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.     

Development and Applications of a Solid-Phase Radioimmunoassay for the P0 Protein of Peripheral Myelin

This is the first report of a quantitative radioimmunoassay for PO. The assay uses antigen-coated plastic microwells, with antibody binding detected by 125I-labeled protein A. Either peripheral myelin proteins or purified PO may be used as the antigen. Optimal extraction of tissue samples for PO immunoassay requires careful attention to the sodium dodecyl sulfate-to-protein ratio. Sodium dodecyl sulfate interference with antibody binding can be minimized by adding an excess of nonionic detergent and carrier protein to the incubation buffer. This method allows the detection of 0.8 ng of PO (20 ng/ml). Results from this assay showed little or no immunoreactivity in extracts of brain, centra myelin, liver, purified myelin basic proteins, cultured, purified secondary Schwann cells, or membrane preparations from these cells. PO was clearly detectable in Schwann cell cultures from 3- to 4-day-old rats at 12-18 h after dissociation (4% of the level in adult sciatic nerve) and in extracts of one-day-old rat sciatic nerve (2% of the level in adult nerve). Myelin basic protein radioimmunoassays showed that the ratio of PO to myelin basic protein is essentially constant in extracts of sciatic nerve from ne-day-old, four-day-old, and young adult rats. Another result was that PO levels are reduced in the trembler mouse sciatic nerve.     

Phosphorylation and Fucosylation of Myelin Protein In Vitro by Sciatic Nerve from Developing Rats

Abstract: Proteins of the paniculate fraction of sciatic nerve of rats ranging from 1 to 55 days of age were analyzed by polyacrylamide gel electrophoresis. The major myelin protein, P₀, could not be detected at 1 day of age, but by 10 days it comprised from 15 to 20% of the particulate protein, the same proportion as in adult rats. Growth of nerve continued throughout the period studied. Rat sciatic nerves were incubated with [³²P]orthophosphate or [³H]fucose. Particulate matter proteins from sciatic nerve (and in certain cases proteins of myelin purified from sciatic nerve) were separated by polyacrylamide disc gel electrophoresis and the distribution of protein and of radioactivity along the gels was determined. [³²P]Phosphate appeared to label all myelin proteins. Labeling with fucose was more specific; myelin basic proteins were not fucosylated. A developmental study showed that sciatic nerves from 2-day-old rats could incorporate radioactive fucose and [³²P]-phosphate into several proteins at the P₀ region of polyacrylamide gels. Specific radioactivity of [³H]fucose in P₀ protein was highest in preparations from 5-day-old rats and declined by 80% over the next 5 days as it was diluted by accumulating myelin. The specific radioactivity of incorporated [³²P] phosphate was high at the early age points and declined as a result of the accumulation of compact myelin. The results indicate an association of fucosylation and/or phosphorylation with some step in the formation of myelin.     

PROTEIN COMPOSITION OF MYELIN OF THE PERIPHERAL NERVOUS SYSTEM

Abstract— Myelin was purified from the peripheral nervous system (PNS) of several species. The protein composition of these preparations was examined by discontinuous polyacrylamide gel electrophoresis in buffers containing sodium lauryl sulphate. Proteins characteristic of all samples include, in order of increasing mobility: a series of high molecular weight proteins, the major peripheral nerve protein (P₀), two uncharacterized proteins, and two basic proteins (P₁ and P₂). Quantitative results, obtained by densitometry of gels stained with Fast Green showed differences in protein distribution, both between species, and from different types of nerves obtained from the same animal. The relative amounts of P₁ and P₂ proteins were the most variable; e.g. myelin from guinea-pig sciatic nerve had little or no P₂ protein, whereas 15 per cent of the myelin protein of beef posterior intradural root was Pz protein. P₀, P₁ and P₂ proteins from rabbit sciatic nerve and P₀ and P₂ proteins from beef dorsal and ventral intradural roots were purified and their amino acid compositions were determined. Our results indicated that the P₁ protein is very similar in size and amino acid composition to the basic protein of central nervous system myelin, whereas the P₀ and P₂ proteins are unique to the PNS.     

Effect of cycloheximide on palmitylation of PO protein of the peripheral nervous system myelin.

Incubation of rat sciatic nerve slices with Krebs-Ringer bicarbonate buffer containing [3H]palmitic acid resulted in the acylation of the PO glycoprotein and a 24 kDa protein of the peripheral nerve myelin. Radioactivity was removed from PO after treating PO with hydroxylamine (83%) and methanolic KOH (97%). These results provided evidence that the radioactivity incorporated into PO was not due to the metabolic conversion of [3H]palmitic acid into amino acids or sugars. PO was more heavily labelled in the homogenate than in the myelin membrane in 8-day-old rat nerve between 5 min and 2 h of incubation. These results suggested that PO may be primarily acylated in the cell body. Incubation of purified myelin with [1-14C]palmitoyl-CoA resulted in the non-enzymic acylation of PO. This provided evidence of the absence of fatty acyltransferase from the purified peripheral nerve myelin. Glycosylation of PO has been shown to occur in the Golgi complex, and monensin inhibited glycosylation of PO in the homogenate and myelin by 53 and 61% respectively. These results suggest that the processing of PO in the Golgi complex and the assembly of PO into myelin is impaired by monensin. However, fatty acylation of PO was unimpaired by monensin, suggesting that the addition of fatty acids may not occur in the Golgi complex. There was a progressive decrease in the acylation of PO between 5 min (28%) and 2 h (61%) in the presence of cycloheximide, as the pool of previously synthesized PO was gradually depleted. These results also provide evidence that palmitylation of PO is not coupled to protein synthesis, and acylation of this protein probably occurs in the early subcompartment of the Golgi complex, which appears to be insensitive to monensin.     

The PO glycoprotein of peripheral nerve myelin

The PO glycoprotein, the major protein of peripheral nerve myelin, is a hydrophobic glycoprotein which can be isolated in soluble and insoluble forms from rabbit sciatic nerve myelin following extensive defatting and mid acidic extraction. The PO glycoprotein was localized exclusively in peripheral nervous system (PNS) myelin of sciatic nerve and rootlets by the immunofluorescent technique using goat anti-PO serum which showed a single precipitin band in double diffusion and did not cross-react with the myelin basic protein or P2 protein. Central nervous system (CNS) myelin from brain and spinal cord was negative by the immunofluorescent procedure. The major glycoprotein bands in PNS myelin, in addition to the PO glycoprotein at 28K, exist at 23K and 19K, as shown by gel electrophoresis in dodecyl sulfate. These glycoproteins, isolated by gel filtration in 2% dodecyl sulfate, show identity to the PO glycoprotein in their monosaccharide profile and overlapping tryptic peptides on peptide mapping. We conclude that both the 23K and 19K glycoproteins are derived from the PO glycoprotein by in situ proteolysis; the 23K glycoprotein has the identical amino terminal sequence. The 19K glycoprotein, beginning with amino-terminal methionine, is identical with the TPO glycoprotein, shown previously to originate from tryptic hydrolysis of the PO glycoprotein in isolated myelin. A tryptic glycopeptide containing 27 amino acids was isolated from the PO glycoprotein and sequenced. It contained a relatively high proportion of aspartic acid (four residues) and glutamic acid (two residues), thus exhibiting a high negative charge. We conclude that the total carbohydrate of the PO, 23K, and 19K glycoproteins does indeed exist as a single nonasaccharide moiety linked through N-acetylglucosamine to Asp-14 of the glycopeptide in a N-glycosidic linkage. These results further support the role of the PO glycoprotein as a typical amphipathic membrane protein.     

PO Protein and 2'',3''-Cyclic-Nucleotide 3''-Phosphodiesterase Activity in the Peripheral Nerve and Subcellular Fractions of the Trembler Mouse

To investigate the biochemical abnormalities of the Trembler mouse, the level of the PO protein (as % of total protein) and the activity of CNP was compared in the sciatic nerve and subcellular fractions of normal and mutant littermates. There was a significant decrease in both of these myelin markers in total nerve homgenates of the neurological mutant compared with the control animals. Immunoassay of the PO protein and polyacrylamide gel analysis of proteins indicated an accumulation of a protein with an apparent molecular weight of 67K in mutant nerve extracts. The mutant nerve also had relatively decreased levels of a protein of molecular weight about 41K that cross-reacted with antibody to PO protein. The Trembler mouse exhibited a larger percentage recovery of PO protein and CNP activity in subcellular fractions denser than the myelin sheath. Together these results are consistent with the theories that these denser components represent immature forms of myelin and that the Trembler mutant is characterized by hypomyelination.     

Glycopeptide fractions prepared from purified central and peripheral rat myelin

Myelin was purified from rat brain and sciatic nerve after invivo labeling with [³H]fucose and [¹⁴C]glucosamine to provide a radioactive marker for glycoproteins. The glycoproteins in the isolated myelin were digested exhaustively with pronase, and glycopeptides were isolated from the digest by gel filtration on Bio-Gel P-10. The glycopeptides from brain myelin separated into large and small molecular weight fractions, whereas the glycopeptides of sciatic nerve myelin eluted as a single symmetrical peak. The large and small glycopeptide fractions from central myelin and the single glycopeptide fraction from peripheral myelin were analyzed for carbohydrate by colorimetric and gas liquid chromatographic techniques. The glycopeptides from brain myelin contained 2.4 μg of neutral sugar and 0.59 μg of sialic acid per mg total myelin protein, whereas sciatic nerve myelin glycopeptides contained 10 μg of neutral sugar and 3.8 μg of sialic acid per mg total protein. Similarly, the gas-liquid chromatographic analyses showed that the glycopeptides from peripheral myelin contained 4- to 7-fold more of each individual per mg total myelin protein than those from central myelin. Most of the sialic acid and galactose in the glycopeptides from central myelin were in the large molecular weight fraction, and the small molecular weight glycopeptides contained primarily mannose and $\text{N-}\text{acetylglucosamine}$. The considerably higher content of glycoprotein-carbohydrate in peripheral myelin supports the results of gel electrophoretic studies, which indicate that the major protein in peripheral myelin in glycosylated while the glycoproteins in purified central myelin are quantitatevely minor components.     

DHPOSITION OF LIPIDS IN THE DEVELOPING CENTRAL AND PERIPHERAL NERVOUS SYSTEMS OF THE CHICKEN

Abstract— The lipid composition of chick brain and sciatic nerve was determined during development. It was confirmed that the addition of CaCl₂ to solvents during the extraction of lipids from brain results in much higher yields of diphosphoinositides particularly from unmyelinated embryo brain. Unlike the earlier report for rat brain, the recovery of triphosphoinositides was also Substantially increased. The amount of CaCl₂, required to achieve optimal recoveries decreased with increasing age and addition of more than this optimal amount depressed the yields of polyphosphoinositides, particularly triphosphoinositides. CaCl₂, addition did not improve the yield of diphosphoinositides from sciatic nerve of any age but drastically reduced recovery of triphosphoinositidcs. Differenccs in the effect of CaCl₂ were not the result of variation in the tissue concentrations of calcium or magnesium.
The lipid composition of sciatic nerve closely reflected that of the myelin. Both polyphosphoinositides were absent initially and their accumulation paralleled that of cerebrosides and sulfatides. The concentration of diphosphoinositides remained constant after the period of most active myelination while triphosphoinositides and the galactolipids continued to increase suggesting maturational changes in the myelin composition. The pattern of deposition in chick brain was similar except for the much greater contribution of non-myelin structures. Both polyphosphoinositides were present in equimolar amounts in pre-myelination embryonic tissue. The concentration of diphosphoinositides increased during active myelination only while triphosphoinositides continued to increase thereafter.     

Cleavage of the PO glycoprotein of the rat peripheral nerve myelin: Tentative identification of cleavage site and evidence for the precursor-product relationship

The incubation of sciatic nerve slices in Krebs Ringer bicarbonate (KRB) buffer (pH 7.4) at 37°C, or the incubation of freshly isolated myelin in ammonium bicarbonate buffer (pH 8), resulted in the generation of a 24kDa protein with a concomitant decrease of PO protein. The conversion of PO into 24kDa protein was blocked by heating isolated myelin at 100°C for 5 min suggesting that the reaction is enzyme mediated. Inclusion of the protease inhibitors and chelating agent to isolated myelin did not prevent the formation of 24kDa protein. Similarly, addition of CaCl₂ to isolated myelin did not accentuate the formation of 24kDa protein suggesting that the conversion of PO into 24kDa protein may not be due to Ca²⁺ activated protease. It is postulated that the formation of 24kDa protein may be due to neutral protease and/or metalloproteinase associated with the PNS myelin. 24kDa protein was purified and characterized. The N-terminal sequence of 1–17 amino acid residues of 24kDa protein was identical to PO. 24kDa protein was immunostained and immunoprecipitated with anti-PO antiserum indicating the immunological similarities between PO and 24kDa protein. Labeling of 24kDa protein with [³⁵S]methionine provided evidence that PO may be in all probability cleaved between Met-168 and Met-193. Further studies were carried out to demonstrate that 24kDa protein was phosphorylated, glycosylated and acylated like PO. Phosphorylation of 24kDa protein in the nerve slices was increased five-fold by phorbol esters and phosphoserine was the only phosphoamino acid identified after partial acid hydrolysis of 24kDa protein. These results suggested that serine residue phosphorylated by protein kinase C may be located in amino acid residues 1-168. 24kDa protein was stained with periodic Schiff reagent. In addition, 24kDa protein was fucosylated and the fucosylation of 24kDa protein was inhibited (70%) by tunicamycin, providing evidence that it is N-glycosylated. Recently, it was demonstrated that both PO and 24kDa protein were fatty acylated with [³H]palmitic acid in the nerve slices and fatty acids are covalently linked to these proteins (Agrawal, H.C. and Agrawal, D. 1989, Biochem. J. 263:173–177). The time course of inhibition of acylation by cycloheximide of 24kDa protein was identical to PO. Cycloheximide inhibited acylation of PO and 24kDa protein by 61% and 58% respectively, whereas, monensin had little affect on the fatty acylation of these proteins. Less [³H]palmitic acid and¹⁴C-amino acids were incorporated into 24kDa protein when compared to PO between 5–30 min after incubation of the nerve slices. However, more radioactivity was incorporated into 24kDa protein after 60 min when compared to PO under identical conditions. These results provided evidence of a precursor-product relationship between PO and 24kDa protein. Therefore, PO may be cleaved into 24kDa protein in the myelin membrane following its acylation and glycosylation in the Schwann cells.     

MYELIN SYNTHESIS IN VITRO: A COMPARATIVE STUDY OF CENTRAL AND PERIPHERAL NERVOUS TISSUE

Abstract— Brain, spinal cord and sciatic nerve from rats at different ages were incubated for 2 h in a medium containing [¹⁴C]acetate and [¹⁴C]leucine as the precursors for synthesis of lipids and proteins. Myelin was purified from the incubated tissues and the specific and total radioactivites of myelin lipids and protein were determined. The uptake of radioactive precursors decreased with increasing age up to 6 months of postnatal age, the decrease following the same pattern for the three types of myelin. After age 6 months the uptake of the protein and lipid precursors reached a plateau that persisted up to 18 months, the oldest postnatal age studied. The amount of myelin isolated and the total myelin lipids extracted from both the central and peripheral nervous systems increased continuously from age 25 days to 18 months after birth. Consequently we suggest that myelination is a process that continues during the whole life of the rat.
The metabolic activity of peripheral nerve myelin was higher than myelin from the CNS at all ages studied. Although myelination in the sciatic nerve begins before that in brain and spinal cord, the three types of myelin apparently reach maturity at the same age. Lecithin exhibited the highest metabolic activity of the individual myelin lipids at all ages in both the central and peripheral nervous system. The metabolic activity of cholesterol in myelin from the 25-day-old rats was similar to that of lecithin but decreased to very low levels in myelin from the 18-month-old rats.     

An endogenous lectin "CSL" interacts with glycoprotein components in peripheral nervous system myelin

An endogenous mannose binding lectin isolated from the rat cerebellum, CSL, was localized using immunocytochemical techniques in adult and in developing rat sciatic nerve. The lectin is present in Schwann cell cytoplasm and in compact myelin. It is present very early in Schwann cells and persists throughout postnatal sciatic nerve development. Endogenous ligands for the lectin were detected using iodinated-CSL binding to proteins blotted after polyacrylamide gel electrophoresis. Probably PO and MAG glycoproteins are specifically bound by CSL in contrast with numerous other Concanavalin A binding glycoproteins. A 31 kDa glycoprotein identified in purified preparations of axons of young rats also reacts with CSL. Based on the present developmental biochemical and immunochemical studies, an hypothetical scheme is proposed for the molecular basis of axon-Schwann cell interactions and of stabilization of compact myelin.     

Basic proteins of rodent peripheral nerve myelin: immunochemical identification of the 21.5K, 18.5K, 17K, 14K, and P2 proteins

Abstract: Proteins in peripheral nervous system and central nervous system myelin and homogenates of sciatic nerve and brain from young and adult mice and rats were characterized with affinity-purified anti-P₂ and anti-myelin basic protein sera after electrophoretic transfer from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose sheets. Using this method we have identified a component of rodent peripheral nervous system myelin as P₂ protein. Peripheral nervous system myelin also showed the presence of four basic proteins in addition to P₂ protein. These were found to be analogous to the 14, 17, 18.5, and 21.5K species found in the central nervous system myelin. A number of high-molecular-weight proteins were also detected with anti-myelin basic protein serum in peripheral nervous system, as well as central nervous system myelin. In addition, we report the presence of a high-molecular-weight P₂ cross-reactive protein in rodent brain stem homogenates, but not in central nervous system myelin.     

Protein kinases associated with peripheral nerve myelin. 1. Phosphorylation of endogenous myelin proteins and exogenous substrates.

When highly purified myelin from rat sciatic nerve was incubated with [gamma-32P]ATP, protein components of the membrane were phosphorylated indicating the presence of both the substrate (receptor protein) and an endogenous kinase in the membrane. Polyacrylamide gel electrophoresis of the phosphorylated membrane proteins followed by scintillation counting of gel slices and autoradiography showed that the polypeptides of molecular weights 28000, 23000 and 19000 were phosphorylated, and 32P from [gamma-32P]ATP having been incorporated into serine residues of the substrate proteins. Phosphorylation of purified myelin was Mg2+-dependent, was optimal at pH 6.5 and was not stimulated by adenosine 3',5'-monophosphate. We found that proteins other than those in myelin, such as phosvitin, casein, protamine and histones, can also act as a substrate for the membrane associated kinase. Muscle protein kinase inhibitor had no effect on the endogenous phosphorylation of myelin proteins or on the phosphorylation of phosvitin by peripheral nerve myelin protein kinase. However, the phosphorylation of histone by peripheral nerve myelin protein kinase was inhibited by the protein kinase inhibitor. After washing the membrane with 150 mM KCl the protein kinase that utilizes histone as substrate was found in the supernatant. In contrast, the endogenous phosphorylation of membrane proteins or the phosphorylation of phosvitin by the membrane associated kinase was not affected by washing. From these findings we conclude that at least two protein kinase systems exist in purified peripheral nerve myelin. One system is not inhibited by muscle kinase inhibitor, is tightly bound to the membrane and utilizes as its receptor proteins either exogenous phosvitin or endogenous membrane proteins. The second system is inhibited by muscle kinase inhibitor, is removable from the membrane and utilizes histones as its receptor proteins.     

Tumor promoters accentuate phosphorylation of PO: Evidence for the presence of protein kinase C in purified PNS myelin

The effects of carbon tetrachloride, methylene chloride and chloroform on phosphorylation of PO was examined. The results of the dose response curve revealed that carbon tetrachloride (0.67%), methylene chloride (2%) and chloroform (1%) induced phosphorylation of PO by approximately 4, 6, and 12-fold, respectively. PO was found to be phosphorylated on the serine residue, and the phosphorylation of the serine residue was markedly increased when PO was phosphorylated in the presence of these compounds. Since tumor promoters, carbon tetrachloride and chloroform, have been shown to activate protein kinase C in platelets it is postulated that the increased phosphorylation of PO may result from the activation of myelin associated protein kinase C. The presence of phospholipid sensitive Ca²⁺-dependent protein kinase (protein kinase C) in purified nerve myelin was demonstrated by increased phosphorylation of PO in the presence of Ca²⁺ and phosphatidylserine.     

Protein kinase associated with peripheral nerve myelin. 1. Phosphorylation of endogenous myelin proteins and exogenous substrates

When highly purified myelin from rat sciatic nerve was incubated with [γ-³²P]ATP, protein components of the membrane were phosphorylated indicating the presence of both the substrate (receptor protein) and an endogenous kinase in the membrane. Polyacrylamide gel electrophoresis of the phosphorylated membrane proteins followed by scintillation counting of gel slices and autoradiography showed that the polypeptides of molecular weights 28000, 23000 and 19000 were phosphorylated, and ³²P from [γ-³²P]ATP having been incorporated into serine residues of the substrate proteins. Phosphorylation of purified myelin was Mg²⁺-dependent, was optimal at pH 6.5 and was not stimulated by adenosine 3′,5′-monophosphate. We found that proteins other than those in myelin, such as phosvitin, casein, protamine and histones, can also act as a substrate for the membrane associated kinase. Muscle protein kinase inhibitor had no effect on the endogenous phosphorylation of myelin proteins or on the phosphorylation of phosvitin by peripheral nerve myelin protein kinase. However, the phosphorylation of histone by peripheral nerve myelin protein kinase was inhibited by the protein kinase inhibitor. After washing the membrane with 150 mM KCl the protein kinase that utilizes histone as substrate was found in the supernatant. In contrast, the endogenous phosphorylation of membrane proteins or the phosphorylation of phosvitin by the membrane associated kinase was not affected by washing.From these findings we conclude that at least two protein kinase systems exist in purified peripheral nerve myelin. One system is not inhibited by muscle kinase inhibitor, is tightly bound to the membrane and utilizes as its receptor proteins either exogenous phosvitin or endogenous membrane proteins. The second system is inhibited by muscle kinase inhibitor, is removable from the membrane and utilizes histones as its receptor proteins.     

Purification and partial characterization of two glycoproteins in bovine peripheral nerve myelin membrane

Two major glycoproteins of bovine peripheral nerve myelin were isolated from the acid-insoluble residue of the myelin by a procedure involving delipidation with chloroform/methanol (2:1, v/v) and chromatography on Sephadex G-200 column with a buffer containing sodium dodecyl sulfate. The separation patterns of the proteins on the gel were affected considerably by the dodecyl sulfate concentration in the elution buffer. At above 2% dodecyl sulfate concentration in the elution buffer, the glycoproteins could be separated clearly on the gel and were purified. The purified proteins, the BR protein (mol. wt. 28 000) and the PAS-II protein (mol. wt. 13 000), were homogeneous on dodecyl sulfate-polyacrylamide gel electrophoresis. The NH₂-terminal amino acids of the BR and the PAS-II proteins were isoleucine and methionine, respectively. The BR protein contained glucosamine, mannose, galactose, fucose and sialic acids and the PAS-II protein contained glucosamine, mannose, galactose, fucose and glucose. Neither the BR protein nor the PAS-II were a glycosylated derivative of a basic protein of bovine peripheral nerve myelin, a deduction based on the results of amino acid analysis. The two major glycoproteins were observed commonly in the peripheral nerve myelin of cows, pigs, rabbits and guinea pigs, using dodecyl sulfate-polyacrylamide gel electrophoresis.     

Protein composition and synthesis in the adult mouse spinal cord

Propepties of spinal cord proteins were studied in adult mice subjected to unilateral crush or electrical stimulation of sciatic nerve. The protein composition of spinal tissue was determined using SDS-polyacrylamide gel electrophoresis coupled with subcellular fractionation. Comparisons of mouse spinal cord and brain revealed similarities in the types but differences in the concentrations of myelin associated proteins, nuclear histones and other proteins. Comparisons with sciatic nerve proteins demonstrated differences in types of proteins but similarities in the concentration of myelin proteins and nuclear histones. The short term (<2 hrs.) incorporation of radioactive amino acids into spinal cord proteins revealed heterogeneous rates of incorporation. Neither nerve crush six days prior to testing nor sciatic nerve stimulation had a significant effect on the protein composition or amino acid incorporation rates of spinal cord tissue. These observations suggest that known differences in spinal cord function following alterations in nerve input may be dependent upon different mechanisms than have been found in the brain.     

Age-Related Changes of Myelin Proteins in the Rat Peripheral Nervous System

Age-related changes in amounts of myelin proteins from rat sciatic nerve or spinal root were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). In the aged peripheral nerve myelin, the relative amounts of band 105K and proteins X and Y increased, whereas those of proteins P0 and P1 and band 190K decreased. Band 105K purified by preparative SDS-PAGE exhibited three bands of 105K, 28K, and 21K at the second electrophoresis. A repeated SDS-PAGE did not improve the purity of bank 105K, but increased the ratio of 21K to 28K. Compared with P0 protein, band 105K has a very similar peptide map pattern and amino acid composition, as well as the identical NH2 terminal residue, isoleucine. These findings suggest that band 105K is an aggregate form of P0 protein and its fragment, 21K. The 21K protein is a distinct entity from X protein. The quantitative and qualitative alterations in myelin proteins, as we report here, may reflect continuing demyelination and remyelination in aged peripheral nerves.