Distribution of lipid synthesizing enzymes, 2′,3′-cyclic nucleotide 3′-phosphodiesterase,and myelin proteins in rat forebrain subfractions during development

The distribution of UDP-galactose: ceramide galactosyltransferase (CGalT) was studied in subcellular fractions of rat forebrain during development using zonal centrifugation on linear gradients. Specialized subfractions: SN 1, a microsomal fraction, SN 4, a myelin-related fraction, and purified myelin were also used for this study. For comparison, two microsomal lipid synthesizing enzymes, a myelin-specific enzyme, 2,3-cyclic nucleotide 3-phosphodiesterase and myelin proteins were measured in the same subfractions. UDP-glucose: ceramide glucosyltransferase and cerebroside sulfotransferase were confined to microsomes. CGalT was ferase and cerebroside sulfotransferase were confined to microsomes. CGalT was localized in microsomes, but also in myelin and myelin-related fractions. The developmental change in distribution of CGalT in adult animals toward myelin containing fractions could indicate that the replacement of galactosylceramide in compact myelin could be carried out in close proximity to compact myelin (mesaxon, paranodal loops) rather than in the distant oligodendrocyte perikaryon.     

QUAKING MOUSE MYELIN: BIOCHEMICAL CHARACTERIZATION OF ZONAL GRADIENT SUBFRACTIONS

The brains of Quaking and littermate control mice were fractionated by differential and density gradient centrifugation into soluble, microsomal, myelin and related (SN 4) fractions. There were no apparent differences in protein composition between any Quaking and control fraction with the exception of myelin and SN 4. Analysis of CNP activity indicated that in Quaking animals a high proportion of the total activity was localized in microsomal fractions, while in controls a large percentage of activity was found in myelin and SN4; in contrast, there were no marhcd differences in the distribution of AChE activity between Quaking and control fractions. The yield of myelin isolated from Quaking animals was 3.6%, of that from controls by electron microscopy myelin fractions from both Quaking and controls consisted of compact myelin whorls. Zonal centrifugation on continuous sucrose gradients demonstrated that both control and Quaking myelin was distributed in a bell-shaped mode with peak densities at 0.66 0.68 and 0.71-0.75 M-sucrose, respectively. The specific activity of CNP was generally lower in mutant subfractions than in controls. Protein analysis revealed that there were similar qualitative trends between light and heay myelin subfractions from both mutant and control animals, although the levels of proteolipid and small basic proteins were substantially lower in all Quaking fractions. These results indicate that. although all mutant myelin subfractions are compositionally abnormal, the type of particle heterogeneity in Quaking myelin is similar to that observed in controls.     

Calcium ion stimulated endogenous protein kinase catalyzed phosphorylation of basic proteins in myelin subfractions and myelin-like membrane fraction from rat brain

Polypeptide composition and endogenous phosphorylation were investigated in the subfractions of rat brain myelin isolated by either discontinuous or continuous sucrose density gradient centrifugation of myelin. Similarly, a myelin-like membrane fraction (SN4) was also studied. Observations were made that strongly indicated the presence of a calcium-stimulated protein kinase in a highly purified myelin preparation and which exclusively phosphorylated myelin basic proteins of the membrane preparation. Adenosine cyclic 3',5'-phosphate (cAMP) stimulated kinase on the other hand was found to be considerably enriched in the myelin-like membrane fraction. Although this latter enzyme is also capable of phosphorylating the basic proteins, its effect was at least 5 times weaker compared to the calcium-stimulated myelin protein kinase. Within the gradient subfractions there appeared a close relation between the amount of basic proteins and their calcium-stimulated phosphorylation; a similar relationship, however, was not obtained in the case of cAMP-dependent phosphorylation of myelin basic proteins. The former (i.e., Ca2+-stimulated phosphorylation) was found to require a protein factor that functionally resembled calmodulin. The results thus raises an interesting possibility of the presence of calmodulin-like proteins and a calcium-stimulated protein kinase in adult myelin membrane from mammalian brain, both of which have been hitherto unrecognized constituents of myelin membranes.     

Characterization of sulphatide-containing lipoproteins in rat brain

—(1) Water-soluble [³⁵S]sulphatide is found in the 105,000 g supernatant (SN) of rat brain after intraperitoneal injection of Na₂³⁵SO₄. This labelled sulphatide has a density between those of free lipid and free protein. (2) Fractionation of SN by preparative acrylamide gel electrophoresis indicates that the [³⁵S]sulphatide is not distributed among all SN proteins, but is associated with certain specific proteins. One of the isolated [³⁵S]sulphatide-containing proteins appears homogeneous by analytical acrylamide gel electrophoresis at several pH values. (3) Comparison of the turnover of [³⁵S]sulphatide in microsomes, SN, and myelin indicates that these three subcellular fractions behave as distinct metabolic pools, which meet the requirements for a precursor-product relationship between microsomes and SN and between SN and myelin. (4) These results suggest that sulphatide, synthesized in the microsomes, is transported to the myelin membrane as water-soluble sulphatide containing Iipoproteins in SN.     

2'', 3''-Cyclic Nucleotide 3''-Phosphohydrolase and Lipids of Myelin from Multiple Sclerosis and Normal Brains

Abstract: A study of purified myelin samples from normal-appearing white matter of 10 multiple sclerosis (MS) brains was undertaken and the results were compared with 10 age-matched control brains. Statistical evaluations were carried out with Student's r-test for differences. In pathological samples the yield of myelin came to only two-thirds of the corresponding controls. Enzyme assays of the 2', 3'-cyclic 3'-phosphohydrolase revealed an obviously significant reduction of specific activity to one-half in MS myelins. In myelin the contents of protein, lipid classes as cholesterol, glycolipids and phospholipids did not differ significantly. No cholesterol esters or any lysophospholipid were detectable either in MS or in controls. Within the individual phospholipids the main components were in the same order, while a significant decrease of the acidic representatives and of sphingomyelin occurred. Analysis of the fatty acid pattern of phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylethanolamine (PE), including the aldehydes from the last, revealed quite similar values with no significant differences, except C22: 4 fatty acid in the PE fraction and C20: 1 fatty acid in PS, which were reduced in MS myelin samples.     

Subcellular distribution of 2′,3′-cyclic nucleotide-3′-phosphodiesterase in cat peripheral nerve

Subcellular distribution of 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase) in desheathed, saline perfused cat sciatic nerve is reported. CNPase specific activity was enriched in the total particulate (P₂) fraction and was low in the soluble (S₂) fraction. “Light-myelin” floating above the 0.60 M sucrose phase had the highest CNPase activity, 2.5-fold over the crude homogenate (CH). By contrast, enzyme activity in “heavy myelin” floating above the 0.85 M sucrose interface was equal to that of the CH and accounted for only 12% of total activity. CNPase activity in the membranes floating above the 0.25 and 0.60 and 0.85 M sucrose phases comprised nearly 70% of the total CNPase activity. The “light myelin” fraction floating above the 0.60 M sucrose accounted for approx. 51% of the total CNPase activity. SDS-PAGE of membranes individually harvested from above the 0.25 and 0.60 and 0.85 M sucrose phases revealed the presence of myelin-specific proteins (P₀, P₁; and P₂). Electron microscope examination demonstrated the presence of myelin in each membrane fraction. The results of this study show that the majority of CNPase activity is associated with “light myelin” in cat peripheral nerve.     

The identity of a myelin-like fraction isolated from developing brain

1. A myelin-like membrane fraction was isolated from developing rat brain by a new method. 2. The chemical composition and morphology of the fraction are described. 3. The myelin-like fraction is similar to myelin in characteristic enzyme activity but differs in the absence of basic protein and cerebrosides. No similarity to other subcellular fractions was observed. 4. It is suggested that the myelin-like fraction is a stage in the formation of compact myelin from glial plasma membrane. 5. ;Early' myelin consists of the myelin-like and compact myelin fractions from developing brain.     

UDP-galactose-ceramide galactosyltransferase in rat brain myelin subfractions during development.

The localization and activity of the enzyme UDP-galactose-hydroxy fatty acid-containing ceramide galactosyltransferase is described in rat brain myelin subfractions during development. Other lipid-synthesizing enzymes, such as cerebroside sulphotransferase, UDP-glucose-ceramide glucosyltransferase and CDP-choline-1,2-diacylglycerol cholinephosphotransferase, were also studied for comparison in myelin subfractions and microsomal membranes. The purified myelin was subfractionated by isopycnic sucrose-density-gradient centrifugation. Four myelin subfractions, three floating respectively on 0.55 M- (light-myelin fraction), 0.75 M- (heavy-myelin fraction) and 0.85 M-sucrose (membrane fraction), and a pellet, were isolated and purified. At all ages, 70--75% of the total myelin proteins was found in the heavy-myelin fraction, whereas 2--5% of the protein was recovered in the light-myelin fraction, and about 7--12% in the membrane fraction. Most of the galactosyltransferase was associated with the heavy-myelin and membrane fractions. Other lipid-synthesizing enzymes studied appeared not to associate with purified myelin or myelin subfractions, but were enriched in the microsomal-membrane fraction. During development, the specific activity of the microsomal galactosyltransferase reached a maximum when the animals were about 20 days old and then declined. By contrast the specific activity of the galactosyltransferase in the heavy-myelin and membrane fractions was 3--4 times higher than that of the microsomal membranes in 16-day-old animals. The specific activity of the enzyme in the heavy-myelin fraction sharply declined with age. Chemical and enzymic analyses of the heavy-myelin and membrane myelin subfractions at various ages showed that the membrane fraction contained more proteins in relation to lipids than the heavy-myelin fraction. The membrane fraction was also enriched in phospholipids compared with cholesterol and contrined equivalent amounts of 2':3'-cyclic nucleotide 3'-phosphohydrolase compared with heavy- and light-myelin fractions. The membrane fraction was deficient in myelin basic protein and proteolipid protein and enriched in high-molecular-weight proteins. The specific localization of galactosyltransferase in heavy-myelin and membrane fractions at an early age when myelination is just beginning suggests that it may have some role in the myelination process.     

CHARACTERIZATION AND PROTEIN ANALYSIS OF MYELIN SUBFRACTIONS IN RAT BRAIN: DEVELOPMENTAL AND REGIONAL COMPARISONS

—Myelin preparations from the whole brains of 16-day-old rats and from cortical regions and brainstem, respectively, of 40-day-old rats were separated into light, medium and heavy subfractions on a discontinuous sucrose gradient by a procedure previously used for whole adult rat brain (Matthieu, et al., 1973). The total dry weight of myelin recovered from the 16-day-old rats was only 2·4mg/g fresh brain in comparison to 20 mg from adult brains. In 16-day-old rat brains, the percentage of the total myelin protein in the light fraction was higher than that found in adult brains; the percentage in the medium fraction was only one-third that in adults; while the percentage in the heavy fraction was about the same at both ages. The heavy fraction from the 16-day-old rats contained less basic protein and proteolipid than the light fraction, and the levels of the 2′3′-cyclic nucleotide 3′-phosphohydrolase (CNP) and glycoprotein were less than half those in the light and medium fractions. Double labelling experiments with radioactive fucose indicated that the major labelled glycoprotein in the heavy and medium fractions had a slightly higher apparent mol. wt than that in the light fraction. Electron microscopy showed much readily identifiable, compact myelin in the light and medium fractions from the 16-day-old rats, whereas the heavy fraction contained more single membranous structures and much less multilamellar myelin. The yield of myelin/g fresh wt from brainstem of 40-day-old rats was 4-fold higher than from cortical regions, and the percentage recovered in the light fraction was greater in the brainstem. In both regions basic proteins decreased from the light to the heavy fraction, whereas high mol. wt proteins, the glycoprotein and CNP increased. The biochemical and morphological results suggest that in both 16-day-old and young adult rats the light fraction is enriched multilamellar, compact myelin. In contrast, the heavy fraction at both ages is enriched in loose, uncompacted myelin and myelin-related membranes, although the heavy fraction from 16-day-old rats also may be substantially contaminated with membranes which are unrelated to myelin.     

Synthesis and turnover of cerebroside sulfate of myelin in adult and developing rat brain

The turnover of cerebroside sulfate (sulfatide) was followed in both microsomal and myelin fractions of developing and adult rat brains after an intracerebral injection of Na(2)(35)SO(4). In the adult rats, the specific radioactivity of sulfatide of the microsomal fraction reached a maximum 12 hr after the injection, and after 3 days it was reduced to less than 30% of the maximum. In contrast, the specific radioactivity of the myelin sulfatide did not reach a peak until 3 days after the injection and remained essentially at the same level for as long as 6 months. In the case of 17-day-old rats, the specific radioactivity of myelin sulfatide reached a maximum level around 12 hr after the injection and then appeared to decline. The decline was most marked 2-6 days after the injection, suggesting an apparently rapid turnover of myelin sulfatide. When a correction was made for deposition of newly formed sulfatide, the results indicated that the turnover of myelin in the developing animals was also relatively slow. In vitro experiments with purified myelin and 3'-phosphoadenosine-5'-[(35)S]phosphosulfate showed that myelin does not catalyze the galactocerebroside sulfotransferase reaction. This enzyme was found mainly in the microsomal fraction. In vivo studies suggested that a transfer of sulfatide molecules from the endoplasmic reticulum to myelin might occur. In order to obtain direct evidence for such a transfer, rat brain slices after pulse labeling with Na(2)(35)SO(4) were washed free of the isotope and reincubated with nonlabeled Na(2)SO(4). The specific radioactivity of the microsomal sulfatide declined, with a concomitant rise in the specific radioactivity of the myelin sulfatide. These observations are therefore consistent with the postulate that myelin sulfatide is probably synthesized in the endoplasmic reticulum.     

Enzyme activity and composition of myelin and subcellular fractions in the developing rat brain

1. Subcellular fractions and myelin were isolated from developing and adult rat brain. 2. Measurements of chemical composition and enzyme activities indicate the presence of a second myelin-like fraction mainly in the brain of developing rats. 3. This membrane fraction has a different lipid composition from myelin, but resembles myelin in its content of phosphohydrolase and aminopeptidase activity. 4. It is suggested that the second myelin-like fraction may be a submicrosomal contaminant or it may be derived from oligodendroglial plasma membrane during myelinogenesis.     

Neurochemical Characteristics of Myelin-like Structure in the Chick Retina

Abstract: Certain characteristics of myelin-like structures in the chick retina were examined morphologically and biochemically. Developmental changes of 2', 3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) in the chick retina and optic nerve were examined. The measurable activity in the retina was first detected at 16 days of incubation and thereafter, it increased rapidly until 4 weeks post-hatching. By contrast, CNPase activity in the optic nerve reached the maximum level at 4 days post-hatching and maintained a constant level thereafter. The purifed myelin fraction from the chick retina showed higher activity of CNPase, whereas its activity in the retinal homogenate was very low. Hence, it was considered that the myelin fraction from the chick retina is similar to that of CNS myelin with respect to CNPase. Protein profiles of the purified myelin fractions isolated from the chick optic tectum, optic nerve, retina and sciatic nerve were analysed by SDS-polyacrylamide gel elec-trophoresis. Myelin fractions from the chick optic tectum and optic nerve contained basic protein (BP) and Folch-Lees proteolipid protein (PLP). Myelin fraction from the chick sciatic nerve contained BP, P2 and two glycoproteins (PO and 23K). In contrast, retinal myelin fraction contained only BP. PLP, PO, 23K and P2 proteins were definitely undetectable. Electron micrographs revealed that some axons in the optic nerve fiber layer of the chick retina were wrapped by a spiral-structured myelin-like sheath, which showed some differences from those of CNS and PNS myelin sheaths. It was suggested that the origin of the myelin-like structure in the chick retina is other than from oligodendroglia or Schwann cells.     

Localization of 2',3'-cyclic nucleotide-3'-phosphohydrolase of rabbit brain by sedimentation in a continuous sucrose gradient.

Abstract— The enzyme 2′,3′-cyclic nucleotide-3′-phosphohydrolase (CNP) has been assayed in fractions from a continuous sucrose density gradient zonal centrifugation of rabbit brain homogenates. Basic protein (BP) was also assayed by a radioimmunomethod. Fractions were examined by SDS-polyacrylamide gel electrophoresis and by electron microscopy. These studies show that the major membrane fractions in the gradient differ greatly in the content of CNP and BP, and of high molecular weight proteins (HMW). The lightest membrane fractions contained numerous multilamellae, the highest content of BP and the lowest content of CNP and HMW, while the heaviest membrane fractions contained single membrane fragments and vesicles of unknown origin, the lowest content of BP and the highest content of CNP and HMW. The fraction containing the largest amount of membrane measured by turbidity, protein content, and water-washed dry weight contained only half the CNP specific activity of a denser fraction in the gradient. CNP specific activity in the lightest fractions was insignificant compared to that of denser fractions. Thus, we conclude that this enzyme may be absent from the typical multilamellar myelin structures but present in the single-membrane structures associated with myelin, such as the glial membrane and the paranodal segments of myelin adjacent to the axon. BP appears to occupy the opposite positions, highest in the multilamellae and lowest in the single-membrane structures of myelin. These studies do not exclude the possibility that CNP may not be bound to myelin membranes, but rather to a membrane of different origin. Evidence that this enzyme is a myelin-marker enzyme is circumstantial. Our evidence indicates the enzyme could be present either in a unique portion of myelin membranes or in another membrane structure.     

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.     

Study of a floating fraction obtained during preparation of myelin from degenerating goldfish optic tract

A floating fraction that layers on top of 0.25 sucrose has been obtained during the preparation of myelin from intact and 9 day degenerating goldfish optic tracts. The proportion of total tract protein isolated in floating fraction rises from 6.6% to 11.0% during degeneration. This increase is paralleled by a morphologically observed splitting of myelin lamellae. Floating fraction contains all of the major myelin proteins but shows a 40% increase in the proportion of basic protein and a 2–3 fold decrease in the proportion of IP proteins (intermediate molecular weight glycoproteins) and a 36 Kd (X) protein. The lipid to protein ratio is slightly higher in floating fraction than myelin. Lipid composition is characterized by 1/2–1/3 the myelin levels of galactolipids and twofold increased levels of triglycerides and cholesterol esters. Electron microscopy of floating fraction shows a mixture of myelin fragments with few lamellae and single membrane fragments. Taken together the results indicate that floating fraction in the degenerating goldfish optic tract is at least partially derived from the breakdown of myelin.     

Immunohistochemical Localization of 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase and Myelin Basic Protein in the Chick Retina

A GTP-dependent regulatory component of adenylate cyclase was found in myelin from rat brain. The fraction solubilized from myelin contained a component that reconstituted guanine nucleotide-responsive adenylate cyclase activity when combined with the catalytic unit of adenylate cyclase prepared from rat brain. Purified myelin demonstrated little adenylate cyclase activity, even in the presence of F- or Mn2+. The reconstituted activity was dependent on the amount of the solubilized myelin fraction and required the presence of 5'-guanylylimidodiphosphate, a hydrolysis-resistant analog of GTP. The elution pattern of the component solubilized from myelin in gel filtration was very similar to that of a GTP-dependent regulatory component from synaptic plasma membranes. The content of the regulatory component-like activity in myelin was estimated to be 50-60% of that in synaptic plasma membranes. Cholera toxin ADP-ribosylated proteins having molecular weights of 48,000, 38,000, 23,000, 20,000, and 15,000 and other minor peptides in myelin, some of which were also present in synaptic plasma membranes. We conclude that myelin contained a GTP-dependent regulatory component of adenylate cyclase despite the apparent lack of adenylate cyclase activity in myelin.     

Incorporation of injected [32P]phosphate into the phosphoinositides of subcellular fractions from young rat brain

1. Six subcellular fractions were prepared from the brain of 3-week-old rats after the injection of [(32)P]orthophosphate into the subarachnoidal space. The fractions were characterized by chemical and enzymic determinations and by electron microscopy. 2. The highest concentration of phosphoinositides was found in the microsomal fraction. 3. After an exchange period of 4hr. in vivo, the specific radioactivity of phosphatidylinositol was high in the nuclear, mitochondrial and supernatant fractions, and that of diphosphoinositide was high in the nuclear, microsomal and supernatant fractions. Triphosphoinositide specific activity was highest in the myelin fraction. 4. Specific activities (counts/100sec./mug. of P) were in the following order for all fractions except myelin: diphosphoinositide>phosphatidylinositol>triphosphoinositide. For myelin the order was: diphosphoinositide>triphosphoinositide>phosphatidylinositol. 5. Phosphatidylinositol extracted from a tissue fraction by acidified chloroform-methanol had roughly twice the specific activity of that extracted by neutral solvents. The same applied to diphosphoinositide.     

EFFECTS OF DIFFERENT DIETARY LEVELS OF ESSENTIAL FATTY ACIDS ON THE FATTY ACID COMPOSITION OF ETHANOLAMINE PHOSPHOGLYCERIDES IN MYELIN AND SYNAPTOSOMAL PLASMA MEMBRANES

Abstract— Three dietary levels of essential fatty acids (EFA), 3 0, 0 75 and 0 07 calorie-% were fed to rats for two generations or more. Myelin was isolated at the ages of 18, 30, 45 and 120 days and synaptosomal plasma membranes at 18, 30 and 45 days. No difference was found in the lipid composition between the dietary groups in either subcellular fraction. The fatty acid patterns of ethanolamine phosphoglycerides (EPG) were analysed. In myelin the proportions of 18:1 and 20:1 increased with age, while those of 20:4 (n-6) and 22:6 (n-3) decreased, in synaptosomal plasma membranes the proportions of 20:4 (n-6) decreased with age, but 22:6 (n-3) increased and the sum of the polyunsaturated fatty acids was constant. At no age were significant differences found between the proportions of saturated and monounsaturated fatty acids, in either myelin or the synaptosomal plasma membrane fraction, when the different dietary groups were compared. In myelin from rats fed 007 calorie-% EFA the proportions of 20:4 (n-6) were slightly lower than in the two other groups, while those of 22 6 (n-3) were considerably lower. The synaptosomal plasma membranes fraction of rats fed O-07 calorie-% EFA had equal or slightly larger amounts of 20:4 (n-6) than in the two other groups, while 22:6 (n-3) was considerably smaller. In both subcellular fractions the decreased proportion of fatty acids of linoleic and linolenic acid series was compensated for by an increase in 20:3 (n-9) and 22:3 (n-9). The sum of these two fatty acids was equal in the EPG of myelin and synaptosomal plasma membranes at 18 days of age. At 30 and 45 days of age a lower value was found in the synaptosomal plasma membranes, while in the myelin fraction a slight decrease was found only at 120 days of age.     

SIMULTANEOUS ISOLATION OF PURIFIED MICROSOMAL and MYELIN FRACTIONS FROM RAT SPINAL CORD

Abstract— The fraction that sediments between 2 × 10⁵ g -min and 6 × 10⁶ g -min from dilute dispersions of rat brain in 0.32 m -sucrose is a microsomal fraction with very little contamination by myelin. A crude microsomal fraction prepared in the same way from rat spinal cord contains more myelin than microsomes. Centrifugation of the crude microsomal fraction in 0.85 m -sucrose gave a floating fraction, an infranatant fraction (purified microsomes) and a small pellet. The purified microsomes contained very little myelin as judged by electron microscopy and polyacrylamide gel electrophoresis. The lipid composition resembled that of spinal cord myelin except that the purified microsomes contained relatively less cholesterol and ethanolamine plasmalogens. The content of galactolipids was much greater in spinal cord microsomes than in brain microsomes. The spinal cord CDP-ethanol-amine:diglyceride ethanolaminephosphotransferase activity (EC 2.7.8.1) was concentrated in the purified microsomes.
A spinal cord myelin fraction isolated from the 2 × 10⁵ g -min pellet was quite pure as judged by electron microscopy, enzyme activities and polyacrylamide gel electrophoresis. No NADPH-cyto-chrome c reductase activity (EC 1.6.2.3) could be detected in the purified myelin. The ethanolaminephosphotransferase specific activity was about 5% of that found in the purified microsomal fraction. The protein content was 25% by weight for spinal cord myelin and 31% for brain myelin. Of the total spinal cord 2',3'-cyclic nucleotide-3'-phosphohydrolase activity, 16% was lost from the crude myelin during purification, 21% was recovered in the purified myelin, and 11% was found in the floating fraction from the crude microsomes. The purified myelin and microsomal fractions from spinal cord were relatively pure. Additional myelin was recovered in the floating fraction from the crude microsomes.     

Protein determinants of myelination in different regions of developing rat central nervous system.

Measurements of several different protein determinants correlated with the time and rate of myelination in five areas of the central nervous system are presented. The deposition of protein in the subcellular fraction corresponding to the density of adult myelin, the appearance of basic protein characteristic myelin, the change in proportions of the individual myelin proteins, the appearance and distribution of the myelin marker 2':3'-cyclic nucleotide3'-phosphohydrolase, and the results of morphological studies of purified myelin are compared. According to these various criteria, and in agreement with the morphological observations of others, myelin appears earliest in the spinal cord, then in the brain stem, and latest in the cerebral hemispheres. Multilamellar myelin was observed in the rat brain stem and spinal cord as early as 5 days of age. The relative proportion of the individual myelin proteins changed with myelin maturation in all areas, with the larger basic protein decreasing reciprocally with increase of the smaller basic protein. The proportion of Wolfgram protein also decreased with maturation. Larger proportions of the enzyme 2':3'-cyclic nucleotide 3'-phosphohydrolase were located in the microsomal fraction at early ages. During development the enzyme activity gradually became associated more with a fraction of a density corresponding to adult myelin, suggesting the presence of precursor membrane fragments in microsomal fractions in the early stages of myelination before compact myelin formation. A significant proportion of the total nucleotide phosphohydrolase activity of the homogenate could not be recovered in subcellular fraction at early ages, but the recovers of the enzyme increased with maturation and the activity was found more in the myelin fraction.