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.     

THE INCORPORATION OF LABELLED ACETATE INTO CEREBROSIDE AND OTHER LIPIDS OF THE DEVELOPING MOUSE BRAIN

—Cerebroside in the brain is highly localized in myelin and has a relatively slow turnover rate. The aim of this study was to evaluate the true cerebroside biosynthetic activity under conditions in which the degradation and reutilization of brain lipids were as small as possible. The 3-week-old mice were decapitated at 0·5, 1, 2·5, 5 and 15 min after the intraperitoneal injection of labelled acetate and the incorporation of radioactivity into each lipid class was examined. Even at 0·5 min, a considerable amount of radioactivity was found in simple lipids, especially in the free fatty acid fraction, and in the course of time the radioactivity of complex lipids increased. On the other hand, the incorporation of radioactivity into cerebrosides was extremely small throughout the experimental period. Results indicated that the low radioactivity of cerebroside might be due to its high content of long-chain fatty acids which were weakly labelled. The radioactivity of the sphingosine moiety was also low. In short, one of the rate-limiting steps of cerebroside synthesis in brain might exist in long-chain fatty acid and sphingosine synthesis. In addition, the incorporation curves of each component of cerebroside were compared with each other and the difference of the incorporation pattern of non-hydroxy fatty acids of cerebroside was noted.     

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.     

Divalent cation-mediated interaction between cerebroside sulfate and cerebrosides: an investigation of the effect of structural variations of lipids by electrospray ionization mass spectrometry.

Divalent cations mediate a carbohydrate-carbohydrate association between the two major glycolipids, galactosylceramide (GalCer) and its sulfated form, cerebroside sulfate (CBS), of the myelin sheath. We have suggested that interaction between these glycolipids on apposed extracellular surfaces of myelin may be involved in the stability or function of this multilayered structure. A mutant mouse lacking galactolipids because of a disruption in the gene that encodes a galactosyltransferase forms myelin that initially appears relatively normal but is unstable. This myelin contains glucosylceramide (GlcCer) instead of GalCer. To better understand the role of GlcCer in myelin in this mutant, we have compared the ability of divalent cations to complex CBS (galactosyl form) with GlcCer or GalCer in methanol solution by using positive ion electrospray ionization mass spectrometry. Because both the alpha-hydroxylated fatty acid species (HFA) and the nonhydroxylated fatty acid species (NFA) of these lipids occur in myelin, we have also compared the HFA and NFA species. In addition to monomeric Ca2+ complexes of all three lipids and oligomeric Ca2+ complexes of both GalCer and GlcCer, Ca2+ also caused heterotypic complexation of CBS to both GalCer and GlcCer. The heterotypic complexes had the greatest stability of all oligomers formed and survived better at high declustering potentials. Complexes of CBS with GlcCer were less stable than those with GalCer. This was confirmed by using the free sugars and glycosides making up the carbohydrate headgroups of these lipids. HFA species of CBS and GalCer formed more stable complexes than NFA species, but hydroxylation of the fatty acid of GlcCer had no effect. The ability of GlcCer to also complex with CBS, albeit with lower stability, may allow GlcCer to partially compensate for the absence of GalCer in the mouse mutant.     

Turnover rate of molecular species of sphingomyelin in rat brain

Turnover rate of individual molecular species of sphingomyelin of adult rat brain myelin and microsomal membranes was determined after an intracerebral injection of 100 Ci of [C³H₃]choline. Myelin and microsomal membrane sphingomyelins were isolated from the rest of the lipids. The individual molecular species of benzoylated sphingomyelin were separated and quantitated by reversed-phase high performance liquid chromatography. All individual major molecular species of microsomal and myelin sphingomyelin had maximum incorporation at 6 and 15 days, respectively, after the injection. The specific radioactivity of all the various molecular species of both myelin and microsomal sphingomyelin declined at a similar rate after reaching a maximum. There was no significant difference in the turnover rate of short chain (16:0, 18:0) and long chain (>22:0) fatty acid containing sphingomyelin. The average apparent turnover rate of myelin and microsomal sphingomyelin molecular species was about 14–16 days for the fast pool and about 45 days for the slow pool. It is concluded that individual molecular species of sphingomyelin of myelin and microsomal membranes turned over at a similar rate. Thus, turnover rate of sphingomyelin in myelin and microsomal membranes is not affected by the fatty acyl composition of the lipid.     

OBSERVATIONS ON THE BIOSYNTHESIS OF RAT BRAIN CERAMIDE AND CEREBROSIDE IN VIVO

Abstract— The incorporation in vivo of l -[¹⁴C]serine into ceramide and cerebroside of young rat brain has been studied. Acid hydrolysis of labelled ceramide and galactosyl-ceramide followed by selective partitioning of the resulting components indicated that 88 per cent of the radioactivity was present in the long-chain base portion. At early time points (10 min, 20 min) the precursor was incorporated into ceramide and to a lesser degree into glucosyl-ceramide. During time intervals of 5 and 10 h, the specific activity values (d.p.m./μmol) for ceramide and glucosyl-ceramide decreased, while values for galactosyl-ceramide, containing either unsubstituted fatty acids (NFA) or α-hydroxy fatty acids (HFA), increased 50 and 30 per cent, respectively. Analysis of labelled ceramide at all time points studied (10 min-10 h) indicated that l -[¹⁴C]serine was incorporated onto the NFA type. This observation suggests that HFA-ceramide may not be the physiological precursor of HFA-galactosyl-ceramide. In this context, the postulated precursor roles of both ceramide and psychosine in the biosynthesis of brain cerebrosides are discussed.     

Lipid metabolism and structure-activity features of the endoplasmic reticulum membrane in regenerating rat liver]

The relationship between the neutral lipid and phospholipid metabolism and some structure-function peculiarities of regenerating rat liver endoplasmic reticulum membranes (13 hours after surgery, i.e., corresponding to the G1-period of the cell cycle) was studied. There was an increase in the degree of the endoplasmic reticulum membrane development and the nonesterified fatty acid (NFA) and triglyceride (TG) content in regenerating rat liver microsomes. The relative specific radioactivity of neutral lipid and phospholipid fractions in regenerating rat liver microsomes was lower than in control animals, presumably due to the high rate of the microsomal lipid exchange in the regenerating liver with other cell organelles. The changes in the lipid content and rate of their metabolism in the regenerating rat liver were associated with the increase in the membrane microviscosity and the decrease in the activity of the membrane-bound enzyme (glucose-6-phosphatase). The differences in the time-dependent changes in the synthesis and metabolism of lipids in the NFA and TG fractions may be regarded as an endogenous factor determining the structure-function peculiarities of endoplasmic reticulum membranes.     

Transport of sulfatides towards myelin. Effect of colchicine,monensin and calcium on their intracellular translocation

The in vitro effect of colchicine or monensin upon sulfatide delivery from microsomes and perikarya of oligodendroglial cells and its further incorporation into myelin was studied using brain slices obtained from 18-day-old rats incubated during 20 min with [³⁵S]sulfuric acid and reincubated for different times with unlabeled precursor. Labeled sulfatides were measured in a total homogenate, myelin, microsomes and perikarya of oligodendroglial cells. Neither colchicine nor monensin depressed the incorporation of [³⁵S]sulfate into sulfatides of the total homogenate. However, these drugs inhibited by 50% the incorporation of labeled sulfatides into myelin. Furthermore, while the specific radioactivity present in microsomes and perikarya of oligodendroglial cells isolated from controls at 120 min decreased to about 40% of the value at 20 min, no decrease was observed in fractions obtained from slices incubated with colchicine or monensin. Similar results were obtained when the slices were incubated in “Ca²⁺ free” medium. The perturbed delivery of [³⁵S]sulfatides from microsomes and perikarya of oligodendroglial cells and the diminished incorporation into myelin, in the presence of monensin and colchicine, are consistent with a possible involvement of the Golgi complex and of the cytoplasmic microtubules in the transport of sulfatides towards myelin. Moreover, the transport of these galactolipids appears to require calcium.     

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

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

Distribution of radioactivity in myelin lipids following subcutaneous injection of [14C]stearate

Blood fatty acids are an important parameter for the synthesis of brain myelin as exogenous stearic acid is needed: after subcutaneous injection to 18-day-old mice this labelled stearic acid is transported into brain myelin and incorporated into its lipids. However the acid is partly metabolized in the brain by elongation (thus providing very long chain fatty acids, mainly lignoceric acid) or by degradation to acetate units (utilized for synthesis of medium chain fatty acids as palmitic acid, and cholesterol). These metabolites are further incorporated into myelin lipids. The myelin lipid radioactivity increases up to 3 days; most of the activity is found in phospholipids; their fatty acids are labelled in saturated as well as in polyunsaturated homologues but sphingolipids, especially cerebrosides, contain also large amounts of radioactivity (which is mainly found in very long chain fatty acids, almost all in lignoceric acid). The occurrence of unesterified fatty acids must be pointed out, these molecules unlike other lipids, are found in constant amount (expressed in radioactivity per mg myelin lipid).     

Levels of sulfatide synthesis distinguish oligodendroglia in different stages of maturation

In the central nervous system, oligodendroglia elaborate extensive quantities of membranes to form the multilamellar myelin sheath. Whether the production of extensive networks of processes by oligodendroglia in culture is a similar type of phenomenon as the formation of myelin is an unanswered question. Rat oligodendroglia, prepared by a modification of a differential shaking and plating method, elaborate extensive processes in culture. In contrast, bovine oligodendroglia, obtained by a bulk-isolation method, produce whorls of membrane lamellae, adjacent to the cell soma. The incorporation of various radiolabeled substrates into specific lipids was compared with the two cell types. It was found that rat oligodendroglia do produce myelin specific lipids, but at a lower level than bovine oligodendroglia which are actively synthesizing myelin lipids, especially cerebrosides, from a variety of substrates. Interestingly sulfatides are produced at a higher level in the cells not producing myelin, rat oligodendroglia. Other lipids that are associated with myelination (cerebrosides with -hydroxy fatty acids and phosphatidylinositides) are produced at higher levels in bovine oligodendroglia. Thus it appears that the extension of processes by oligodendroglia in culture is a different phenomenon than the production of myelin membranes and requires lower levels of myelin lipids.     

Flip-flop of hydroxy fatty acids across the membrane as monitored by proton-sensitive microelectrodes

Hydroxyl group-containing fatty acids play an important role in anti-inflammatory action, neuroprotection, bactericide and anti-cancer defense. However, the mechanism of long-chain hydroxy fatty acids (HFA) transport across plasma membranes is still disputed. Two main hypotheses have been suggested: firstly, that protonated HFAs traverse across the membranes spontaneously and, secondly, that the transport is facilitated by proteinaceous carriers. Here, we demonstrate that the protonated HFA are able to move across planar lipid bilayers without protein assistance. This transport step is accompanied by the acidification of the buffer in receiving compartment and the pH augmentation in the donating compartment. The latter contained liposomes doped with HFA. As revealed by scanning pH-sensitive microelectrodes, the pH shift occurred only in the immediate vicinity of the membrane, while bulk pH remained unchanged. In concurrence with the theoretical model of weak acid transport, the pH value at maximum proton flux was almost equal to the pK of the studied HFA. Intrinsic pKi values were calculated from the electrophoretic mobilities of HFA-containing liposomes and were 5.4, 6.5, 6.9 and 6.3 for 2-hydroxyhexadecanoic, 16-hydroxyhexadecanoic, 12-hydroxydodecanoic and 9,10,16-trihydroxyhexadecanoic acids, respectively.     

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

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

High rates of [1-14C]acetate incorporation into the lipid of isolated spinach chloroplasts.

Spinach chloroplasts, isolated by techniques yielding preparations with high O2- evolving activity, showed rates of light-dependent acetate incorporation into lipids 3-4 fold higher than any previously reported. Incorporation rates as high as 500 nmol of acetate/h per mg of chlorophyll were measured in buffered sorbitol solutions containing only NaHCO3 and [1-14C]acetate, and as high as 800 nmol/h per mg of chlorophyll when 0.13 mM-Triton X-100 was also included in the reaction media. The fatty acids synthesized were predominantly oleic (70-80% of the total fatty acid radioactivity) and palmitic (20-25%) with only minor amounts (1-5%) of linoleic acid. Linolenic acid synthesis was not detected in the system in vitro. Free fatty acids accounted for 70-90% of the radioactivity incorporated and the remainder was shared fairly evenly between 1,2-diacylglycerols and polar lipids. Oleic acid constituted 80-90% of the free fatty acids synthesized, but the diacylglycerols and polar lipids contained slightly more palmitic acid than oleic acid. Triton X-100 stimulated the synthesis of diacylglycerols 3-6 fold, but stimulated free fatty acid synthesis only 1-1.5-fold. Added glycerol 1-phosphate stimulated both the synthesis of diacylglycerols and palmitic acid relative to oleic acid, but did not increase acetate incorporation into total chloroplast lipids. CoA and ATP, when added separately, stimulated acetate incorporation into chloroplast lipids to variable extents and had no effect on the types of lipid synthesized, but when added together resulted in 34% of the incorporated acetate appearing in long-chain acyl-CoA. Pyruvate was a much less effective precursor of chloroplast fatty acids than was acetate.     

Flip-flop of hydroxy fatty acids across the membrane as monitored by proton-sensitive microelectrodes

Hydroxyl group-containing fatty acids play an important role in anti-inflammatory action, neuroprotection, bactericide and anti-cancer defense. However, the mechanism of long-chain hydroxy fatty acids (HFA) transport across plasma membranes is still disputed. Two main hypotheses have been suggested: firstly, that protonated HFAs traverse across the membranes spontaneously and, secondly, that the transport is facilitated by proteinaceous carriers. Here, we demonstrate that the protonated HFA are able to move across planar lipid bilayers without protein assistance. This transport step is accompanied by the acidification of the buffer in receiving compartment and the pH augmentation in the donating compartment. The latter contained liposomes doped with HFA. As revealed by scanning pH-sensitive microelectrodes, the pH shift occurred only in the immediate vicinity of the membrane, while bulk pH remained unchanged. In concurrence with the theoretical model of weak acid transport, the pH value at maximum proton flux was almost equal to the pK of the studied HFA. Intrinsic pK_i values were calculated from the electrophoretic mobilities of HFA-containing liposomes and were 5.4, 6.5, 6.9 and 6.3 for 2-hydroxyhexadecanoic, 16-hydroxyhexadecanoic, 12-hydroxydodecanoic and 9,10,16-trihydroxyhexadecanoic acids, respectively.     

A calorimetric study of the thermotropic behaviour of mixtures of brain cerebrosides with other brain lipids

We have used a computer-controlled differential scanning calorimeter to determine the phases present in mixtures of the brain galactocerebrosides with other representative brain lipids. There are two types of brain galactocerebroside, those which possess an alpha-hydroxy substituent on the acyl chain (HFA) and those that do not (NFA). In the liquid crystalline state both cerebrosides were miscible with all the lipids studied, but in the gel state they were immiscible with cholesterol and the brain phosphatidylcholines. However, cholesterol mixtures in which the cholesterol mole fraction exceeded one third formed homogeneous metastable gel states on cooling from above the melting point of the cerebroside. Relaxation to the stable two phase state took place slowly over several hours. The solubilities of the galactocerebrosides in the other main brain sphingolipid, sphingomyelin, were much higher. Only in the case of the NFA galactocerebroside and at low mole fractions of sphingomyelin was immiscibility detected. Ternary mixtures of the two cerebrosides with sphingomyelin/cholesterol and phosphatidylcholine/cholesterol (PC/Chol) showed different miscibility characteristics. On cooling from 80 degrees C all mixtures formed homogeneous gel states. However, on standing the cerebrosides separated into discrete gel phases in all mixtures but one, that in which HFA galactocerebrosides were mixed with sphingomyelin and cholesterol. The cerebroside in the mixture with the composition closest to that of myelin, HFA/PC/Chol, melted at 38 degrees C. On scanning guinea pig CNS myelin which had been equilibrated at 5 degrees C a transition was detected with Tmax 33 degrees C. On the basis of comparison with the HFA/PC/Chol mixture we propose that the transition in myelin at this temperature is due to the melting of a galactocerebroside gel phase.     

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.     

Studies on the submicrosomal fractions of bovine oligodendroglia: Lipid composition and glycolipid biosynthesis

Oligodendroglia were isolated from bovine brain, and a crude, microsomal fraction obtained from cell homogenates was subfractionated into myelin (MP), plasma membranes (PM), Golgi (GF), smooth (SER) and rough (RER) endoplasmic membranes using discontinuous-sucrose gradient centrifugation. The submicrosomal fractions were characterized by ultrastructural examination and analysis of the specific organelle markers. The myelin and plasma membrane rich fractions contained characteristically the highest amounts of the lipid with lower mole percentages of total phospholipids and phosphatidylcholine, and higher concentrations of phosphatidylethanolamine (+plasmalogens), cholesterol and galactolipids. Considerable amounts of the typical myelin galactolipids (galacto-cerebrosides, sulfatides and monogalactosyl diglycerides) were also found in the Golgi fraction (GF). The GF fraction had the greatest enrichment of glycolipid-forming galactosyltransferases, and the distribution of these enzymes correlated well with that of the Golgi marker enzymes. The results give evidence that intracellular Golgi apparatus of oligodendroglia is rich in the myelin-specific lipids, and suggest its involvement in the synthesis and processing of myelin lipids.     

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.     

Simultaneously entry of palmitic acid into proteolipid protein in different myelin subfractions of rat brain

Rats of 20-days of age were injected intracranially with radioactive palmitic acid to study its incorporation into proteolipid protein (PLP) of myelin and myelin subfractions. At short times (120 min), the radioactivity present in PLP was shown to be due to palmitic acid bound to the protein by ester linkages. The specific radioactivity of palmitic acid labeled PLP was identical in all the myelin subfractions except the myelin-like fraction, in which it was lower, suggesting that the entry of the fatty acid into PLP of the different subfractions occurs simultaneously.Experiments using time staggered injections of ¹⁴C- and ³H-labeled palmitic acid also showed that entry of the fatty acid into PLP of the various subfractions was simultaneous. These results seem to indicate that the acylation of PLP occurs in the myelin membrane and that synthesis and transport of this protein are events unrelated to the acylation process.