The in vivo synthesis of myelin proteins in rabbit sciatic nerve.

Rabbits were injected into the sciatic nerves with either (35)S-methionine, or (3)H-fucose. After times ranging from 45 min to 15 days the nerves were removed and the total particulate material from the nerves fractionated to give seven subfractions with densities between 0.2 and 1.2 M sucrose. The patterns of radio-labelled proteins were examined by SDS-PAGE and quantitative fluorography. The results showed that the P(2) basic protein was metabolically far more active than either the major P(0) glycoprotein, or the basic protein BP. The P(2) protein also entered the myelin fractions more rapidly than either P(0), or BP components. The net synthesis of P(0) was slower than P(2) and BP and this intrinsic membrane protein remained associated with the denser membrane fractions (>0.7 M sucrose) for longer than the basic proteins prior to entering myelin. Newly synthesized high molecular weight proteins remained concentrated in the denser membrane fractions and turned over faster than the myelin proteins. A low density myelin fraction (B) was detected in which both the P(2) protein and certain high molecular weight proteins became more rapidly labelled than in compact myelin. In this fraction the specific activity remained higher than that of compact myelin for up to five days after the injection of (35)S-methionine into the nerve. The results indicate that the major PNS myelin proteins are incorporated into and turn over in the various compartments of the Schwann cell plasma membrane-myelin continuum at very different rates.

Myelin-associated glycoprotein and myelin basic protein are present in central and peripheral nerve myelin throughout phylogeny.

This phylogenetic study of central and peripheral nervous system myelin proteins demonstrates that important changes occur in the composition of certain myelin proteins during evolution. Only two components, myelin basic protein (MBP) and myelin-associated glycoprotein (MAG) are present in all Gnathostomata representatives investigated. While MBP components varied considerably even among the representatives of a given order, the apparent molecular weight of MAG showed little variation indicating that the conservation of the molecular structure could be important for the function of MAG in glia axon interactions.

Appearance of Myelin proteins during vertebrate evolution.

Myelin, defined as an arrangement of spirally fused unit membranes, is an acquisition of vertebrates and first appeared during evolution in Gnathostomata. In all species studied PNS and CNS myelins contain the myelin-associated glycoprotein (MAG) and the myelin basic protein (MBP). Throughout phylogeny PNS myelin is characterized by the major P(0) glycoprotein which is called IP in fishes. The PNS myelin proteins did not evolve further except for the addition of P(2) protein from reptiles onward. In Elasmobranchii and Chondrostei, PNS and CNS myelin proteins are similar. CNS myelin of actinopterygian fishes possesses a 36,000 Da protein (36K) in addition to P(0)-like IP glycoproteins. In tetrapod CNS myelin, P(0) is replaced by the proteolipid protein (PLP) and the Wolfgram protein (WP). Of particular interest in a transitional phylogenetic sense are the lungfish Protopterus, carrying glycosylated PLP (g-PLP) but no P(0), 36K or WP, and the bichir Polypterus, showing simultaneous presence of P(0), 36K and PLP. These results indicate that myelin proteins could be valuable molecular markers in establishing vertebrate phylogenetic relationships and in reconstructing the fish-tetrapod transition.

Density distribution of 2?,3?-cyclic nucleotide 3?-phosphodiesterase and myelin proteins in particulate material from myelin deficient (mld) mutant and control brains.

Total particulate material from control and myelin deficient (mld) brains was subjected to density centrifugation on a continuous sucrose gradient. Particles from control brains distributed in a bell-shaped mode with a peak density near 0.64 M-sucrose. In mld material only a slight elevation of optical density was observed near 0.8 M-sucrose. The highest specific activities of 2?,3?-cyclic nucleotide 3?-phosphodiesterase were observed at densities of 0.63 and 0.71 M-sucrose for mld and control brains, respectively. The peak of myelin basic protein in control fractions was near 0.60 M-sucrose. In mld fractions no peak was observed. Proteolipid and Wolfgram proteins had a maximum near 0.65 and 0.73 M-sucrose in control and mld fractions, respectively. The absence of myelin basic proteins in all the fractions makes it unlikely that, in mld mice, myelin basic proteins are synthesized but not incorporated into myelin.

Nervous system myelin in the electric ray, Torpedo marmorata: Morphological characterization of the membrane and biochemical analysis of its protein components.

In Torpedo, PNS as well as CNS myelines are characterized by clearly separated double intraperiod lines. CNS myelin of Torpedo contains two glycosylated hydrophobic proteins labelled T1 (25,800 Da1) and T2 (29,700 Da1), and two basic proteins BP1 and BP2, migrating like mammalian large basic protein (BP2) and pre-small basic protein (BP1) (Barbarese et al., 1977). PNS myelin of Torpedo carries only BP1 and is characterized by a closely spaced doublet of the glycosylated hydrophobic proteins Con A+ (29,700 Da1) and Con A? (31,000 Da1); the latter does not bind Concanavalin A. These glycosylated proteins (T1, T2, Con A+, Con A?) contain mannose, N-acetylglucosamine and galactose, but lack fucose and sialic acids. They have isoleucine at their amino terminus. They bind anti-rat PNS myelin P(0) antibodies but do not react with anti-rat CNS myelin PLP antibodies. Limited proteolyses of isolated proteins suggest sequence homologies between T1 and T2, and possibly between Con A+ and Con A?. The two basic proteins BP1 and BP2 bind antibodies directed against human myelin basic protein. All Torpedo myelin proteins electrofocus in pH regions characteristic of their mammalian counterparts.

Protein and enzyme distribution in microsomal and myelin fractions from rat and Jimpy mouse brain.

The protein, glycoprotein and enzyme composition of myelin and myelin-related fraction (SN 4) from rat forebrain was compared with that of microsomal fractions. Acetylcholinesterase was largely confined to the microsomal fractions, wheras 2',3'-cyclic nucleotide-3'-phosphohydrolase (CNP) showed a high specific activity in myelin and SN 4 fractions. Nevertheless, the total specific activities of CNP present in microsomal membranes and in a water-soluble form were not negligible, and suggest that this enzyme has a wide distribution among subcellular particles. A high molecular weight protein was identified in myelin and all the other fractions studied. This protein (X), which co-migrates with the major myelin glycoprotein, was present in myelin and in fractions lacking typical myelin components as well as in fractions from a myelin deficient mutant, the Jimpy mouse. The results suggest that the X protein is probably a contaminant in isolated myelin, although the occurrence of this protein as an intrinsic component of several different membranes cannot be ruled out. Despite substantial overlap in density upon zonal centrifugation between SN 4 and microsomal fractions, the enzyme patterns of the fractions were different.

Biochemical characterization of the central nervous system myelin proteins of the rainbow trout, Salmo gairdneri.

Central nervous system myelin isolated from the rainbow trout (Salmo gairdneri) displays a very low median density on zonal gradient centrifugation, banding at approximately 0.32 M sucrose. Its proteins consist of a 36 K (36,000 mol.wt.) component, two Concanavalin A-reactive intermediate proteins IP1 (23,000 mol.wt.) and IP2 (26,200 mol.wt.), and two basic proteins BP1 and BP2, of which the latter co-migrates with rat SBP while BP1 is of slightly smaller size. The trout myelin proteins electrofocus at pH positions similar to those of their mammalian counterparts. Immunoblotting shows that antibodies against rat PNS myelin P0 glycoprotein are bound by IP1 and IP2, but not by 36K. None of the trout myelin proteins react with anti-rat CNS myelin proteolipid protein (PLP) antiserum. The basic proteins BP1 and BP2 bind strongly to antibodies directed against human myelin basic protein. In vivo injection of tritiated fucose or palmitate leads to radiolabeling of IP1 and IP2. Under autolytic in situ conditions the appearance of a glycosylated 20,000 mol.wt. component (IP0) is noted, with parallel reduction of both IP1 and IP2, indicating sequence homologies between IP1 and IP2. The 36K protein is not affected by autolysis.

Characterization of a myelin-related fraction (SN 4) isolated from rat forebrain at two developmental stages.

A myelin-related fraction (SN 4) was isolated from forebrain of 17- and 40-day-old rats. Fraction SN 4 was obtained as a supernatant in a slow speed differential centrifugation of a myelin fraction. In contrast to multilamellar myelin fraction, SN 4 consisted of small vesicular profiles of a mixture of single membranes and some triple-layered structures. All typical myelin components were found in the SN 4 fraction from adult rat brain but their relative proportion was different from that of myelin: Wolfgram protein, myelin glycoproteins and 2',3'-cyclic nucleotide 3'-phosphohydrolase were increased, while basic proteins and proteolipid protein were decreased significantly. In contrast, the lipid composition appeared very similar to the one found in myelin. SN 4 from 17-day-old rat brains was essentially similar to that from adults, except that the major myelin glycoprotein was not enriched in comparison to myelin. Developmental changes found in myelin were also present in the SN 4 fraction. The specific radioactivity of the fucose-labeled major myelin glycoprotein was similar in SN 4 and in myelin. The particular composition of fraction SN 4 suggests that this material is not significantly contaminated by non-myelin-related membranes but rather supports the hypothesis that it could be enriched in a membrane representing a zone of transition during the formation of myelin and which is subjected to a remodelling of its protein components.

Central nervous system myelin proteins and glycoproteins in vertebrates: a phylogenetic study.

CNS myelin was isolated by a conventional method from a wide range of vertebrate classes and analyzed by SDS-PAGE for proteins (Coomassie blue) and glycoproteins (concanavalin A (Con-A)-peroxidase). Mammalian, avian and reptilian myelin shared similar protein patterns (basic protein, BP; intermediate protein, DM-20; proteolipid protein, PLP; Wolfgram protein, W). Amphibians lacked DM-20 but were characterized by specific activities of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) higher than those of the other classes examined. The Con A-binding profiles were similar in the high molecular weight (HMW) regions of the classes listed above, while the typical myelin proteins in the low molecular weight (LMW) regions were devoid of Con A-binding properties. In teleost myelin a putative BP band ran well ahead of rat small basic protein (SBP), whereas the region corresponding to rat PLP was covered by several closely spaced bands, most of which bound Con A. In elasmobranch myelin, apart from bands corresponding to BP, Con A-binding glycoproteins were detected migrating in the region of rat DM-20 and PLP as well as with mammalian PNS P0 protein. Cyclostomates yielded only very small amounts of material in the myelin preparation and displayed undifferentiated Coomassie blue- and Con A-binding in the HMW region, while typical LMW myelin proteins were absent. These results demonstrate that CNS myelin from bony and cartilaginous fishes is characterized by containing several major Con A-binding proteins of low molecular weight. This is in striking contrast to myelin from phylogenetically higher classes.

Changes of myelin proteins during Wallerian degeneration in situ and in millipore diffusion chambers preventing active phagocytosis.

Changes of myelin proteins in mouse sciatic nerves were studied comparing nerves degenerating in situ with nerves enclosed in millipore diffusion chambers which eliminate invasion of non-resident cells. Nerves kept in chambers showed nearly complete preservation of myelin sheaths with a very slow degradation of myelin proteins. Nerves degenerating in situ showed rapid myelin phagocytosis by macrophages with almost complete disappearance of myelin proteins after 28 days. These data elucidate the role of macrophages for removal of myelin proteins.

Distribution of PNS myelin proteins and membrane enzymes in fractions isolated by continuous gradient zonal centrifugation.

Myelin was purified from adult rabbit sciatic nerve by two procedures: discontinuous gradient centrifugation and continuous gradient zonal centrifugation. Two fractions were obtained from the discontinuous gradient. The fraction floating on 0.32 M sucrose and the fraction recovered from the 0.32/0.85 M sucrose interface showed typical myelin membranes by electron microscopy and typical myelin proteins by gel electrophoresis. The specific activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) decreased from the top to the bottom of the discontinuous gradient. The myelin separated by zonal centrifugation on a continuous sucrose gradient showed three distinct peaks (on monitoring optical density) at 0.10, 0.30 and 0.57 M sucrose. The latter peak yielded 92% of the material applied. The two minor peaks of low density exhibited high CNP and acetylcholinesterase (AChE) activities but the specific activity of both enzymes increased markedly at the heavy end of the gradient. The zonal fractions showed typical myelin proteins in all fractions by polyacrylamide gel electrophoresis but with important quantitative differences. These results indicate that PNS myelin shows significant heterogeneity.

Expression of myelin proteins characteristic of fish and tetrapods by Polypterus revitalizes long discredited phylogenetic links.

CNS myelin constituents were used as evolutionary markers to study the controversial relationships of Polypterus with bony fishes, lungfishes and amphibians. The occurrence in Polypterus CNS of myelin proteins similar to those observed in the sterlet confirms its close relationship to Chondrostei. However, the simultaneous presence of proteolipid protein (PLP) demonstrates the existence of a long discredited relationship between Polypterus and tetrapods, and also with the lungfish Protopterus, an ally of tetrapods. As in the lungfish Protopterus, Polypterus cerebrosides and sulfatides contained alpha-hydroxy fatty acids. In contrast to Protopterus CNS myelin which carries glycosylated PLP but lacks Po-like component and the 36 kDa protein, Polypterus possesses these two bony fish myelin components. Furthermore, the presence of aglycosylated PLP in Polypterus, as in higher vertebrates, differentiates it from Protopterus. The simultaneous presence in Polypterus of myelin constituents from fish and land vertebrates indicates that Polypterids occupy an unusual intermediate phylogenetic position. The near absence of 2',3'-cyclic nucleotide 3'-phosphodiesterase activity in Polypterus. Protopterus, and in other fishes, confirmed by the lack of Wolfgram protein, establishes this myelin enzyme as the only CNS myelin constituent specifically expressed by tetrapods.

The lipid composition of light and heavy myelin subfractions isolated from rabbit sciatic nerve.

Centrifugation of adult rabbit sciatic nerve homogenates on continuous sucrose density gradients yields three distinct maxima at 0.12, 0.33 and 0.57 M sucrose (labeled A, B and C), respectively. Fraction A is absent in homogenates of immature rabbit sciatic nerve and the material isolated from adult animals is found to contain large amounts of triglycerides. It is proposed that fraction A results from the presence of adipose tissue adhering to the adult nerve preparations and should not be regarded as a true myelin subfraction. The concentration of the major membrane lipid classes (phospholipid, cholesterol and galactosyl ceramide) decreases from the light to the heavy side of the gradient. The ethanolamine phosphatide to sphingomyelin ratio increases from 1.06 to 1.42 between fractions B and C, due to an increase in the phosphatidal ethanolamine content at the expense of sphingomyelin.

Myelination in rabbit optic nerves is accelerated by artificial eye opening.

Artificial opening of the eyes of young rabbits on the 5th postnatal day led to accelerated myelination: the myelin-specific basic and proteolipid proteins nearly doubled between the 7th and the 10th postnatal days when compared to controls; 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) activity also increased by about 60%. Conversely, lowered AChE activities presumably reflected elevated myelin/axolemma ratios. Myelination in treated animals normalized during later ontogenetic stages (greater than 20th postnatal day).

Phylogenetic examination of vertebrate central nervous system myelin proteins by electro-immunoblotting.

Central nervous system (CNS) myelin proteins from vertebrate classes were examined by immunoblotting with antisera against mammalian CNS myelin proteins. Higher vertebrates possessed proteolipid (PLP), DM-20 and Wolfgram (WP) proteins, except that DM-20 was missing in amphibia. Fish CNS myelins contained neither PLP nor WP; instead they bound antisera to mammalian peripheral nervous system P0 protein. All classes carried myelin basic protein, but only mammals exhibited a component equivalent to rat 21.5K (21,500 dalton). These phylogenetic data are consistent with major changes in CNS myelin protein composition at the transition from fishes to higher vertebrates.

Carbonic anhydrase activity in myelin fractions from rat optic nerves.

The carbonic anhydrase activity of myelin fractions isolated from the optic nerves of adult and immature (20-day-old) rats was examined. The specific activity in both total homogenate and myelin fractions was about 2-fold higher in adult than in immature animals and at both ages, the activity in the homogenate was higher than in myelin. After subfractionation by zonal gradient centrifugation, it was shown that carbonic anhydrase activity was greatest in the heaviest myelin particles at both ages. These data are consistent with the hypothesis that a small proportion of the total enzyme activity is localised in myelin.

Density and protein profiles of myelin from two regions of young and adult rat CNS.

Myelin, isolated from forebrain and spinal cord of young and adult rats, was distributed by zonal centrifugation on linear (0.32--1.00 M) sucrose gradients in a bell-shaped mode. The peak position of forebrain myelin shifted from the density of 0.58 M sucrose in young animals to that of 0.67 M sucrose in adult rats, while in spinal cord no such pronounced shift was noticed (approximately 0.58 M sucrose). Morphologically, the preparations appeared very similar across the density ranges. Specific activities of acetylcholinesterase were substantially below the total homogenates, while those of 2', 3'-cyclic nucleotide 3'-phosphohydrolase were higher in all fractions, except in the light myelin subfractions from adult spinal cord. Basic proteins decreased from the light to the heavier fractions; higher molecular weight proteins increased, together with proteolipid protein, which in spinal cord reached a plateau and in forebrain decreased towards the heavy side. The ratio of the small basic protein/large basic protein showed higher values in the light myelin subfractions in the regions and ages examined, pointing to a higher degree of maturation.

Sodium dodecyl sulfate in protein chemistry.

This review summarizes in a brief manner the main aspects of the application of sodium dodecyl sulfate (SDS) to protein chemistry. The principal problems of SDS-polyacrylamide gel electrophoresis are described, as well as the anomalous behavior of protein-SDS complexes and the inactivation of enzymes due to variable binding of SDS to the polypeptides studied. The particular value of SDS in elucidating the protein composition of biological membranes and in membrane-reconstitution experiments is discussed.

Protein heterogeneity in rat CNS myelin subfractions.

Microsomal fraction-free myelin from forebrain and spinal cord of young and mature rats, when subjected to hypo-osmotic shock and slow speed centrifugation, yielded a myelin pellet and a supernatant fraction (SN 4). Fraction SN 4 consisted of small vesicular profiles in which the major myelin proteins were reduced whereas high molecular weight material such as Wolfgram protein, myelin-associated glycoprotein and CNP were substantially increased over myelin. A close correlation of the SN 4 fraction to the myelin-like fraction of Davison and coworkers was suggested. The myelin pellets were subfractioned on zonal sucrose gradients to yield bell-shaped particle distributions. Besides shifts in densities of the maxima between myelin of young and mature forebrain and spinal cord, a decrease was observed from the light to the heavy gradient end in basic proteins, and an increase in Wolfgram protein and other high molecular weight proteins. Proteolipid protein took an intermediate position. Light fractions from adult spinal cord displayed CNP activities below those of the total homogenate. This result, together with the very high CNP activities in fraction SN 4 casts some doubt on CNP being a marker for compact myelin; rather it appears that CNP is a marker for the process of myelin formation.

The preparation and analysis of myelin from small quantities of central nervous tissue: regional studies of the quaking mouse.

Myelin of considerable purity may be isolated from small (minimum 1 mg wet weight) samples of central nervous tissue, using a 4-step centrifugation procedure. The separation of myelin proteins by micro-linear gradient polyacrylamide gel electrophoresis yields similar results to those obtained by macro-scale (homogeneous) gel systems. These techniques have been employed for a preliminary study of the regional composition of myelin fractions from the Quaking mouse.