Turnover of Axonally Transported Phospholipids in Nerve Endings of Retinal Ganglion Cells

We have investigated the metabolic turnover of axonally transported phospholipids in myelinated axons (optic tract) and nerve endings (superior colliculus) of retinal ganglion cells. One week following intraocular injection of [2-3H]glycerol, turnover rates for individual phospholipid classes in the retina (which contains a number of other cell types in addition to the ganglion cells) were all very similar to each other, with apparent half-lives of approximately 7 days. Apparent half-lives of labeled phospholipids in superior colliculus (presumably primarily in retinal ganglion cell nerve endings) were 10 days for both choline and inositol phosphoglycerides and 13 days for both serine and diacylethanolamine phosphoglycerides. Subcellular fractionation data obtained from superior colliculus at various times after injection suggested that apparent turnover rates determined for nerve ending phospholipids probably were not significantly affected by transfer of axonally transported 3H lipids into myelin. Apparent half-lives for phospholipids in optic tract were somewhat longer than in superior colliculus, ranging from 11 to 18 days. The slower turnover rates in optic tract may, in part, reflect the transfer of some axonal lipids to the more metabolically stable pool of lipids in the myelin ensheathing the retinal ganglion cell axons. In both optic tract and superior colliculus, apparent half-lives for axonally transported phospholipids labeled with [32P]phosphate were only slightly longer than for [2-3H]glycerol, while those for [14C]choline and [3H]acetate were markedly longer, indicating differing degrees of metabolic conservation or reutilization of these precursors relative to glycerol.     

AXONAL TRANSPORT OF LIPIDS IN THE RABBIT OPTIC SYSTEM

Abstract— Axonal transport of lipids was demonstrated in the rabbit optic system using [2-³H]glycerol and [3-¹⁴C]serine. Following intraocular injection of these precursors, radioactive lipids were detected in the optic tract, superior colliculus and lateral geniculate body over a 31 day period. The bulk of lipid appeared to migrate at a rate equivalent to that of rapidly transported protein which, when combined with a prolonged period of release into the axon, led to a peak of transported radioactivity at 6-10 days for the 3 tissues. The suggestion of a second peak at 17 days indicated the possibility of a smaller slow component, although another interpretation is suggested. Analysis of individual transported lipids revealed [2-³H]glycerol to label phosphoglycerides preferentially and [3-¹⁴C]serine to be an effective precursor for sphingolipids and certain of the phosphoglycerides. [3-¹⁴C]Serine labeled axonally transported proteins to an even greater extent than lipids, revealing the same fast and slow components previously shown with other amino acids.     

Kinetics of entry of galactolipids and phospholipids into myelin.

Abstract— Brain slices from 17 day rats were incubated with [³H]galactose and [³⁵S]sulphate to label cerebroside and sulphatide. Myelin was isolated by centrifugation on a sucrose density gradient. Following lipid extraction and alkaline methanolysis, cerebroside and sulphatide were isolated by tic, and radioactivity was measured. Appearance of [³H]cerebroside and [³H]sulphatide in myelin showed a lag of less than 15min, while appearance of [³⁵S]sulphatide in myelin showed a longer lag of about 30min. In chase experiments, the rate of appearance of [³H]cerebroside and [^3SS]sulphatide in the non-myelin fraction and of [³H]cerebroside in the myelin fraction slowed markedly after the chase. In contrast, [³⁵S]sulphatide continued to increase in myelin at a normal rate for 30min after the chase, then stopped, while ³H from galactose continued to accumulate in myelin sulphatides for 60 min. These data are interpreted to demonstrate an interval of 30 min between synthesis of cerebroside and its sulphation in the non-myelin fraction, and another delay of 30 min between sulphation and appearance in myelin. The distribution of newly synthesized cerebroside and sulphatide between myelin and non-myelin fractions also supported the concept that a complex metabolic pool of cerebroside in the non-myelin fraction is precursor to sulphatide of myelin. For comparison, entry of phosphatidyl choline and phosphatidyl ethanolamine into myelin was followed with [2-³H]glycerol as precursor. Like cerebroside, both phospholipids showed little delay in their initial appearance in myelin, and prompt cessation of their addition after a chase with unlabeled precursor. These results are consonant with either rapid entry of these three lipids into myelin after synthesis at an extra-myelin site, or synthesis of the lipids within myelin itself.     

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.     

METABOLIC RELATIONSHIPS BETWEEN MYELIN SUBFRACTIONS: ENTRY OF GALACTOLIPIDS AND PHOSPHOLIPIDS 1

Abstract— Partially purified myelin from the brains of 17-day-old rats was separated into 4 subfractions on a three-step sucrose gradient by virtue of heterogeneity in density and particle size. Precursor-product relationships between different membrane fractions were investigated by determining the specific radioactivity of individual lipids in each subcellular fraction 15 min after intracranial injection of an appropriate precursor. Rats were injected with [2-³H]glycerol. myelin subfractions prepared, and individual lipids separated by TLC. For choline and ethanolamine phospholipids, specific radioactivity was highest in the densest fraction (D), intermediate in the next densest fraction (C), and lowest in the lighter fractions (B and A). Similar results were observed for cerebroside and sulphatide when [³H]galactose was the precursor. These data are consistent with (but do not prove) a precursor-product relationship for individual lipids from the densest to the lightest subfraction. Another experimental design involving time staggered injections of [³H] and [¹⁴C] precursors was developed which enables a more definitive result with regard to precursor-product relationships to be obtained. A precursor-product relationship between a given lipid in a dense myelin membrane fraction, and the same lipid in a lighter subfraction, would be indicated by a change in isotope ratio. If there is no precursor-product relationship. Ihe isotope ratio should be constant. Such experiments were done with [³H] and [¹⁴C]glycerol. The data indicated that phosphatidyl ethanolamine and its plasmalogen analog were added first to the densest subfraction and then in turn to the lighter subfractions. In contrast, phosphatidyl choline and its plasmalogen analog were added “simultaneously” (i.e. with delays of much less than 15min) to each of the subfractions. Similar experiments with [³H] and [¹⁴C]galactose showed that cerebroside, sulphatide and galactosyl diglyceride also entered the subfractions simultaneously rather than in sequential order. Thus the assembly of the myelin sheath involves an obligate order of addition of certain lipids. while other lipids are probably added in a random order.     

Axonal transport of phospholipids in rat visual system.

Abstract— Seventeen day old rats were injected intraocularly with a phospholipid precursor, [³²P]phosphate, and a glycoprotein precursor, [³H]fucose. Animals were killed between 1 h and 21 days later, and structures of the visual pathway (retina, optic nerve, optic tract, lateral geniculate body, and superior colliculus) were dissected. Radioactivity in phospholipids ([³²P] in solvent-extracted material) and in glycoproteins ([³H] in solvent-extracted residue) was determined. Incorporation of [³H]fucose into retinal glycoproteins peaked at 6–8 h. Labelled glycoproteins were present in superior colliculus by 2h after injection, indicating a rapid rate of transport; maximal labelling was at 8–10 h after injection. Incorporation of [³²P]phosphate into retinal phospholipids peaked at 1 day after injection. Phospholipids were also rapidly transported since label was present in the superior colliculus by 3 h after injection: however, maximal labelling did not occur until 5–6 days. These results indicate that newly synthesized phospholipids enter a preexisting pool, part of which is later committed to transport at a rapid rate. Transported phospholipids were catabolized at the nerve endings with a maximum half-life of several days; there was minimal recycling of precursor label. Lipids were fractionated by thin-layer chromatography, and radioactivity in individual phospholipid classes determined. Choline and ethanolamine phosphoglycerides were the major transported phospholipids, together accounting for approx 85% of the total transported lipid radioactivity. At early time points, the ratio of radioactivity in choline phosphoglycerides to that in ethanolamine phosphoglycerides increased in structures progressively removed from the site of synthesis (retina) but by 2 days approached a constant value. In each structure, choline phosphoglyceride-ethanolamine phosphoglyceride radioactivity ratios decreased with time, rapidly at first, but plateaued by 2 days. These results indicate that choline phosphoglycerides are committed to transport sooner than ethanolamine phosphoglycerides. Some experiments were also conducted using [2-³H]glycerol as a phospholipid precursor. Results concerning incorporation of this precursor into individual phospholipid classes and their subsequent axonal transport were comparable to those obtained using [³²P]phosphate, with the following exceptions: (a) incorporation of [2-³H]glycerol into retinal phospholipids was relatively rapid (near-maximal levels at 1 h after injection) although transport to the superior colliculus showed an extended time course very similar to [³²P]-labelled lipids; (b) [2-³H]glycerol was somewhat less efficient than [³²P]phosphate in labelling lipids committed to transport relative to labelling those which remained in the retina; and (c) [2-³H]glycerol did not label plasmalogens.     

HE TURNOVER RATE OF TUBULIN IN RAT BRAIN

The turnover rate of tubulin in rat brain was determined from the decay in specific radioactivity of the protein after pulse-labeling. When precursors were administered by a parenteral route, the shortest half-life, 9.8 days, was obtained with [¹⁴C]NaHCO₃; the longer half-lives obtained with [U-¹⁴C]glucose or [4,5-³H]leucine suggest significant reutilization of label. Furthermore, with leucine as precursor maximal specific radioactivity of tubulin was not obtained until eight days after administration of label. Labeling and decay kinetics obtained with [4,5-³H]leucine were markedly different when the isotope was administered directly into the lateral ventricle. The difference between the turnover rates of the -α and β subunits of tubulin purified by means of high resolution polyacrylamide gel electrophoresis was not statistically significant. A half-life for tubulin of 6.2 days was measured by continuous intravenous infusion of [U-¹⁴C]tyrosine.     

Lipid metabolism in brain tissue explants

Abstract— Tissue explants from frontal lobes of rat brain were used for the study of cerebral fatty acid metabolism. After tissues had been maintained in serum-supplemented medium, a lipid-free medium was substituted and metabolic studies were carried out. Under these conditions explants continued to take up lipid precursors for at least 48 h, as judged by incorporation of dl -[2-¹⁴C]mevalonic acid into cellular lipids. [l-¹⁴C]Stearic acid and [l-¹⁴C]palmitic acid were bound to cells as the free fatty acids, or incorporated into neutral lipids (particularly triglycerides), glycolipids and phospholipids. In the galactolipid fraction, cerebrosides were the principal radioactive lipids. Choline phosphoglycerides, ethanolamine phosphoglycerides, inositol phosphoglycerides and serine phosphoglycerides were the principal radioactive phospholipids. Fatty acids were incorporated into cellular lipids either unchanged or after desaturation, chain elongation, or both. Maximum incorporation of stearate occurred in tissues derived from 3-day-old animals. With increasing age the uptake of fatty acid dropped sharply. When the labelling of lipids as a function of time was followed in 3-day-old animals, triglycerides and choline phosphoglycerides were the first fractions to take up labelled stearate. Labelling of cerebrosides occurred slowly, only becoming evident after 24 h. These studies exemplify the usefulness of tissue explants for prolonged metabolic studies in normal and pathological specimens of brain.     

In vivo incorporation of l-[14C]serine into phospholipids and proteins of the subcellular fractions of developing rat brain

A study was conducted on the in vivo incorporation of l -[¹⁴C]-serine into the lipids and proteins of the various subcellular fractions of the developing rat brain before and during the stage of active myelination. The total radioactivity in the various fractions at 12 days of age was higher than that at 3 days, while the radioactive specific activity was reversed. The specific activities of the proteins and lipids were higher at 3 days of age with the exception of the subcellular fraction containing myelin. At both ages the lipids of the various cellular fractions had similar specific activities, a finding that suggests a common source for lipid biosynthesis. Incorporation of radioactivity into the various phospholipids was in the following order: phosphatidyl serine > phosphatidyl ethanolamine > phosphatidal serine > sphingomyelin and phosphatidyl choline. Of all the phospholipids, the plasmalogens increased most in total radioactivity during the period when meylination was most active. Serine-containing phospholipids appear to be most tightly bound to proteins. The brain mitochrondrial fraction contained most of the phosphatidyl serine decarboxylase activity with some activity in the nuclei. Biosynthesis of phosphatdyil ethanolamine through decarboxylation of phosphatidyl serine could take place in rat brain. Four unidentified radioactive metabolites were found in the acid-soluble fraction in addition to l -[¹⁴C]serine.     

Effect of pyridoxine deficiency on nucleic acid metabolism in the rat

1. The nucleic acid metabolism in the pyridoxine-deficient rat has been investigated through studies on the incorporation of radioactivity from various isotopically labelled compounds into liver and spleen DNA and RNA. 2. In pyridoxine deficiency, the incorporation of radioactivity from sodium [¹⁴C]formate was apparently increased. The magnitude of this effect on incorporation into liver RNA and DNA and spleen RNA was approximately the same. The incorporation into spleen DNA was enhanced to a much greater degree. Administration of pyridoxine 24hr. before the rats were killed reversed the changes in incorporation of radioactivity from [¹⁴C]formate. 3. In pyridoxine deficiency, the incorporation of radioactivity from dl-[3-¹⁴C]serine, [8-¹⁴C]adenine, [Me-³H]thymidine and [2-¹⁴C]deoxyuridine was decreased. The incorporation of radioactivity from l-[Me-¹⁴C]methionine was not affected. No noteworthy differences in the effect of pyridoxine deficiency on the incorporation of radioactivity from dl-[3-¹⁴C]serine into DNA and RNA were observed, whereas the effect of the deficiency on the incorporation of radioactivity from [8-¹⁴C]adenine into spleen DNA was somewhat greater than that into spleen RNA. Administration of pyridoxine 24hr. before the rats were killed reversed the changes in incorporation of radioactivity from [3-¹⁴C]serine and [8-¹⁴C]adenine. 4. The adverse effects of pyridoxine deficiency on the biosynthesis of nucleic acids and cell multiplication are discussed in relation to the role of pyridoxal phosphate in the production of C₁ units via the serine-hydroxymethylase reaction.     

Axonal transport of glycerophospholipids following intracerebral injection of glycerol into substantia nigra or lateral geniculate body

[2-³H]Glycerol was injected into one substantia nigra of adult rats. Incorporation of radioactivity into lipids at the injection site was maximal by 2 hr, after which it declined. Rapidly transported³H-labeled lipids were just beginning to accumulate in the primary projection site, the ipsilateral corpus striatum by 2 hr, as evidenced by 20-fold higher levels of lipid radioactivity in the projection site relative to control regions. However, the bulk of labeled lipid arrived between 6 hr and 3 days postinjection, suggesting either a prolonged period of release of rapidly transported lipids from the nerve cell bodies or a slow rate of transport for the later arriving lipids. Colchicine applied locally to the fibers of this tract blocked the axonal transport of lipids to the striatum almost completely. Choline and ethanolamine phosphoglycerides were the major transported lipids, accounting for approximately 60% and 25%, respectively, of the total. Similar results were obtained in studies of [2-³H]glycerol-labeled lipids synthesized in the lateral geniculate body and transported to the visual cortex. The rapid axonal transport of lipids labeled with [³²P]phosphate (injected simultaneously with [2-³H]glycerol) could also be demonstrated in both tracts. However, in contrast to [2-³H]glycerol, considerable amounts of³²P soluble label were present in the projection sites, and colchicine only partially blocked the accumulation of³²P-labeled lipid. These results demonstrate the relative utility of [2-³H]glycerol as a lipid precursor for examination of axonal transport in intrabrain tracts. Characteristics of lipid axonal transport in these two intrabrain tracts are similar to each other and are also similar to those previously described for retinal ganglion cells, indicating a common requirement for the axonal transport of these membrane constituents to axons and nerve endings in widely divergent CNS tracts.Presented in part at the 11th meeting of the American Society for Neurochemistry, Houston, Texas, March 1980.     

Serine and ethanolamine incorporation into different plasmalogen pools: Subcellular analyses of phosphoglyceride synthesis in cultured glioma cells

In cultured glioma cells, plasma membrane (PM) is enriched in phosphatidylserine (PtdSer) and plasmalogens (1-O-alk-1-enyl-2-acyl-sn-glycero-3-phosphoethanolamine). Serine can be a precursor of headgroups of both ptdSer and ethanolamine phosphoglycerides (PE) including plasmalogens and non-plasmalogen PE (NP-PE). Synthesis of phospholipids was investigated at the subcellular level using established fractionation procedures and incorporation of [³H(G)]L-serine and [1,2-¹⁴C]ethanolamine. Specific radioactivity of PtdSer from [³H]serine was 2-fold greater in PM than in microsomes, reaching maximum by 2–4 h. Labeled plasmalogen from [³H]serine appeared in PM by 4 h and increased to 48 h, whereas almost no plasmalogen accumulated in microsomes within 12 h. In contrast, labeled plasmalogen from [1,2-¹⁴C]ethanolamine appeared in both PM and microsomes at early incubation times and became enriched in PM beyond 12 h. Thus, in glioma cells: (1) greater and faster accumulation of labeled PtdSer in PM may reflect direct synthesis from serine within PM; (2) PM is a major source of PtdSer for decarboxylation and PE synthesis; (3) NP-PE in both PM and microsome provides headgroup for synthesis of plasmalogen; and, (4) plasmalogen synthesis may involve different intracellular pools depending on headgroup origin.Abbreviations NP-PE nonplasmenylethanolamine phosphoglycerides including both diacyl and alkylacyl species - PE total ethanolamine phosphoglycerides: plasmalogen-plasmenylethanolamine or alkenylacyl ethanolamine phosphoglyceride (1-O-alk-1-enyl-2-acyl-sn-glycero-3-phosphoethanolamine) - PL phospholipid - PM plasma membrane - PtdCho phosphatidylcholine - PtdSer phosphatidylserine     

Quantitative evaluation of two pathways for phosphatidylcholine biosynthesis in rat brain in vivo

[Me-³H] choline and [³²P] orthophosphate were injected intraventricularly into adult female rats. After variable intervals from injection (1–10 min) the animals were sacrificed by means of a microwave apparatus, and phosphorylcholine and choline phosphoglycerides extracted from brain and counted for radioactivity content after separation. The kinetic constants (K) for phosphorylcholine incorporation into lipids were determined both for [³²P] and [³H] labeling. From the data obtained by these procedures it is concluded that base-exchange reactions for choline incorporation into lipids are operating in rat brain in vivo and that they represent a rapidly equilibrating system.     

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.     

The in vivo incorporation of linolenic acid into neuronal and glial cells and myelin

Abstract— Neurons, astrocytes, oligodendrocytes, and myelin were prepared from 21-day-old rat brain at various times after intracerebral injection of [1-¹⁴C]linolenate. Comparisons of phospholipid specific radioactivity demonstrated that the oligodendrocytes were much more active than neuronal, astroglial, or myelin fractions. This is consistent with the concept that the oligodendrocyte is responsible for synthesis of the relatively large mass of myelin sheath. Initially the phosphatidylcholine fraction was more active than the phosphatidylethanolamine fraction, but during the 36 h after injection the former decreased in radioactivity while the latter fraction showed an increase. Fatty acid elongation occurred rapidly. Within 2h after injection, 2/3 of the label had been converted to elongated products (20:4. 20:5, 22:5 and 22:6). All three cell types apparently contained the enzymes necessary to incorporate, elongate, and desaturate linolenic acid and this occurred at similar rates in each cell type. No direct precursor-product relationship was found between the lipids of oligodendrocytes and myelin. There was, however, a lag in the appearance of elongated fatty acids in the phosphoglycerides of myelin. indicating that the polyunsaturated fatty acids in myelin were synthesized elsewhere and transported into the myelin sheath.     

PROVENANCE OF THE ACETYL GROUP OF ACETYLCHOLINE AND COMPARTMENTATION OF ACETYL-CoA AND KREBS CYCLE INTERMEDIATES IN THE BRAIN IN VIVO

—The origin of the acetyl group in acetyl-CoA which is used for the synthesis of ACh in the brain and the relationship of the cholinergic nerve endings to the biochemically defined cerebral compartments of the Krebs cycle intermediates and amino acids were studied by comparing the transfer of radioactivity from intracisternally injected labelled precursors into the acetyl moiety of ACh, glutamate, glutamine, ‘citrate’(= citrate +cis-aconitate + isocitrate), and lipids in the brain of rats. The substrates used for injections were [1-¹⁴C]acetate, [2-¹⁴C]acetate, [4-¹⁴C]acetoacetate, [1-¹⁴C]butyrate, [1, 5-¹⁴C]citrate, [2-¹⁴C]glucose, [5-¹⁴C]glutamate, 3-hydroxy[3-¹⁴C]butyrate, [2-¹⁴C]lactate, [U-¹⁴C]leucine, [2-¹⁴C]pyruvate and [³H]acetylaspartate. The highest specific radioactivity of the acetyl group of ACh was observed 4 min after the injection of [2-¹⁴C]pyruvate. The contribution of pyruvate, lactate and glucose to the biosynthesis of ACh is considerably higher than the contribution of acetoacetate, 3-hydroxybutyrate and acetate; that of citrate and leucine is very low. No incorporation of label from [5-¹⁴C]glutamate into ACh was observed. Pyruvate appears to be the most important precursor of the acetyl group of ACh. The incorporation of label from [1, 5-¹⁴C]citrate into ACh was very low although citrate did enter the cells, was metabolized rapidly, did not interfere with the metabolism of ACh and the distribution of radioactivity from it in subcellular fractions of the brain was exactly the same as from [2-¹⁴C]pyruvate. It appears unlikely that citrate, glutamate or acetate act as transporters of intramitochondrially generated acetyl groups for the biosynthesis of ACh. Carnitine increased the incorporation of label from [1-¹⁴C]acetate into brain lipids and lowered its incorporation into ACh. Differences in the degree of labelling which various radioactive precursors produce in brain glutamine as compared to glutamate, previously described after intravenous, intra-arterial, or intraperitoneal administration, were confirmed using direct administration into the cerebrospinal fluid. Specific radioactivities of brain glutamine were higher than those of glutamate after injections of [1-¹⁴C]acetate, [2-¹⁴C]acetate, [1-¹⁴C]butyrate, [1,5-¹⁴C]citrate, [³H]acetylaspartate, [U-¹⁴C]leucine, and also after [2-¹⁴C]pyruvate and [4-¹⁴C]acetoacetate. The intracisternal route possibly favours the entry of substrates into the glutamine-synthesizing (‘small’) compartment. Increasing the amount of injected [2-¹⁴C]pyruvate lowered the glutamine/glutamate specific radioactivity ratio. The incorporation of ¹⁴C from [1-¹⁴C]acetate into brain lipids was several times higher than that from other compounds. By the extent of incorporation into brain lipids the substrates formed four groups: acetate > butyrate, acetoacetate, 3-hydroxybutyrate, citrate > pyruvate, lactate, acetylaspartate > glucose, glutamate. The ratios of specific radioactivity of ‘citrate’ over that of ACh and of glutamine over that of ACh were significantly higher after the administration of [1-¹⁴C]acetate than after [2-¹⁴C]pyruvate. The results indicate that the [1-¹⁴C]acetyl-CoA arising from [1-¹⁴C]acetate does not enter the same pool as the [1-¹⁴C]acetyl-CoA arising from [2-¹⁴C]pyruvate, and that the cholinergic nerve endings do not form a part of the acetate-utilizing and glutamine-synthesizing (‘small’) metabolic compartment in the brain. The distribution of radioactivity in subcellular fractions of the brain after the injection of [1-¹⁴C]acetate was different from that after [1, 5-¹⁴C]citrate. This suggests that [1-¹⁴C]acetate and [1, 5-¹⁴C]citrate are utilized in different subdivisions of the ‘;small’ compartment.     

Biosynthesis of Choline and Ethanolamine Phospholipids in Crithidia fasciculata*

The incorporation of radioactivity from [1,2-³⁴C]choline, [1,2-³⁴C]ethanolamine, [3-¹⁴C]serine and [methyl-¹⁴C]methionine into lipids was studied in growing cultures of Crithidia fasciculata. Lecithin was formed both from choline and by the methylation of phosphatidylethanolamine. Mono- and dimethylphosphatidylethanolamines were present in no more than trace amounts. Growth of the protozoa in media containing choline (1 mM) did not decrease synthesis by the methylation pathway. Phosphatidylethanolamine was formed from ethanolamine. Radioactivity from serine also was present in both phosphatidylethanolamine and lecithin; however, the presumed intermediate, phosphatidylserine, could not be detected.     

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.     

Metabolism of triacylglycerol in developing rat brain

Metabolism of triacylglycerol (TAG) in developing brain has been examined. TAG is a relatively minor fraction of brain lipid in both suckling and adult rats and cannot be accounted for as entrapped blood. When glycerol tri[1-¹⁴C]oleate and [2-³H]glycerol trioleate were simultaneously injected intracerebrally into suckling rats, both labels appeared in diacylglycerol and the major phospholipids; acyl chain label was incorporated more extensively at early time points, with choline phosphoglycerides being most actively labeled. With [1-¹⁴C]fatty acids and [2-³H] glycerol administration, the specific activity of TAG was much greater than that of the more abundant phospholipids. Although direct acyl exchange between TAG and phospholipids was not demonstrated, relationships of TAG to selective mechanisms of phosphoglyceride synthesis were indicated.Abbreviations used TAG triacylglycerol - DAG diacylcerol - HPLC high performance liquid chromatography - CoA coenzyme A - BSA bovine serum albumin - TLC thin layer chromatography - DPM disintegrations per minute - ATP adenosine triphosphate - GLC gas liquid chromatography - PC choline, phosphoglyceride - PE ethanolamine phosphoglyceride - PS serine phosphoglyceride - PI inositol phosphoglyceride     

Appearance of newly synthesized protein in myelin of young rats.

Abstract— Seventeen-day-old rats were injected intracranially with [³H]leucine, then sacrificed between 1 and 24 h. Myelin was prepared from the brains on discontinuous sucrose gradients and the proteins were separated by discontinuous gel electrophoresis in buffers containing sodium dodecyl sulphate. Proteins were stained with acid Fast Green and the distribution was quantitated by densitometry. The gels were then sliced and the radioactivity in each slice was determined. Between 1 and 24 h, the radioactivity in proteolipid protein increased from 18% to 37% of the total radioactivity in the proteins of isolated myelin. During this same period, the per cent distribution of radioactivity in basic and Wolfgram proteins remained constant while that in the remaining high molecular weight proteins decreased. Similar results were also obtained with [³H]glycine as a precursor. The relative specific activity of all of the myelin proteins increased between 1 and 6 h, then remained constant between 6 and 24 h. At 1 h, proteolipid protein reached only 25% of its maximal (6 h) relative specific radioactivity, while the other two proteins reached 50% of maximum. These results indicate a lag in the appearance of labelled amino acids in proteolipid protein relative to the other myelin proteins.