ASSOCIATION OF AXONALLY TRANSPORTED PROTEINS WITH GOLDFISH BRAIN MYELIN FRACTIONS

—The presence of rapidly transported axonal proteins in purified preparations of myelin has been investigated in the goldfish visual system. Fish were injected intraocularly with ³H proline and contralateral optic tecta were pooled 8–12 h later for purification of myelin. Three purification procedures were employed using continuous and discontinuous gradients of sucrose and continuous gradients of CsCl. All of the myelin preparations were found to have physical, chemical and enzymatic properties attributable to relatively pure preparations of myelin. The goldfish myelin differed from mammalian preparations in having a slightly lower density and in containing an additional major protein of approx. 45,000 mol. wt. All of the myelin preparations retained relatively high levels of axonally transported radioactivity with specific radioactivities which ranged from 70 to 80 per cent of that of the whole tectal homogenate. Acrylamide gel analysis showed the myelin-associated radioactivity to be confined to the higher molecular weight proteins with very little radioactivity associated with basic protein or proteolipid protein. Both the axonally transported radioactivity and the group of higher molecular weight proteins were found to be more concentrated in a myelin subfraction of relatively high density than in a subfraction of low density. The possible significance of the association of axonally transported proteins with myelin is discussed.     

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.     

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.     

Maternal deficiency of protein or protein and calories during lactation: effect upon CNS myelin subfraction formation in rat offspring.

The present study has examined the effects of maternal protein and protein-calorie deficiency during lactation on the development of CNS myelin subfractions in rat offspring. The offspring of both the protein and protein-calorie deficient rats had decreased brain and body weights, as well as delayed CNS myelination. Delayed active CNS myelination was demonstrated by the fact that 53-day-old nutritionally stressed pups incorporated significantly more [³H]leucine and [¹⁴C]glucose into all myelin subfractions than age-matched controls. Delayed myelination was also supported by the tremendous accretion of myelin proteins in the nutritionally deprived pups between 25 and 53 days of age. Despite the delayed active synthesis of myelin, the myelin deficit persisted in the offspring of protein deficient rats. These offspring had a deficiency of light + medium myelin throughout the study. At 17 days, both groups of nutritionally stressed rats had an excess of the high molecular weight proteins in heavy myelin. Heavy myelin from 17 day offspring of protein-calorie deficient rats had a deficiency of basic proteins, while that from the offspring of protein deficient rats had a deficiency of proteolipid protein. The protein composition of all myelin subfractions was normal at 53 days.     

ANOMALIES OF MYELIN-ASSOCIATED GLYCOPROTEINS IN''QUAKING MICE

Abstract— Proteins and glycoproteins in a myelin fraction isolated from Quaking mutant mice were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and stained with Fast Green or with periodic acid-Schiff reagents. Double labelling experiments with [³H]fucose and [¹⁴C]fucose were also used to compare glycoproteins in myelin from the mutant mice with those from control mice. In the myelin fraction from the Quaking mice the basic proteins and proteolipid protein were decreased relative to the high molecular weight proteins. Some glycoproteins which are present in small amounts in myelin from normal mice were increased relative to the major glycoprotein in the myelin fraction of the Quaking mice. Furthermore, the major myelin-associated glycoprotein was shifted toward higher apparent molecular weight in comparison with controls of the same age or even with 9-day-old controls. The abnormal glycoproteins in the mutant myelin fraction could be a factor in the impairment of myelination.     

KINETICS OF ENTRY OF PROTEINS INTO THE MYELIN MEMBRANE1

Brain slices were prepared from 17-day old rats, and incubated with [³H]glycine or [³H]-leucine to label proteins. Myelin was isolated from the slices, and the proteins were separated by discontinuous gel electrophoresis in buffers containing sodium dodecyl sulfate. Radioactive basic and Wolfgram proteins appeared in myelin at similar initial rates, and their entry was nearly linear between 15 and 120 min with no detectable lag. Radioactive proteolipid protein appeared in myelin at one-fourth the rate of the basic and Wolfgram proteins between 0 and 30 min, then entered at a rate comparable to the other proteins between 45 and 120 min. When cycloheximide (0.2 mM) or puromycin (1.0 mM) was added, appearance of newly labeled basic and Wolfgram proteins in myelin stopped while proteolipid protein continued to appear in myelin at a normal rate for at least 30 min. Chase experiments with unlabeled glycine had similar effects. These results indicate the existence of a previously synthesized precursor pool of proteolipid protein with a 30-min interval between synthesis of proteolipid protein and its appearance in myelin. Incorporation of [³H]fucose into glycoprotein of the myelin sheath was studied, as was inhibition of incorporation of radioactivity by the use of either cycloheximide, or dilution with unlabeled fucose. The results indicated fucosylation of a sizable pool of presynthesized protein and a delay of 30 min between fucosylation of these polypeptides and their subsequent appearance in myelin as glycoproteins.     

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

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

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.     

Metabolism of three subfractions of myelin in developing rats.

Purified myelin, isolated from rat brain, was subfractionated into light, medium and heavy myelin. The metabolism of [³H] leucine in myelin subfractions was studied at intervals from 1 to 24 hours and from 18 hours to 85 days after the injection of 12-day-old rats. The metabolism of [¹⁴C] glucose in myelin subfractions was also examined during the 85 day interval. In addition, the development of each of these subfractions, as reflected by protein accretion, was determined.Between 13 and 97 days of age, the amount of the three myelin subfractions increased 10- to 44-fold. At 13 days of age the heavy subfraction accounted for the greatest percentage of myelin protein. However, beyond 13 days, light myelin predominated.The total ³H-radioactivity in the light, medium and heavy subfractions increased throughout most of the 85 day interval examined. The ³H specific radioactivity (³H dpm/μgram protein) of light myelin peaked at 12 hours after injection. The specific radioactivity of both ³H and ¹⁴C (¹⁴C dpm/μgram lipid) in light myelin declined beyond the initial time point in the long term (18 hour – 85 day) study. In contrast, the specific radioactivity of both ³H and ¹⁴C peaked in the medium and heavy subfractions at 4 days after injection of radioactive precursor.The possible existence of a membranous precursor to myelin is discussed.     

PROTEIN AND GLYCOPROTEIN COMPOSITION OF MYELIN AND MYELIN SUBFRACTIONS FROM BRAINS OF ''QUAKING'' MICE

Abstract— Quaking mutants in mice are known to be affected by an arrest of myelinogenesis and to have a purified myelin which is more dense than that of controls. Their myelin has been shown to demonstrate a striking decrease in proteolipid protein, a lesser decrease in the small myelin basic protein and changes in glycoproteins comprising reduction in the major peak and shift of this peak towards a higher apparent molecular weight. The possibility that these findings might reflect merely contamination of myelin with other membranes was tested by subfractionation. Light myelin (floats on 0.62 m -sucrose) is generally accepted as more compact and mature than the heavier subfraction (floating on 0.85 m -sucrose). The changes previously found were present in both subfractions and even more marked in the light myelin. These results indicate that the anomalies of myelin proteins and glycoproteins were not caused by contaminants and are present in compact myelin as well as in membranes which are transitional between the glial plasma membrane and the myelin sheath. Therefore, we suggest that the Quaking mutation results in dysmyelination rather than hypomyelination.     

Stoichiometric relation of protein components in cerebral myelin from different species

Abstract— In cerebral myelin from man, ox, rabbit, guinea pig and chicken, the amounts of proteolipid protein, basic protein and the fraction of further protein components were found to be present in a fixed ratio of 5·0: 3·5: 2·0 by weight. The molecular weights of 25,000 and 35,000 as obtained for the basic protein and proteolipid protein might indicate that cerebral myelin contains one molecule of basic protein per molecule of proteolipid protein. This fixed ratio of protein components was found to be changed in myelin from the PNS and in cerebral myelin from rat and carp, with their exceptional basic proteins. Using the polyacrylamide-gel electrophoresis it was possible to demonstrate that a homogeneous structural protein (the Folch-Lees proteolipid protein) constitutes about 50 percent of the total amount of myelin proteins in all species studied. An attempt was made to correlate myelin protein and lipid patterns from various species.     

METABOLIC ACTIVITY OF CNS PROTEINS IN RATS AND MONKEYS WITH EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS (EAE)

—The metabolic activity of proteins from myelin and non-myelin fractions of slices of lesions in monkey brains and in spinal cords of Lewis rats with acute experimental allergic encephalomyelitis was investigated using [1-¹⁴C]leucine as a protein precursor. The uptake in vitro of [1-¹⁴C]leucine into the monkey EAE lesions was greatly increased in both the myelin and non-myelin fractions. Similar findings were made in spinal cord slices of the EAE rat with an average specific activity 341 per cent of control measured in proteins of purified myelin and 415 per cent of control in the non-myelin protein. The increased uptake appeared with the onset of paralytic symptoms 10–14 days after injection. The increased uptake did not appear to be a result of an increased amino acid pool size as measured with uniformly labelled l -leucine, valine, arginine and phenylalanine. The increase in specific activity of the myelin protein of the EAE rats was shown to be associated with the peaks characteristic of myelin protein when separated on polyacrylamide gels and the serial slices counted. Most of the radioactivity of both the control and EAE myelin protein migrated with the high molecular weight fraction, and the largest increase in radioactivity in myelin protein appeared in this fraction. Some increase in specific activity was also found in the basic and proteolipid proteins. Four different guinea-pig antigens were used to induce EAE: whole spinal cord, purified basic protein, purified myelin and basic protein + cerebroside. All caused paralytic symptoms and greatly increased incorporation in vitro of [1-¹⁴C]leucine into spinal cord proteins. The incorporation of [1-¹⁴C]leucine into slices of the inguinal and popliteal lymph nodes of the EAE and Freund's adjuvant control rats were measured and compared with the incorporation into the spinal cord non-myelin fractions. The specific activity of lymph node proteins was of the order of 10 × that of the non-myelin protein of the control spinal cord. Invasion of a moderate number of cells of the order of activity of these lymph nodes could account for the large increase in rate of protein synthesis in the EAE nervous tissue. It is concluded that much of the increased protein synthesis could be due to the inflammatory cells, although a small amount of the total increase appears to be associated with myelin protein. Other changes in metabolism of the CNS tissue of the EAE rat include a lower rate of lipid synthesis and a decreased activity of the tricarboxylic acid cycle.     

In vivo acylation of proteolipid protein and DM-20 in myelin and myelin subfractions of developing rat brain: Immunoblot identification of acylated PLP and DM-20

The acylation of proteolipid protein (PLP) was examined in myelin and myelin subfractions from rat brain during the active period of myelination. Proteolipid protein and DM-20 in myelin and myelin subfractions were readily acylated in developing rat brain 22 hours after intracerebral injection of [3H]palmitic acid. No differences in the relative specific activity of PLP in myelin from 9-, 15-, and 30-day-old rat brains was observed; however, the relative specific activity of PLP in the heavy myelin subfraction tended to be higher than that in the light myelin subfraction. The acylation of PLP was confirmed by fluorography of immuno-stained cellulose nitrate sheets, clearly establishing that the acylated protein is in fact the oligodendroglial cell- and myelin-specific protein, PLP. Since PLP is acylated in the 9-day-old animal, when little compact myelin is present, it is possible that the acylation of PLP is a prerequisite for the incorporation of this protein into the myelin membrane.     

ARE MYELIN PROTEINS SYNTHESIZED IN RETINAL GANGLION CELLS?1

—We studied the incorporation of radioactivity into individual proteins of myelin by sodium dodecyl sulfate polyacrylamide gel electrophoresis after the injection of [³H]tryptophan into the right eye of developing rabbits. We found that the specific activity of basic protein (c.p.m./mg of basic protein) and the specific activity of DM-20 and proteolipid protein (c.p.m./mg total myelin protein applied to the gel) did not approach the ratio predicted by decussation of the fibres of the rabbit optic nerve. The specific activity of Wolfgram protein, however, approached an expected ratio of 15:1. We therefore concluded that myelin basic protein, DM-20 and proteolipid protein were probably not synthesized in retinal ganglion cells.     

Lipid and Protein Alterations of Spinal Cord and Cord Myelin of Multiple Sclerosis

Abstract: A comprehensive study was carried out to clarify the chemical compositions of spinal cord, cord myelin, and myelin subfractions of multiple sclerosis (MS). The protein compositions of normal-appearing cerebral white matter and cerebral plaque and periplaque tissues were also analyzed for comparison. MS whole cord samples were found to contain higher amounts of water compared with normal samples. The total lipid contents were below normal. Among the individual lipids, cholesterol content remained unchanged, whereas cholesteryl esters appeared increased in MS cords. The acidic phospholipid concentrations were found to be lower than normal. Glycolipids, such as cerebrosides G_M4, G_M1, and G_D1b, which are abundant in myelin, were all decreased. However, the concentrations of G_M3 and G_D3, which are more characteristic of reactive astrocytes, were highly elevated. The total protein content of MS cord samples was decreased, and the decrease was attributable to the loss of myelin proteins as evidenced by the low recovery of myelin. The concentrations of myelin-specific proteins, such as proteolipid protein and myelin basic protein, were significantly reduced. Other changes in the protein compositions included the accretion of two low molecular weight proteins of approximately 11,000 and 12,000, and the appearance of a periodic acid-Schiff-positive protein with the same electrophoretic mobility as the P₀ protein. Analysis of the isolated myelin indicated that it had a grossly normal protein composition. However, the two low molecular weight proteins and the P₀ protein appeared to be enriched in an upper-phase cord subtraction. We attribute the appearance of the two low molecular weight proteins to the breakdown of proteolipid protein and/or myelin basic protein as a result of demyelination, and the appearance of P₀ to the involvement of PNS myelin. The latter finding provides the first biochemical evidence that in MS cord, remyelination can be achieved in part by invading Schwann cells and/or by the small number of Schwann cells that may be present in the cord.     

Differential turnover of axonally transported glycoproteins

Metabolic turnover of axonally transported glycoproteins has been examined in membranous and soluble subfractions of goldfish optic tectum following intraocular injection of [³H]fucose. Radioactivity in total transported glycoproteins reached a maximum in the tectum after 24–30 hr, then declined with a half-life of approximately 20 days. Radioactivity in the total membranous subfraction declined with a similar half-life of 20–21 days while radioactivity in the soluble fraction showed a significantly shorter half-life of approximately seven days. Various sized glycopeptides derived from the membranous subfraction showed differential rates of loss of radioactivity with the lower molecular weight nondialyzable molecules displaying the most rapid turnover. In contrast, the glycopeptides derived from the soluble fraction showed relatively uniform rates of turnover. The results are discussed in the context of metabolic compartmentalization between membranous and soluble glycoproteins and among the carbohydrate chains of the membranous molecules.Supported by NIH grant NS 11456.     

Relationship of ATP Turnover, Polyphosphoinositide Metabolism, and Protein Phosphorylation in Sciatic Nerve and Derived Peripheral Myelin Subfractions from Normal and Streptozotocin Diabetic Rats

Sciatic nerve from streptozotocin-induced diabetic rats has previously been shown to incorporate more 32P into phosphatidylinositol-4,5-bisphosphate (PIP2) and the principal myelin proteins than normal nerve. In the present study, labeling of ATP and PIP2 was compared. Using nerve segments, [gamma-32P]ATP specific activity reached a plateau after incubation for 4 h with [32P]orthophosphate, whereas the specific activity of [32P]PIP2 rose much more slowly and was still increasing after 8 h. The rate of disappearance of radioactivity from prelabeled ATP was biphasic, with 75% being lost within 30 min and the remainder declining much more slowly for several hours thereafter. In contrast, no decrease in prelabeled PIP2 radioactivity could be detected for up to 4 h. The kinetics of ATP metabolism were not appreciably different for normal and diabetic nerve. However, after incubation with [32P]orthophosphate for 2 h, the specific activity of PIP2 was 50-120% higher in diabetic nerve. This phenomenon, therefore, cannot be ascribed to altered specific activity of the ATP precursor pool. Greater labeling of PIP2 in 32P-labeled diabetic nerve was present in purified myelin isolated using a simple discontinuous sucrose density gradient, but not in a "nonmyelin" fraction. When nerve homogenate was fractionated on a more complex gradient, three myelin-enriched subfractions were obtained which were heterogeneous as judged by morphological appearance, protein profile, and lipid metabolic activity. The proportion of total lipid radioactivity accounted for by PIP2 was elevated in all the subfractions relative to the homogenate. As compared to myelin subfractions from normal nerve, an increased percentage of 32P in PIP2 was obtained only in the major myelin subfraction from diabetic nerve. The phosphorylation of P0 relative to the other myelin proteins was also enhanced in this subfraction in nerve from diabetic animals.     

Labelling by axonal transport of myelin-associated proteins in the rabbit visual pathway.

After intraocular injections of [3H]leucine, six regions of the visual pathway of adult rabbit were used to study the spatio-temporal pattern of the slow anterograde axonal transport of radioactive proteins associated with the particulate fraction, the water-soluble fraction and the myelin fraction. Unlike other fractions, myelin-associated labelled proteins represented a time-constant (for a given region) percentage of total tissue radioactivity. This percentage increased from the first half to the second half of the optic nerve and remained high in the chiasma and tract. The peak specific radioactivity of myelin decreased in the same direction. Myelin proteins were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and the labelling patterns obtained in different regions and at different survival times were compared. At the peak of myelin radioactivity of a given region the label was typically associated with four protein bands, L1, L2, L3 and L4, of 40000, 44000, 62000, and 68000 mol.wts. respectively. The basic protein, the proteolipid protein and the W1 component (mol.wt. 51000-53000) of the Wolfgram proteins were not significantly labelled. The radioactivity associated with the W2 component (mol.wt 60000) of the Wolfgram proteins could be derived from the closely migrating L3 component. At shorter survival times no clear labelling pattern could be detected. At longer survival times radioactivity was almost totally localized around band L3. The results presented underline the importance of choosing appropriate experimental conditions to obtain a consistent labelling pattern of myelin-associated proteins and to investigate the possible mechanism responsible for this phenomenon.     

Synthesis of the myelin proteolipid protein in the developing mouse brain

Mice ranging in age from 16 to 44 days were injected intracerebrally with ³H-leucine, and incorporation into total brain proteolipids and the myelin proteolipid protein was measured. All proteolipids were isolated from whole brain by ether precipitation and separated into their individual components by SDS polyacrylamide gel electrophoresis. Two major proteolipids with apparent molecular weights of 20,700 and 25,400 were observed in these preparations, and their proportion increased over the developmental period examined. A Ferguson plot analysis comparing these proteins with those of isolated myelin showed that the 25,400-dalton proteolipid component from whole brain was the myelin proteolipid protein. Rates of incorporation of ³H-leucine into total brain proteolipids peaked at 22 days of age. Synthesis of the myelin proteolipid protein increased rapidly to a maximum value at 22 days and decreased rather slowly until at 44 days it was about 83% of its maximum rate of synthesis. The data indicate that the developmental pattern of synthesis of the myelin proteolipid protein is unlike that of the myelin basic proteins. Synthesis of the major myelin proteins is developmentally asynchronous in that peak synthesis of the myelin proteolipid appears to occur several days later than the basic proteins. In addition, it maintains its maximum rate of synthesis over a longer period of time than do the basic proteins.     

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.