and myelin required 7–14 days.     

Levels and syntheses of cerebrosides and sulfatides in subcellular fractions of jimpy mutants

During the period of brain development, the levels of nonhydroxy- and hydroxy-cerebrosides in the cytosol from brains of jimpy mutants were determined by high-performance liquid chromatography and compared to those in the rest of the particulates from the same brains as well as those in the littermate controls. The concentrations of cerebrosides in jimpy brain preparations were much lower than in controls at all ages. In another experiment, [U-¹⁴C]glucose was injected intraperitoneally into jimpy mutants and their littermate controls. The amounts of radioactivity incorporated into cerebrosides and sulfatides in brain cytosol, the microsome-rich fraction, and the rest of the heavier particles were determined. Although the total radioactivity incorporated into these lipids was much lower in jimpy, the specific activities were 2–3 times the control value in all subcellular fractions.     

THE METABOLISM OF PALMITIC ACID IN THE PHOSPHOLIPIDS, NEUTRAL GLYCERIDES AND GALACTOLIPIDS OF MOUSE BRAIN

Abstract— Following intracerebral injection, [¹⁴C]palmitic acid was rapidly incorporated into a variety of brain lipids. After 12 hr, 78 per cent of the lipid radioactivity was in phospholipids, 15 per cent was in triacylglycerols, 1 per cent each was in free fatty acids and galactolipids, and the remainder was in other neutral glycerides. Over 65 per cent of the phospholipid radioactivity was found in the choline phosphoglycerides but this proportion decreased substantially with time. At later times, increasing portions of the radioactivity were present in the monounsaturated acyl groups and the alkenyl groups but no radioactivity was detected in cholesterol or polyunsaturated acyl groups. These results indicate that most of the extensive recycling of radioactivity took place without oxidative degradation of the palmitoyl groups. The relative rates of incorporation of radioactivity were compared at 12 hr after injection. The specific radioactivities of the serine, ethanolamine, and choline phosphoglycerides had ratios of 6:3:2 based on the palmitoyl group content and 1:2:4 based on their phosphorus content. The specific radioactivities of galactolipids with O -acyl groups were higher than the specific radioactivitiesof cerebrosides or cerebroside sulphates. A new solvent mixture for thin-layer chromatography of brain galactolipids was described (chloroform-acetone-methanol-water, 60:20:20:1, by vol.).     

THE DISTRIBUTION AND BIOSYNTHESIS OF THE MYELIN -GALACTOLIPIDS IN THE SUBCELLULAR FRACTIONS OF BRAINS OF QUAKING AND NORMAL MICE DURING DEVELOPMENT

Abstract— The biosynthesis and accumulation of monogalactosyl diglyceride, galacto-cerebrosides and sulfatides were studied in the brain of quaking mouse during myelination. The specific activity of monogalactosyl diglyceride synthesis of the mutant mouse was reduced to 50% of the control of the same age, comparable to the reduction in the biosynthesis of galactosylcerebrosides and sulfatides. The three galactolipids were largely associated with the myelin and microsomal fractions in the normal and quaking mice at the ages studied. Although the concentrations of microsomal galactolipids (expressed as nmol/g wet wt of brain) were lower in quaking mice than in the controls at all ages, the percentage of total brain monogalactosyl diglyceride recovered in the microsomes of the mutant mouse was always larger than in the microsomes of the controls. Between 16 and 41 days, the monogalactosyl diglyceride content of the control myelin increased 10-fold, whereas the concentrations in the mutant increased only 2-fold. In normal animals, the percentage of total myelin galactolipids in the 'small myelin' decreased over the age of 1841 days with concomitant increase in the 'large myelin'. In contrast, in the mutant, large percentages of these compounds remained associated with the small myelin even at late periods of myelin development. These findings indicate that the slow rate of deposition of myelin in the brain of quaking mouse may be due to a defective transport mechanism of the galactolipids from the site of synthesis (microsomes) to the site of deposition (myelin), or to a defect in the mechanism of final myelin assembly, rather than to a lipid-specific genetic error.     

INCORPORATION OF AXONALLY TRANSPORTED SUBSTANCES INTO MYELIN LIPIDS

Abstract— The possibility that axonally transported lipids and/or proteins might undergo transaxonal migration and become incorporated into surrounding myelin lamellae was studied by isolating myelin from optic tracts of myelinating rabbits at various times following intraocular injection of [3-¹⁴C]-serine and [2-³H]glycerol. Myelin isolated by a procedure employing ethylene glycol-bis(β-aminoethyl ether)-.N,N'-tetraacetic acid had relatively constant specific radioactivity with respect to both isotopes over a 21 day period. Myelin lipids showed a gradual increase in ¹⁴C specific radioactivity, attributed to reutilization of [¹⁴C]serine from the axon by a compartment of the oligodendrocyte. Free serine is postulated to arise in the axon from catabolism of axonally transported proteins (and possibly lipids) and to migrate transaxonally into the neighboring oligodendroglia. This reutilization mechanism resulted in synthesis of myelin cerebrosides, sphingomyelin, ethanolamine phosphoglycerides and possibly sulfatides, but not gangliosides or serine phosphoglycerides. The data for choline- and inositol-phosphoglycerides are inconclusive. [³H]Glycerol-labeled myelin lipids decreased slowly in ³H specific radioactivity with time, indicating either that [2-³H]glycerol does not participate in the reutilization pathway or that the label is lost in the process. Evidence is presented that ³H- and ¹⁴C-labeled lipids are true myelin constituents. Lipids from the myelin, axolemma- and axon-enriched fractions tended to converge in specific radioactivity over the 21 days, especially the former two fractions. These results together with isotope ratio changes point to an equilibration process whereby lipids are able to transfer. (or exchange) between the 3 compartments. Protein radioactivity in isolated myelin was suggested to arise from residual axon/axolemma contamination, and no evidence was found for transaxonal migration of protein into myelin. The 2 mechanisms elucidated here are believed to account for a quantitatively small portion of myelin lipid and are considered to represent a form of axon-glia interaction.     

Synthesis and turnover of cerebrosides and phosphatidylserine of myelin and microsomal fractions of adult and developing rat brain.

The synthesis and turnover of cerebrosides and phospholipids was followed in microsomal and myelin fractions of developing and adult rat brains after an intracerebral injection of [U-14C]serine. The kinetics of incorporation of radioactivity into microsomal and myelin cerebrosides indicate the possibility of a precursor-product relationship between cerebrosides of these membranes. The specific radioactivity of myelin cerebrosides was corrected for the deposition of newly formed cerebrosides in myelin. Multiphasic curves were obtained for the decline in specific radioactivity of myelin and microsomal cerebrosides, suggesting different cerebroside pools in these membranes. The half-life of the fast turning-over pool of cerebrosides of myelin was 7 and 22 days for the developing and adult rat brain respectively. The half-life of the slowly turning-over pool of myelin cerebrosides was about 145 days for both groups of animals. The half-life of the rapidly turning-over microsomal cerebrosides was calculated to be 20 and 40 h for the developing and adult animals respectively. The half-life of the intermediate and slowly turning-over microsomal cerebrosides was 11 and 60 days respectively, for both groups of animals. The amount of incorporation of radioactivity into microsomal cerebrosides from L-serine was greatly decreased in the adult animals, and greater amounts of the precursor were directed towards the synthesis of phosphatidylserine. In the developing animals, considerable amounts of cerebrosides were synthesized from L-serine, besides phosphatidylserine. The time-course of incorporation indicated that a precursor-product relationship exists between microsomal and myelin phosphatidylserine. The half-life of microsomal phosphatidylserine was calculated to be about 8 h for the fast turning-over pool in both groups of animals.     

The effect of chronic alcoholic intoxication on the level and metabolism of glycolipids in the rat brain

The chronic alcohol intoxication has been studied for its effect on the content of glycolipids in the rat brain and incorporation of [I-14C]acetate into them. It is established that administration of ethanol to animals (2 g per 1 kg of body weight daily for 7 days) rises the content of cerebrosides I in the brain tissue. The specific radioactivity of sulphatides I falls as a result of a decrease of the [I-14C]acetate into fatty acids and galactose. The specific radioactivity of sulphatides II, cerebrosides II and III falls as a result mainly of a decrease of the specific radioactivity in the galactose components.     

Decreased Metabolism of Cerebrosides and Sulfatides in Rat Sciatic Nerve After Intraneural Injection of Colchicine

To obtain an understanding of the importance of the neuronal cytoskeleton in Schwann cell metabolism, an antimicrotubular agent (colchicine) was injected into the rat sciatic nerve 24 or 48 h before incubation of the nerve with labeled precursor: [35S]sulfate, [14C]galactose, or [3H]-galactose. Colchicine inhibited the incorporation of 35S radioactivity into sulfatides and, to a lesser extent, into proteins. With galactose as the radioactive precursor, synthesis of cerebrosides was reduced by colchicine injection, whereas incorporation of radioactivity into phosphatidylserine and phosphatidylcholine increased. Intraneural injection of lumicolchicine had no effect. The effects of colchicine on the metabolism of the Schwann cell are discussed in relation to its action on microtubules.     

The Lipid Composition of Urodele Myelin Which Lacks Hydroxycerebroside and Hydroxysulfatide

The concentrations of cerebrosides and sulfatides were measured in the nervous systems of urodeles and related orders with a high performance liquid chromatographic technique. The peripheral and central nervous systems of all three urodele species, Necturus maculosis (mud puppy, a salamander), Notophthalmus viridescens (eastern red spot newt), and Desmognathus ochropheus (mountain salamander), were found to be completely devoid of alpha-hydroxy fatty acid-containing cerebrosides and sulfatides. All species of reptiles and fish classes close to urodeles contain these galactolipids. The levels of nonhydroxy fatty acid-containing cerebrosides and sulfatides are essentially similar in both urodeles and reptiles. Myelin isolated from Necturus spinal cord had a specific density of 1.07, lighter than mammalian myelin. Except for the absence of hydroxycerebrosides and hydroxysulfatides, the lipid composition of Necturus spinal cord myelin is essentially similar to that of frog and rat myelin. The fatty acids of nonhydroxycerebrosides are rich in monounsaturated homologs of C22-C25, and the sphingoid base consists of both sphinganine and sphingosine. Electron microscopic examination of the sciatic nerve showed that the general structure and interlamellar distances of salamander and newt myelin are identical to those of frog, chameleon, and rat. Necturus myelin, therefore, can be used as a model for the study of the functional and structural role of hydroxygalactolipids.     

Synthesis and Transport of Cerebrosides and Sulfatides in Rat Brain During Development

Synthesis and transport of nonhydroxy fatty acid (NFA)- and hydroxy fatty acid (HFA)-containing ceramides, cerebrosides, and sulfatides were studied in vivo in rat brain during development. After an intracerebral injection of [3H]serine, incorporation into these lipids of microsomal and myelin membranes was analyzed after HPLC. Distribution of amounts and incorporation of radioactivity were also determined in individual molecular species of these lipids. The results showed that HFA-ceramides and long-chain NFA-ceramides have small pool sizes and rapid turnover rates in the microsomal membranes and are preferentially utilized for the synthesis of long-chain (greater than or equal to 20:0) HFA- and NFA-galactocerebrosides of both microsomal and myelin membranes. Glucocerebrosides are not expressed in myelin and their synthesis in microsomal membranes is predominant before the onset of myelination. With development, synthesis and accumulation of HFA-cerebrosides increase over NFA-cerebrosides in both microsomal and myelin membranes. In myelin, incorporation of radioactivity into HFA-cerebrosides is even higher than that expected by transport alone from microsomal membranes and it is possible that part of the HFA-cerebrosides in myelin could be due to de novo synthesis by myelin itself. The amount of NFA- and HFA-sulfatides is about equal, both in myelin and microsomal membranes, and this relative proportion does not change with development. Similar relative rates of incorporation of radioactivity into sulfatides of microsomal and myelin membranes are consistent with the notion that both NFA and HFA sulfatides are synthesized in the microsomal (Golgi) membranes and are transported to myelin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of the ethanolamine phosphoglycerides of mouse brain myelin and microsomes

Abstract— Three groups of six mice each were killed 1, 4 and 7 days after an intracerebral injection of [1,2-¹⁴C]ethanolamine. The specific radioactivities of the acid-labile ethanolamine phosphoglycerides (ethanolamine plasmalogens) and of the acid-stable ethanolamine phosphoglycerides (diacyl and alkyl acyl glycerophosphoryletholamines) from myelin and microsomal fractions were determined. All of these brain ethanolamine phosphoglycerides turn over rapidly with an apparent half-life of less than 3 days. The biosynthesis of alkenyl acyl glycerophosphorylethanolamines from diacyl glycerophosphorylethanolamines in mouse brain myelin or microsomes is unlikely.     

Neonatal malnutrition in the rat affects the delivery of sulfatides from microsomes and their entry into myelin

Brain slices from 18 day old normal and malnourished rats were incubated in the presence of [³⁵S]sulfate to explore its incorporation into sulfatides of a total brain homogenate and the appearance of labeled sulfatides in different subcellular fractions. While the incorporation of label into sulfatides of the total homogenate was similar in both groups of animals, in subcellular fractions separated on a linear sucrose density gradient, labeling of sulfatides in malnourished animals was relatively higher in the region corresponding to the microsomal fraction. Time course incorporation and pulse-chase experiments were carried out to explore the kinetics of labeling of microsomal and myelin sulfatides. In pulse-chase experiments, normal controls showed a decrease in the specific radioactivity of sulfatides in the microsomal fraction after the chase, which was not observed in malnourished animals, while the appearance of labeled sulfatides in the myelin fraction of the latter group of animals was found to be lower than in normals. These results suggest that in neonatal malnutrition there is a defect in the transport of de novo synthesized sulfatides towards myelin or/and a problem in the assembly of these lipids into the myelin membrane.     

Intracellular transport of a newly synthesized asialoglycoprotein receptor in rat liver

Intracellular transport of a newly synthesized asialoglycoprotein receptor was studied biochemically using a monospecific antibody for the receptor. Pulse-labeling by intravenous injection of [3H]leucine and pulse-chasing after 10 min by cycloheximide injection resulted in the maximal labeling of the receptor in the rough microsomes at 15 min, in the smooth microsomes and the heavy Golgi subfraction (GF3) at 25 min and in the intermediate plus light Golgi subfraction (GF1+2) at 30 min. By 60 min, the labeling in GF1+2 had decreased and leveled off. In the plasma membrane fraction, the labeled receptor first appeared at 20 min, increased rapidly and also reached a constant level at 40-60 min. Intracellular movement of the newly synthesized receptor in the GF1+2 and plasma membrane fractions was also investigated by purifying the receptor protein from the GF1+2 and plasma membrane fractions by affinity chromatography. It was revealed that the specific radioactivities of the receptor in the two fractions become equilibrated after 60-120 min. The receptor of the various membrane fractions was also pulse-labeled in vivo for 20 min simultaneously with [3H]glucosamine and [14C]leucine, and pulse-chased for the following 40 min. After pulse-labeling for 20 min, the ratio of the radioactivity of [3H]glucosamine or [3H]sialic acid to [14C]leucine of the receptor from the rough and smooth microsomes, and GF3, GF2, and GF1 increased in that order. That of the receptor from the plasma membrane fraction was infinitely higher, because, while a significant amount of 3H-radioactivity was incorporated into the receptor in the Golgi apparatus, only a negligible amount of 14C-radioactivity was incorporated into the same receptor in the plasma membrane due to the delay in the arrival of [14C]leucine labeled receptor to the plasma membrane. After chasing for 40 min, however, the same radioactivity ratios of the GF1 and plasma membrane fractions approached each other. All these results strongly suggest that the distribution of the newly synthesized receptor becomes rapidly equilibrated between the trans-Golgi components and plasma membranes probably by repeated recycling of the receptor protein between the two membranes.     

METABOLISM IN VIVO OF BRAIN GALACTOLIPIDS: THE JIMPY MUTANT

Abstract— The incorporation in vivo of [U-¹⁴C]glucose into the galactolipids of the brain of control and Jimpy mutant mice was examined. Over a 24-h period of incorporation there was no indication of an increased rate of turnover of brain galactolipids in the mutant. The Jimpy mutant was identified at ages prior to and at the inception of myelination (7–10 days post partum) with a coat marker (Tabby). There was similar total radioactivity in galactolipids of the Jimpy at these ages but a reduction to 13 per Cent of control at 13 days and to 6 per cent at 16 days of postnatal age. This devetopmental pattern of galactolipid synthesis in Jimpy brain is not in accord with a primary defect in the biosynthesis of cerebrosides and sulphatides.     

Structure of sulfatides biosynthesized in vitro

Starting from galactose-(14)C-labeled phrenosine and 3'-phosphoadenosine-5'-phosphosulfate, radioactive sulfatides have been obtained in vitro with a biosynthetic system similar to the one described by McKhann and Ho (Ref. 6). It has thus been proved that exogenous cerebrosides can act as acceptors of sulfate. The specific radioactivity of the synthetic phrenosine used as precursor was sufficiently high to permit the proof of the structure of the resulting sulfatides to be done by methylation on an amount estimated at 0.1 micro g. The sulfate group was found only at C-3 of galactose, the position at which it is located in sulfatides isolated from tissues. This observation indicates the specificity of the sulfotransferase involved in the in vivo synthesis of sulfatides.     

The disposition of [24-3H]cerebrosterol in developing rat brain.

—The uptake into subcellular fractions of developing rat brain in vivo of intracerebrally injected [4-¹⁴C]cholesterol, [24-³H]cerebrosterol, and [24-³H]24-epicerebrosterol was measured for periods up to 30 days following administration. [4-¹⁴C]cholesterol was accumulated rapidly in nuclei, nerve endings, and microsomes, more slowly in myelin and mitochondria. [24-³H]cerebrosterol was accumulated rapidly in myelin, nerve endings, and microsomes, more slowly in nuclei and mitochondria. The uptake of [24-³H]24-epicerebrosterol was essentially the same as that of [24-³H]cerebrosterol. Ratios of radioactivities of [24-³H]cerebrosterol and [4-¹⁴C]cholesterol accentuated the early accumulation of [24-³H]cerebrosterol in myelin, nerve endings, and microsomes, and declining ³H:¹⁴C ratios disclosed the rapid elimination of [24-³H]cerebrosterol and [24-³H]24-epicerebrosterol relative to [4-¹⁴C]cholesterol in nerve endings and microsomes. The data suggest that the removal of [24-³H]cerebrosterol from brain results from an enzymic metabolism of the sterol, therefore that cerebrosterol exists in brain in a dynamic state of biosynthesis and catabolism.     

RNA metabolism in rabbit brain: study with neuron-glia and subcellular fractions

—Uridine incorporation into RNA of rabbit brain was studied by using an in vitro system for incubation of brain slices for up to 180 min. Neuron-enriched and glia-enriched fractions were prepared by ficoll density gradient centrifugation, and various subcellular fractions were prepared by sucrose density gradient centrifugation. Although the difference was not as great as in the case of l -leucine incorporation into protein, the neuron-enriched fraction consistently showed a higher specific radioactivity than the glia-enriched fraction. The specific radioactivity of the nuclei increased promptly and remained high at 180 min; the increase in the microsomes was gradual. Comparison of these data suggests that both neuron-enriched and glia-enriched fractions retain high radioactivities in their nuclei at 180 min when a considerable portion of the ribosomal RNA in these fractions is not labeled. The sharp diffusion gradient of nucleotides is discussed in relation to the acid-soluble radioactivity.     

Metabolism of cerebrosides and sulfatides in the nervous system ofXenopus tadpole during metamorphosis

Xenopus laevis tadpoles undergoing metamorphosis were used to study the turnover of cerebrosides and sulfatides in the nervous system of the frog. Tadpoles at the beginning of metamorphosis were treated by intraperitoneal injection with [U-¹⁴C]glucose and radioactivity incorporated into galactosphingolipids of brain and tail was measured after various times. The specific activity of brain cerebrosides increased rapidly for the first 24 hr after injection, reached a plateau after 48hr, and then declined 40% by 7 days. The specific activity of sulfatides changed somewhat more slowly. Hydroxy fatty acid-containing galactosphingolipids had nearly twice the specific activity compared with their nonhydroxy counterparts in brain. Despite the complete regression of tail nerve cord, metabolism of glycosphingolipids in this tissue also indicated active synthesis as well as degradation during this period. The specific activities of these lipids were similar and all reached a peak 24 hr after injection. Examination of the components of these galactosphingolipids disclosed that only a small fraction (7–25%) of the radioactivity was in the galactose moiety in both brain and tail. The ratios of the radioactivity in fatty acid to that in the sphingoid base were much higher for hydroxycerebroside and hydroxysulfatide than for the nonhydroxy isomers.Abbreviations used: Cerebroside is N-acyl, 1-0--galactosyl derivative of sphingoid base (D-erythro-2-amino-alkyl-1,3-diol) Sulfatide is the galactose-3-sulfated derivative of cerebroside. The prefixes hydroxy and nonhydroxy indicate cerebroside or sulfatide containing -hydroxy and nonhydroxy fatty acids, respectively     

Intramyelinic conversion of cerebrosides into acylgalactosylceramides

Acylgalactosylceramide (AGC) synthesis was measured in vivo, and in a cell free system. 24 hours post-injection of [³H]palmitic acid into rat brain, more than 60% of the AGC radioactivity was associated with an ester linkage. Isolated rat myelin was incubated in the presence of [¹⁴C]palmitic acid, 2mM ATP, 50 M CoA and 10 mM MgCl₂ and acylation of myelin cerebrosides occurred at a linear rate for at least 60 min. Incubation of isolated myelin under standard conditions with [³H] cerebrosides and [¹⁴C]palmitic acid produced double labeled AGC. Labeling of AGC was maximum at pH 7.5 and 37°C and appeared to be enzyme mediated inasmuch as it was reduced by myelin incubation with trypsin and drastically reduced by preheating the myelin for 5 min at 80°C. Omission of ATP, CoA, MgCl₂ or all three did not reduce fatty acid incorporation into AGC when compared to the values in the complete system. Addition of Triton X100 or Sodium Dodecyl Sulfate had little or no effect on the acylation of cerebrosides. Pulse chase experiments indicated that the reaction involved the net addition of fatty acid to the cerebrosides, rather than a rapid fatty acid exchange.