ISOLATION AND CHARACTERIZATION OF MYELIN-RELATED MEMBRANES

Abstract— Myelin related membrane fractions from rat brain and spinal cord were isolated from material normally discarded during standard myelin isolation procedures. A fraction which floated on 0.32 M-sucrose (F) and the material released after subjecting the myelin fraction to osmotic shock at two stages in the purification (W₁ and W₂) were characterized. These fractions were subjected to subfractionation on three step discontinuous sucrose gradients. Morphologically, the heavier subfrac-tions of W₁ and W₂ were shown to consist mainly of single membranes and vesicles. Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis showed that, relative to myelin, proteolipid and basic protein were reduced in all subfractions, while the high molecular weight proteins were increased. The specific activity of 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP) was up to 2-fold higher than that of myelin in the heavier subfractions of W₁ and W₂. The major myelin-associated glycoprotein was also increased in these subfractions as determined by periodic acid-Schiff staining. Differential centrifugation of the initial tissue homogenate to remove microsomes prior to myelin isolation gave rise to W₁ and W₂ subfractions with a CNP specific activity 3–4 times that of myelin. The high molecular weight proteins and glycoproteins were enriched in these microsome-depleted subfractions, but were qualitatively similar to those of myelin. Some of the membranes in these fractions may be derived from the continuum between the plasma membrane of the oligodendrocyte and compact myelin. Fraction F consisted of small membrane fragments and many vesicles, and was particularly deficient in proteolipid. The specific activity of CNP in fraction F was about the same as myelin, while the major myelin associated glycoprotein could not be detected. Fraction F from normal CNS tissue appears to be similar to the floating fractions previously isolated in larger amounts from pathological brain undergoing edematous demyelination.     

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.     

THE BIOSYNTHESIS AND CONCENTRATION OF GALACTOSYL DIGLYCERIDE IN GLIAL AND NEURONAL ENRICHED FRACTIONS OF ACTIVELY MYELINATING RAT BRAIN

—In continuation of our studies on the association of the galactosyl diglycerides of brain with myelination, we have measured the biosynthesis and concentration of these glyceride glycolipids, in oligodendroglial, astroglial, neuronal, and myelin enriched fractions from brains of rats of postnatal age 16, 19 and 29 days. The relative purity of cell fractions and myelin derived from 50 to 60 brains of each age-group was checked by phase contrast microscopy and 2′,3′-cyclic nucleotide-3′-phosphohydrolase activity. The relative purity was comparable to that reported by other investigators for cell fractions from bovine brain. Of the three cell types, the oligodendroglia had the highest and the neurons had the lowest capacity to enzymatically synthesize and to accumulate monogalactosyl diglyceride. The amount of monogalactosyl diglyceride found in myelin compared to that found in oligodendroglial fraction greatly increased during development between 16 and 29 days of age. The biosynthesis of galactosyl ceramide but not glucosyl ceramide was highest in oligodendroglial enriched cell fraction. However, ceramide glucosyl-transferase activity, which was greatly affected by the method used for cellular separation, was highest in a microsomal fraction derived from grey matter. Our results support the contention that the oligodendroglial cells are the site of synthesis of myelin constituents of the central nervous system, and that there is a temporal relationship between this site of synthesis and the site of deposition (myelin).     

Variation of proteins, enzyme markers and gangliosides in myelin subfractions

A discontinuous sucrose gradient was used to separate adult rat brain myelin into light, medium and heavy subfractions. Basic proteins decreased sharply, proteolipid potein changed very little, and high molecular weight proteins increased from the light to the heavy fraction. The concentration of monosialoganglioside G_M1 was the highest in the middle fraction. The amount of carbohydrate in the major myelin-associated glycoprotein per mg total myelin protein increased 3.5-fold from the light to the heavy fraction. 2′,3′-Cyclic nucleotide 3′-phosphohydrolase, which is related to myelin or the oligodendroglial membrane, and acetylcholinesterase, which is in neural membranes such as the axolemma, both increased between the light and the heavy fraction, although their relative distributions among the three fractions were different. The glycoprotein and 2′,3′-cyclic nucleotide 3′-phosphohydrolase had similar distributions suggesting that they were concentrated in similar locations, possibly in the loose myelin and oligodendroglial plasma membrane. Electron microscopic examination of the subfractions was consistent with this interpretation.     

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.     

Myelin-Associated Glycoprotein and Other Proteins in Trembler Mice

The myelin-associated glycoprotein (MAG) and other myelin proteins were quantitated in homogenates of whole sciatic nerve from adult and 20-day-old Trember mice. In the nerves of adult mice, the concentration of MAG was increased from 1.1 ng/micrograms of total protein in the controls to 1.4 ng/micrograms protein in the Tremblers. By contrast, the concentrations of P0 glycoprotein and myelin basic proteins were reduced to 27% and 20% of control levels, respectively. Immunoblots demonstrated that P2 was also greatly reduced in the Trembler nerves. The specific activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) was 65% of the control level. Immunoblot analysis showed that MAG had a higher than normal apparent Mr in the sciatic nerves of the Trembler mice, but its apparent Mr was normal in the brains of these mutants. In 20-day-old Tremblers, the P0 and myelin basic protein were reduced slightly less to about 40% of the level in the nerves of age-matched controls. CNP and MAG levels were not significantly different from those in controls, and MAG exhibited a shift toward higher apparent Mr similar to that in the adults. The maintenance of high MAG levels despite the severe deficit of myelin, as reflected by the decrease of the major myelin proteins, is consistent with the immunocytochemical localization of MAG in periaxonal Schwann cell membranes, Schmidt-Lantermann incisures, lateral loops, and the outer mesaxon and its absence from compact myelin. The abnormal form of MAG in the peripheral nervous system (PNS) of the Trembler mice may contribute to the pathology in this mutant.     

CHARACTERIZATION OF THE FRACTION OBTAINED FROM THE CNS OF JIMPY MICE BY A PROCEDURE FOR MYELIN ISOLATION

Abstract— A subcellular fraction (called the 0·85-fraction) was isolated from the brains of Jimpy mice by a procedure for obtaining myelin of high purity from immature normal brains. The yield of this fraction obtained from 17-day-old Jimpy mice was only 5 per cent of that from age matched controls. In the electron microscope, the O·85-fractions obtained from 9- and 17-day-old control mice showed many multilayered whorls of myelin, whereas the corresponding fraction from the Jimpy mice was free of multilayered structures which could be recognized as myelin. Basic proteins, proteolipid protein and galactocerebrosides could not be detected in the 0·85-fraction from Jimpy mice although they were major components of the 0·85-fractions from both 9- and 17-day-old control mice. The specific activity of 2',3'-cyclic nucleotide 3'- phosphohydrolase in the Jimpy 0·85-fraction was only 15 per cent of the value for controls. These results can be explained either by the 0·85-fraction from Jimpy brain being a very abnormal 'myelin' or by its being primarily non-myelin contaminants. Little or none of the major glycoprotein found in normal myelin fractions was found in the 0·85-fraction from Jimpy brains. This finding is strong evidence indicating that the glycoprotein is closely associated with normal myelin in situ.     

SUBCELLULAR DISTRIBUTION OF 5-HYDROXYTRYPTAMINE IN RAT BRAIN DURING DEVELOPMENT: EFFECT OF DRUGS AND FASTING

Abstract— Distribution of brain 5-HT content between the high-speed supernatant and particulate fractions under normal and experimental conditions was studied in postnatal and adult rats. In adult and 35-day-old rats the 5-HT content of the supernatant fraction was about 25% of that of the total homogenate and significantly higher than that in 1, 7 and 21-day-old rats. In 1-day-old rats fasting caused an increase of 100% in the turnover, 50% in the content and no alteration in the subcellular distribution of brain 5-HT, which suggests that under normal conditions 5-HT stores may be filled near to capacity. After 5-hydroxytryptophan administration, the 5-HT content of the adult rat brain increased 3-fold and that of the supernatant fraction to 35% of 5-HT content of the total homogenate. In postnatal rats, the brain 5-HT content rose to an adult level and the supernatant 5-HT percentage to a markedly higher than adult level, probably because of the known higher than adult 5-hydroxytryptophan decarboxylase activity of brain capillaries. Administration of tranylcypromine to adult rats caused a 2.6-fold increase of brain 5-HT content and a slight increase of the supernatant 5-HT percentage. At various times after the administration of the MAO inhibitors (tranylcypromine or pargyline) and fasting to the 1-day-old rats, brain 5-HT content increased 4, 5 and 7-fold, respectively, and the supernatant 5-HT rose consistently and, as in the adult, to about 30% of the 5-HT content of the total homogenate. After pargyline following reserpine pretreatment, the 5-HT content of the adult and 1-day-old rat brain increased to 2–3 times the control level and that of the supernatant fraction to about 40% of the 5-HT content of the total homogenate. The adult values for 5-HT in the particulate fraction of the 1-day-old rats after the drug treatments are in sharp contradiction to the low endogenous 5-HT content and known lack of nerve terminals and synaptic vesicles in their brains, and suggest that after MAO inhibition brain 5-HT neurons may bind the amine by some other mechanism than the Mg²⁺-ATP-dependent, reserpine-sensitive granular storage.     

Morphological and biochemical characterization of light and heavy myelin isolated from developing rat brain.

Myelin from developing rat brains was separated on a discontinuous sucrose gradient into subfractions of two different densities, i.e. light and heavy myelin. Electron photomicrographs showed that heavy myelin consisted primarily of large compacted multilamellar structures with a distinct intraperiod line characteristic of myelin in situ. Light myelin, on the other hand, was composed of small vesicles having a unilamellar structure. Similar to whole myelin, both membrane subfractions were highly enriched in 2',3'-cyclic nucleotide-3'-phosphohydrolase. The specific activity of the enzyme, however, showed no developmental trend. Both subfractions contained all the four major proteins characteristic of the whole myelin membrane. There were, however, quantitative differences in the relative distribution of these proteins between light and heavy myelin. Basic protein accounted for 55% and proteolipid protein for 46% of the total myelin proteins of light and heavy myelin, respectively. DM-20 (Agrawal, H.C., Burton, R. M., Fishman, M.A., Mitchell, R.F. and Prensky, A.L. (1972) J. Neurochem. 19, 2083-2089) exhibited a developmental "switch" between light and heavy myelin. Light myelin appeared to contain more DM-20 in 15- to 20-day-old rat brain, whereas the concentration of this protein was higher in heavy myelin at subsequent ages studied.     

Myelin changes induced by incubation of brain slices with serum

Forebrain and brain stem slices prepared from adult rats were incubated with pooled normal human serum. Following the incubation, the tissue was homogenized and the fraction floating on 0.32 M sucrose as well as two myelin subfractions (light and heavy) were isolated. Addition of serum into the incubation medium increased generation of the floating fraction by the cerebral slices. Changes in the myelin membrane were also observed. Thus, myelin isolated from forebrain slices revealed pronounced increase in the buoyant density of its particles and loss of basic protein. Furthermore, in spite of the intensive washing employed during the isolation procedure, some serum proteins were found firmly attached to the membraneous fractions. The demonstration of the myelin alterations in the living cerebral tissue exposed to serum during incubation may contribute to understanding the pathogenesis of multiple sclerosis.     

BIOCHEMICAL CHARACTERIZATION OF A MYELIN FRACTION ISOLATED FROM RAT BRAIN AGGREGATING CELL CULTURES

Abstract— Subcellular fractions isolated from rat brain aggregating cell cultures were studied by electron microscopy and showed the presence of typical myelin membranes. The chemical composition of purified culture myelin was similar to the fraction isolated from rat brain in terms of CNP specific activity, protein and lipid composition. The ratio of small to large components of myelin basic protein was comparable in culture and in vivo. These two proteins incorporated radioactive phosphorus. The major myelin glycoprotein was present and during development in culture its apparent molecular weight decreased although it never reached the position observed in myelin isolated from adult rats. In culture, the yield of myelin did not increase substantially between 33 and 50 days and was comparable to that of 15-day-old rat brain. The ratio basic protein to proteolipid protein resembled immature myelin and the cerebroside content was very low. A 'floating fraction' was isolated from the cultures and contained some myelin but mostly single membranes. Although these results indicate that myelin maturation is delayed in vitro this culture system provides substantial amounts of purified myelin to allow a complete biochemical analysis and metabolic studies during development.     

Morphological and biochemical characterization of light and heavy myelin isolated from developing rat brain

Myelin from developing rat brains was separated on a discontinuous sucrose gradient into subfractions of two different densities, i.e. light and heavy myelin. Electron photomicrographs showed that heavy myelin consisted primarily of large compacted multilamellar structures with a distinct intraperiod line characteristic of myelin in situ. Light myelin, on the other hand, was composed of small vesicles having a unilamellar structure. Similar to whole myelin, both membrane subfractions were highly enriched in 2′,3′-cyclic nucleotide-3′-phosphohydrolase. The specific activity of the enzyme, however, showed no developmental trend. Both subfractions contained all of the four major proteins characteristic of the whole myelin membrane. There were, however, quantitative differences in the relative distribution of these proteins between light and heavy myelin. Basic protein accounted for 55 % and proteolipid protein for 46 % of the total myelin proteins of light and heavy myelin, respectively. DM-20 (Agrawal, H. C., Burton, R. M., Fishman, M. A., Mitchell, R. F. and Prensky, A. L. (1972) J. Neurochem. 19, 2083–2089) exhibited a developmental “switch” between light and heavy myelin. Light myelin appeared to contain more DM-20 in 15- to 20-day-old rat brain, whereas the concentration of this protein was higher in heavy myelin at subsequent ages studied.     

QUAKING MOUSE: ISOLATION AND CHARACTERIZATION OF MYELIN PROTEIN

A new technique, involving final purification on a continuous CsCl gradient, was utilized for the isolation of cerebral myelin from adult (4- to 6-month-old) quaking mice, littermate controls and young (10-day-old) normal mice. The yield of myelin from either adult quaking or normal young mice was 5-10 per cent of that from adult controls. After deli-pidation, myelin proteins were separated by polyacrylamide gel electrophoresis in buffers containing sodium dodecylsulphate. Two gel systems were utilized: (1) a high-resolution discontinuous electrophoresis system; and (2) a continuous system utilizing gels cross linked with ethylenediacrylate (EDA). The gels from the discontinuous system were stained with Fast Green and quantified by densitometry. The base lability of the EDA-linked gels permitted direct chemical determination of protein in specific bands. Myelin from brains of normal adult mice contained, as major components, one proteo-lipid and two basic proteins. There were also a number of high-molecular-weight proteins which represented a significant portion of the total. Myelin from quaking mice had qualitatively a similar distribution of proteins but the high-molecular-weight fraction comprised a much greater percentage of the total protein. The ratio of basic to proteolipid protein in preparations from quaking mice was considerably higher than that in the myelin from control mice. The distribution pattern of the myelin proteins from 10-day-old mice was quantitatively similar to that of quaking mice. Altogether the evidence supports the hypothesis that the quaking mutant provides a model of an immature nervous system with respect to myelination.     

Carbonic Anhydrase, 5''-Nucleotidase, and 2'',3''-Cyclic Nucleotide-3''-Phosphodiesterase Activities in Oligodendrocytes, Astrocytes, and Neurons Isolated from the Brains of Developing Rats

The activities of three myelin-associated enzymes, carbonic anhydrase, 5'-nucleotidase, and 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNP), were measured in oligodendrocytes, neurons, and astrocytes isolated from the brain of rats 10, 20, 60, and 120 days old. The carbonic anhydrase specific activity in oligodendrocytes was three- to fivefold higher than that in brain homogenates at each age, and, at all the ages, low activities of this enzyme were measured in neurons and astrocytes. The oligodendrocytes and astrocytes from the brains of rats at all ages had higher activities of the membrane-bound enzyme 5'-nucleotidase than was observed in neurons. In oligodendrocytes from 10- and 20-day-old rats, the 5'-nucleotidase activity was two-to threefold the activity in the homogenates (i.e., relative specific activity = 2.0-3.0), and the relative specific activity of this enzyme in the oligodendrocytes declined to less than 1.0 at the later ages, concomitant with the accumulation of 5'-nucleotidase in myelin. The CNP activity was always higher in oligodendrocytes than in neurons, but not appreciably different from that in astrocytes from 20 days of age onward. The relative specific activity of CNP was highest in the oligodendrocytes from 10-day-old rats but was lower, at all ages, than we had observed in bovine oligodendrocytes. These enzyme activities in oligodendroglia are quite different in amount and developmental pattern from those reported previously for myelin.     

Protein and Glycoprotein Composition of Myelin Subfractions from the Developing Rat Optic Nerve and Tract

Abstract: The rat optic nerve and tract (representing a relatively homogeneous part of the CNS) were utilised for a detailed examination of the protein and glycoprotein composition of developing myelin membranes. Animals aged from 5 days through to adulthood were used. Myelin fractions could first be isolated from the nerve 8 days after birth and the yield increased until 60 days of age, before declining slightly to the adult level; a similar (but possibly slightly delayed) pattern was apparent for the optic tract. The homogeneity of optic nerve myelin (compared with that from brain and spinal cord) was demonstrated by zonal centrifugation on continuous sucrose-density gradients; myelin from both 20-day and adult animals exhibited narrow, Gaussian-like distributions, with 19–22% of the total myelin at the population modes. During development, the myelin density profile was shifted to a denser region of the sucrose gradients. Micro-polyacrylamide gel electrophoretic analyses of "light" and "heavy" myelin subfractions from both optic nerve and tract indicated that the gross developmental changes in protein composition were similar to those previously described for myelin prepared from larger CNS areas, particularly the forebrain. The glycoprotein components of the myelin fractions were stained directly on micro-gels using fluorescein isothiocyanate-labelled concanavalin A. The relative proportion of the major high-molecular-weight glycoprotein decreased rapidly during the early phases of myelination. A number of lower-molecular-weight glycoproteins were also apparent; the proportions of these varied during development and in light and heavy myelin subfractions, but definitive data are not available to determine whether they are components of the myelin sheath or of contaminating membranes.     

Developmental changes in the distribution of 3-hydroxy-3-methylglutaryl coenzyme A reductase among subcellular fractions of rat brain.

—The distribution of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) relative to that of several biochemical markers has been studied in subcellular fractions prepared from the brains of rats, aged 4 days to adult, by differential centrifugation. In the brains of 10-day-old animals fractions which sedimented at 800 g (P₁ and 9000 g (P₂) contained 28% and 65% respectively of the total reductase activity. A similar distribulion of the microsomal marker, NADPH-cytochrome c reductase, suggested that the HMG-CoA reductase activity in the low-speed pellets was due to substantial contamination of these fractions with endoplasmic reticulum. When P₂ was fractionated on a discontinuous sucrose gradient, the distributions of protein, RNA and NADPH-cytochrome c reductase paralleled that of HMG-CoA reductase, indicaling a non-specific association of endoplasmic reliculum and HMG-CoA reductase with all of the structures sedimenting in P₂. As brain maturation proceeded and a greater percentage of total brain protein (primarily associated with myelin) sedimenled in P₁, the subcellular distributions of HMG-CoA reductase and the microsomal marker changed in a parallel way. By 21 days P₁ contained nearly all of the reductase activity. Because the specific activity of HMG-CoA reductase in P₁ decreased steadily between 4 and 21 days, while the specific activity of 2′:3′-cyclic nucleotide 3′-phosphohydrolase in this fraction increased in a coordinate fashion, we conclude that the reductase is not an integral component of myelin, and probably is associated exclusively with the endoplasmic reticulum included in P₁. In view of the developmental changes in the distribution of HMG-CoA reductase among subcellular fraclions, we suggest that whole homogenates (or comparable tissue extracts) should be utilized to evaluate reductase activity in the developing brain.     

THE BIOSYNTHESIS OF CHOLESTEROL AND OTHER STEROLS BY BRAIN TISSUE

Abstract— The distribution of [¹⁴C]labelled material into subcellular fractions of 30-day-old rat brain was studied as a function of time, following intracerebral injection of [2-¹⁴C] mevalonic acid. As in the adult and 15-day-old brain, the microsomal fraction was indicated as the site of sterol synthesis. Unlike the 15-day-old animal, the myelin fraction from the 30-day-old rat was the predominately labelled fraction at 2 weeks after injection of the animal. Significant amounts of [¹⁴C]cholesterol were not present until about 4 h after injection. In order to ascertain whether different populations of cholesterol were being labelled, depending on the age of the animal injected, we compared the labelling of myelin and non-myelin components in animals injected at 15 or at 30 days of age, and sacrificed, respectively, from 14 to 29 days or from 1 to 28 days after injection. Our results indicated that there was an apparent shift of labelled sterol from non-myelin to myelin fractions at about 37–44 days of age.     

Fractionation of Isolated Rat CNS Myelinated Axons by Sucrose Density Gradient Centrifugation in a Zonal Rotor

Myelinated axons isolated from rat CNS brain stem by flotation in a buffered sucrose-salt medium were shocked by vigorous homogenization in hypotonie buffer and then fractionated on a 20-40% (wt/wt) linear sucrose gradient in a Beckman Ti-14 Zonal Rotor. After centrifu-gation to equilibrium, the gradient was fractionated on the basis of sucrose density into 13 individual fractions. The distributions of molecular markers related to myelin [(myelin basic protein, 2’3′-cyclic nucleotide 3′-phos-phodiesterase (EC 3.1.4.37), myelin-associated glycopro-tein (MAG)]; microsomes [CDP-choline:l,2 diglyceride cholinephosphotransferase (EC 2.7.8.2)]; mitochondria [cytochrome c oxidase (EC 1.9.3.1), monoamine oxidase (amine:oxygen oxidoreductase, deaminating, EC 1.4.3.4)], and axolemma [acetylcholinesterase (acetylcho-line hydrolase, EC 3.1.1.7), 5′-nucleotidase (5′-ribonu-cleotide phosphohydrolase, EC 3.1.3.5), Na⁺,K⁺-adeno-sine triphosphatase (EC 3.6.1.3), [³H]saxitoxin binding] were examined, as well as the protein composition and morphological appearance of the fractions. The myelin-related markers were most enriched in the 20-26% region of the gradient, although the MAG was broadly distributed throughout the entire gradient. The axolemma-related markers were most enriched in the 28-32% region of the gradient, whereas the microsomal and mitochondrial-related markers were enriched in the 35-40% region of the sucrose density gradient. Mixing experiments utilizing ¹²⁵I-labeled membrane preparations derived from cultured oligodendroglial and astroglial cells indicated that the constituents of the shocked myelinated axons were not significantly contaminated with glial membranes. The morphology of the fraction was consistent with the membrane molecular marker distribution: the light end of the gradient contained multilamellar myelin; fractions in the center of the gradient were enriched in un-ilamellar membrane fragments; the densest regions of the gradient were enriched in mitochondria. The myelin specific proteins were the prominent polypeptides in the 20-25% regions of the gradient, whereas polypeptides having a molecular weight of 50,000 or greater predominanted in the denser regions of the gradient. The significance of the distribution of these membrane markers and the utility of this fractionation procedure are discussed.     

Myelin glycosphingolipid/cholesterol-enriched microdomains selectively sequester the non-compact myelin proteins CNP and MOG

Plasma membranes are complex arrays of protein and lipid subdomains. Detergent-insoluble, glycosphingolipid/cholesterol-enriched micro-domains (DIGCEMs) have been implicated in protein sorting and/or as sites for signaling cascades in the plasma membrane. We previously identified the presence of DIGCEMs in oligodendrocytes in culture and purified myelin and characterized a novel DIGCEM-associated tetraspan protein, MVP17/rMAL (Kim et al. (1995) Journal of Neuroscience Research 42, 413–422). We have now analyzed the association of known myelin proteins with DIGCEMs in order to provide a better understanding of their roles during myelin biogenesis. We used four well-established criteria to identify myelin DIGCEM-associated proteins: insolubility in a non-ionic detergent Triton X-100 at low temperature (4°C), flotation of the insoluble complexes to low density fractions in sucrose gradients, and TX-100 solubilization at 37°C, or at 4°C following treatment with the cholesterol-binding detergent saponin. We demonstrate that these proteins fall into four distinct groups. Although all tested proteins could be floated to a low-density fraction, proteolipid protein (PLP), myelin basic protein (MBP) and myelin associated glycoprotein (MAG) were solubilized by the detergent extraction, and connexin32 (Cx32) and oligodendrocyte-specific protein (OSP) met only some of the criteria for DIGCEMs. Only the non-compact myelin proteins 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP) and myelin/oligodendrocyte glycoprotein (MOG) satisfied all four criteria for DIGCEM-associated proteins. Significantly, only ～40% of CNP and MOG were selectively associated with DIGCEMs. This suggests that they may have both non-active “soluble”, and functionally active DIGCEM-associated, forms in the membrane, consistent with current views that DIGCEMs provide platforms for bringing together and activating components of the signal transduction apparatus. We therefore propose that CNP and MOG may have unique roles among the major myelin proteins in signaling pathways mediated by lipid-protein microdomains formed in myelin.     

Cerebroside Sulfotransferase in Golgi-Enriched Fractions from Rat Brain

Abstract: Golgi-enriched fractions were prepared from brainstems of 17-day-old rats by first floating off myelin, then fractionating the remaining pellet by a series of differential and density gradient centrifugations in sucrose. Fractions enriched in Golgi membranes were recovered at 0.46/0.76 m and 0.76/0.87 m interfaces on the final sucrose gradient as indicated by morphology and the biochemical markers thiamine pyrophosphatase and [³H]fucose-labeled glycoprotein. Morphology of the two fractions indicated very little contamination with myelin lamellae; however, the presence of significant levels of 2', 3'-cyclic nucleotidase in the lighter fraction suggested a contribution from oligodendroglial or myelin-related membranes. Cerebroside sulfotransferase was highly enriched in the lighter Golgi-enriched fraction relative to the denser fraction, the post-34, 880 x g microsomes, and the myelin-like fraction. In contrast, ceramide galactosyl transferase was more evenly distributed among the fractions. Our results show a more highly localized distribution of sulfatide synthesis than of galactocerebroside synthesis, probably in Golgi membranes or oligodendroglia-related membranes with similar properties.