The PO Protein of Chick Sciatic Nerve Myelin: Purification and Partial Characterization

Abstract: The PO protein of the myelin of chick sciatic nerve was isolated and purified by propanoic acid extraction of peripheral nervous system (PNS) myelin, delipidation, Sepharose CL-6B chromatography in the presence of sodium dodecyl sulfate (SDS), and preparative SDS-polyacrylamide gel electro-phoresis (PAGE). Approximately 15% of the PO protein in the sciatic nerve myelin was recovered in a homogeneous state. The purified protein monomer has an apparent molecular weight of 32.1K as determined by gel electrophoresis. The PO protein undergoes extensive aggregation during exhaustive dialysis and freeze-drying and yields stable dimers, trimers, and tetramers. The aggregation of the PO protein after freeze-drying is independent of the presence of a reducing agent (2-mercaptoethanol) in the solubilizing medium. The PO protein is a glycoprotein. The amino acid composition of the chick PO protein indicates a definite species difference when compared with mammalian PO proteins although the NH₂-terminal isoleucine residue seems to have been retained during evolution.     

SOME PROPERTIES OF A MAJOR STRUCTURAL GLYCOPROTEIN OF SCIATIC NERVE

The major protein of rat sciatic nerve is a glycoprotein which consists of a protein moiety attached to galactose, mannose and perhaps other sugars. On controlled tryptic digestion, it splits into a glycopeptide and a smaller fragment similar in molecular size to peripheral nerve basic proteins. Both the basic proteins of peripheral nervous system (PNS) myelin and the glycoprotein are antigenically active when administered to guinea pigs and produce sciatic nerve lesions similar to those described for experimental allergic neuritis. It is suggested from the amino acid analysis and its antigenic properties that the protein moiety of the glycoprotein may contain a sequence which is similar to the basic proteins of PNS myelin.     

A 42 K protein of chick sciatic nerve is immunologically related to PO protein of peripheral nerve myelin

The major protein (PO) in PNS myelin is an integral membrane glycoprotein with a molecular weight of about 30 K. The level of PO protein in the developing sciatic nerve of the chicken was monitored by a solid-phase immunoassay and densitometry of Coomassie blue stained polyacrylamide gels. The most rapid rate of accumulation of PO protein occurred after 16 days of embryonic development. In addition to the 30 K PO protein, a number of higher molecular weight proteins could be distinctly detected by immunoblotting. Amongst these high molecular weight proteins, a species with an apparent molecular weight of 42 K was specifically immunostained with epitope-selected polyclonal antibodies against PO protein. This 42 K protein could be first detected after 16 days of embryonic development and increased rapidly following the pattern of myelination in the sciatic nerve. The enzyme endoglycosidase F, which specifically removes N-asparagine linked high mannose and complex carbohydrates from glycoproteins, converted the PO and 42 K proteins to lower molecular weight forms, which could be specifically immunostained by epitope selected polyclonal antibodies to the PO protein. Subcellular fractionation of the 17-day embryonic nerve demonstrated that the 42 K protein was enriched in myelin and microsomal subfractions relative to the total homogenate. These results indicate that the 42 K immuno-crossreactive protein might be chemically and functionally related to the PO protein of the PNS myelin.Abbreviations used CNP 2–3-cyclic-nucleotide 3-phosphodiesterase (EC 3.1.4.37) - EDTA ethylene(dinitrilo)tetraacetic acid - Endo F endo--N-acetylglucosaminidase F (EC 3.2.1.96) - NADPH Cyt. Red. NADPH-cytochrome C reductase (EC 1.6.2.4) - 5-NPase 5-ribonucleotide phosphohydrolase (EC 3.1.3.5) - PNS peripheral nervous system - SDS sodium dodecyl sulfate - TPPase thiamine pyrophosphatase (EC 3.1.6.X)     

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.     

A biochemical comparison of Xenopus laevis and mammalian myelin from the central and peripheral nervous systems.

Myelin purified from the central nervous system of Xenopus laevis contained the same major lipid and protein components as human myelin. However, some minor differences in the myelin proteins were noted. The Xenopus basic protein had a higher apparent mol wt. on sodium dodecyl sulfate gels than the corresponding mammalian protein. The absolute specific activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase in the Xenopus myelin was considerably higher than in mammals. There were differences in the high mol wt. proteins, and the glycoproteins in Xenopus myelin were more heterogeneous than those in mammals. Peripheral myelin from Xenopus sciatic nerve was compared with that from the rat. The lipids in the two types of myelin were similar. There was a major glycoprotein in the Xenopus myelin corresponding to the P0 protein and a basic protein of slightly larger mol wt. than the P1 protein of rat myelin.     

Biochemical Demonstration of the Myelin-Associated Glycoprotein in the Peripheral Nervous System

Abstract: Recent immunocytochemical studies indicated that the myelin-associated glycoprotein (MAG) is localized in the periaxonal region of central nervous system (CNS) and peripheral nervous system (PNS) myelin sheaths but previous biochemical studies had not demonstrated the presence of MAG in peripheral nerve. The glycoproteins in rat sciatic nerves were heavily labeled by injection of [³H]fucose in order to re-examine whether MAG could be detected chemically in peripheral nerve. Myelin and a myelin-related fraction, WI, were isolated from the nerves. Labeled glycoproteins in the PNS fractions were extracted by the lithium diiodosalicylate (LIS)-phenol procedure, and the extracts were treated with antiserum prepared to CNS MAG in a double antibody precipitation. This resulted in the immune precipitation of a single [³H]fucose-labeled glycoprotein with electrophoretic mobility very similar to that of [¹⁴C]fucose-labeled MAG from rat brain. A sensitive peptide mapping procedure involving iodination with Bolton-Hunter reagent and autoradiography was used to compare the peptide maps generated by limited proteolysis from this PNS component and CNS MAG. The peptide maps produced by three distinct proteases were virtually identical for the two glycoproteins, showing that the PNS glycoprotein is MAG. The MAG in the PNS myelin and Wl fractions was also demonstrated by Coomassie blue and periodic acid-Schiff staining of gels on which the whole US-phenol extracts were electrophoresed, and densitometric scanning of the gels indicated that both fractions contained substantially less MAG than purified rat brain myelin. The presence of MAG in the periaxonal region of both peripheral and central myelin sheaths is consistent with a similar involvement of this glycoprotein in axon-sheath cell interactions in the PNS and CNS.     

Immunochemical Characterization of Peripheral Nervous System Myelin 170,000-Mr Glycoprotein

A recently described 170,000-Mr glycoprotein, specific to peripheral nervous system (PNS) myelin, was purified from rat PNS myelin by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and used to immunize guinea pigs and rabbits. The resultant antisera proved specific for 170,000-Mr glycoprotein by enzyme-linked immunosorbent assay, by immunoprecipitation of the appropriate peptide from solubilized PNS myelin, and by immunoblot analysis of rat PNS myelin. The anti-rat 170,000-Mr glycoprotein antisera cross-reacted with proteins of similar molecular weight in human and bovine PNS myelin, but such proteins were not detected in human or rat CNS myelin or other rat tissues. The 170,000-Mr glycoprotein was also detected by this immunoblot procedure in recently isolated rat Schwann cells but not in those kept in culture for greater than or equal to 3 days. By indirect immunofluorescent microscopy, anti-rat 170,000-Mr glycoprotein antibody bound to rat PNS myelin sheaths but not to other rat tissues. Together, these studies indicate the 170,000-Mr glycoprotein is specific to PNS myelin of several species and that a neuronal influence may be required for its expression by 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.     

Biochemical demonstration of the myelin-associated glycoprotein in the peripheral nervous system

Recent immunocytochemical studies indicated that the myelin-associated glycoprotein (MAG) is localized in the periaxonal region of central nervous system (CNS) and peripheral nervous system (PNS) myelin sheaths but previous biochemical studies had not demonstrated the presence of MAG in peripheral nerve. The glycoproteins in rat sciatic nerves were heavily labeled by injection of [3H]fucose in order to re-examine whether MAG could be detected chemically in peripheral nerve. Myelin and a myelin-related fraction, W1, were isolated from the nerves. Labeled glycoproteins in the PNS fractions were extracted by the lithium diiodosalicylate (LIS)-phenol procedure, and the extracts were treated with antiserum prepared to CNS MAG in a double antibody precipitation. This resulted in the immune precipitation of a single [3H]fucose-labeled glycoprotein with electrophoretic mobility very similar to that of [14C]fucose-labeled MAG from rat brain. A sensitive peptide mapping procedure involving iodination with Bolton-Hunter reagent and autoradiography was used to compare the peptide maps generated by limited proteolysis from this PNS component and CNS MAG. The peptide maps produced by three distinct proteases were virtually identical for the two glycoproteins, showing that the PNS glycoprotein is MAG. The MAG in the PNS myelin and W1 fractions was also demonstrated by Coomassie blue and periodic acid-Schiff staining of gels on which the whole LIS-phenol extracts were electrophoresed, and densitometric scanning of the gels indicated that both fractions contained substantially less MAG than purified rat brain myelin. The presence of MAG in the periaxonal region of both peripheral and central myelin sheaths is consistent with a similar involvement of this glycoprotein in axon-sheath cell interactions in the PNS and CNS.     

ASTROGLIAL UPTAKE IS MODULATED BY EXTRACELLULAR K+

Abstract— Developmental changes of myelin proteins in chick sciatic nerve were studied at the stage of myelination by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. The myelin of adult hen peripheral nervous system (PNS) contained two glycoproteins (BR-P0 and PASII), both of which are unique to PNS myelin, in addition to the basic encephalitogenic protein, BP, which is common to CNS and PNS myelin. The other basic protein (BF-P2) found in the PNS of other species was not definitely detectable in hen PNS. At the early stages of myelination (from 14 to 18 embryonic days) the amounts of myelin proteins increased rapidly in parallel with the increase in number of layers of the myelin sheath of the PNS. At 14 embryonic days high molecular weight proteins were dominant, while myelin specific proteins were barely detectable in the PNS myelin fraction. At 18 embryonic days, however, BR-PO, BP and PASII proteins became the main protein components of the PNS myelin, whereas high molecular weight proteins decreased in quantitative importance during development. At the early stage of myelination other glycoproteins were also detectable in the PNS myelin. Radioactive fucose was actively incorporated into the two glycoproteins, BR-P0 and PASII, at the early stage of myelination in vivo. These results suggested that myelin proteins especially glycoproteins, may play an important role in PNS myelin formation.     

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.     

High performance liquid chromatography of integral glycoproteins of peripheral nerve myelin

Peripheral nerve myelin contains a large quantity of integral glycoproteins, such as PO and PASII protein. The present paper reports a fast and sensitive method for separation of these glycoproteins. High performance liquid chromatography (HPLC) with TSK-GEL 3000 SW column in the presence of sodium dodecyl sulfate (SDS) or lithium dodecyl sulfate (LDS) was used. Whereas the separation of PO and PASII was inadequate with low concentrations of the detergent, better separation profiles were obtained with high concentrations (1–2%) of the detergent in 0.1 M phosphate buffer. The two glycoproteins were able to be purified by rechromatography. High concentration of the detergent presumably diminished hydrophobic interaction between these glycoproteins. LSD-phosphate, SDS-lithium citrate or SDS-Tris buffer as an eluent was also compared with SDS-phosphate system. This method will be applicable to the detection and purification of proteins from myelin or other organelles.     

Peripheral Nervous System Myelin and Schwann Cell Glycoproteins: Identification by Lectin Binding and Partial Purification of a Peripheral Nervous System Myelin-Specific 170,000 Molecular Weight Glycoprotein

Radioiodinated lectins were used to detect glycoproteins of peripheral nervous system (PNS) myelin (rat, human, bovine) and cultured rat Schwann cells. Proteins were resolved by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and transferred to nitrocellulose filters. The filters were overlaid with radioiodinated lectins of known saccharide affinities. These included concanavalin A, Helix pomatia, Limulus polyphemus, Maclura pomifera, peanut, soybean, Ulex europaeus, and wheat germ agglutinins. Inclusion of the appropriate monosaccharide in the overlay solution (0.2 M) inhibited lectin binding to the nitrocellulose-fixed proteins. Fluorography permitted identification of 26 myelin glycoproteins and many more in Schwann cells. All lectins labeled a band present in myelin, but not Schwann cells, corresponding to the major PNS myelin protein, P0. Our attention focused on a high-molecular-weight myelin glycoprotein [apparent molecular weight (Mr) 170,000], which appeared abundant by Coomassie Blue staining and which was heavily labeled by all lectins except concanavalin A. A protein with approximately this Mr and lectin-binding pattern was present in human and bovine PNS myelin as well, but not detected in rat Schwann cells, CNS myelin, liver and fibroblast homogenates, or cultured bovine oligodendroglia. Hence this 170,000 Mr glycoprotein is apparently unique to PNS myelin.     

Glycoprotein Biosynthesis in Peripheral Nervous System Myelin: Effect of Tunicamycin

Abstract: The effect of an inhibitor of N -glycosylation of glycoproteins, tunicamycin, on synthesis of PNS myelin proteins was investigated in vitro by using chopped sciatic nerves or spinal roots of 21-day-old Wistar rats. Tunicamycin when incubated with these nerves in the presence of ³H-labeled fucose, mannose, or glucosamine inhibited the uptake of radioactivity into myelin proteins including some high-molecular-weight proteins, P₀, 23K protein, and 19K protein by amounts ranging from 42 to 79%. Uptake of ¹⁴Camino acid mixture was inhibited much less by tunicamycin, but a new radioactive protein peak appeared when the protein mixtures had been separated by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. This protein ran directly in front of the P₀ peak, did not correspond to any bands stained by Fast green, and was not labeled by fucose. This peak appeared in increasing larger proportions with progressive time of incubation of nerves with ³H amino acids in the presence of tunicamycin. The new protein, which cross-reacts with P₀ antiserum, was tentatively identified as a nonglycosylated P₀ protein that appears to be almost as well incorporated as P₀ into the subcellular fraction containing myelin. At this time it is not possible to determine whether the unglycosylated P₀ is actually assembled into a site and configuration like that of P₀.     

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.     

The Glycoprotein Composition of Peripheral Nervous System Myelin Subfractions

Two fractions were isolated by continuous density gradient centrifugation from total particulate matter of rabbit sciatic nerves: a minor fraction, B, consisting of small-sized membrane fragments and a major fraction, C, of characteristic multilayered myelin figures, with maxima at 0.33 and 0.58 M-sucrose, respectively. In comparison with C, fraction B was enriched in CNPase and alkaline phosphatase activities and the P0, 23K and Z proteins, but was virtually devoid of basic protein. The glycoprotein composition of all fractions was examined with four fluorescein isothiocyanate-labelled lectins (WGA, Con A, RCA-60, U.E.). These revealed the presence of six glycoproteins in all fractions with similar lectin binding capacities and molecular weights ranging from 35,500 to 16,000, of which P0 was the predominant component. Material found on the heavy side of fraction C was characterized by the presence of a multitude of glycoproteins which bound variable proportions of the four different lectins, suggesting substantial variations in their carbohydrate moieties. Their absence from the central portion of fraction C points to a location other than that of compact PNS myelin.     

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.     

A biochemical comparison of Xenopus laevis and mammalian myelin from the central and peripheral nervous systems

Myelin purified from the central nervous system of Xenopus laevis contained the same major lipid and protein components as human myelin. However, some minor differences in the myelin proteins were noted. The Xenopus basic protein had a higher apparent mol wt. on sodium dodecyl sulfate gels than the corresponding mammalian protein. The absolute specific activity of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in the Xenopus myelin was considerably higher than in mammals. There were differences in the high mol wt. proteins, and the glycoproteins in Xenopus myelin were more heterogeneous than those in mammals. Peripheral myelin from Xenopus sciatic nerve was compared with that from the rat. The lipids in the two types of myelin were similar. There was a major glycoprotein in the Xenopus myelin corresponding to the P₀ protein and a basic protein of slightly larger mol wt. than the P₁ protein of rat myelin.     

Regulation of Myelin P0 Glycoprotein Synthesis in Cultured Rat Schwann Cells and Continuous Rat PNS Cell Lines

We studied the effects of agents that raise intracellular cyclic AMP on synthesis of myelin components by cultured neonatal rat sciatic nerve Schwann cells and by continuous PNS cell lines derived from the fusion of neonatal rat sciatic nerve Schwann cells with rat RN22 Schwannoma. Treatment with N6,2'-O-dibutyryl cyclic AMP (dibutyryl cyclic AMP) caused a fourfold increase in Schwann cell incorporation of 35SO4 into sulfogalactosylceramide (sulfatide), and elicited a 10- to 20-fold increase in such incorporation by the continuous PNS cell lines; a similar effect on PNS cell line sulfatide radiolabelling was obtained with forskolin. Cultured Schwann cells expressed barely detectable levels of myelin P0 glycoprotein (P0) mRNA and myelin basic protein (MBP) mRNA. Treatment of the Schwann cells with axolemmal fragments or with dibutyryl cyclic AMP did not elicit a detectable increase in the levels of these mRNAs. The PNS cell lines constitutively expressed much higher levels of P0 mRNA than did the Schwann cells, and synthesized immunochemically demonstrable P0 glycoprotein, but did not express MBP. Treatment of the PNS cell lines with dibutyryl cyclic AMP markedly reduced expression of P0 mRNA and also diminished immunoreactive P0 glycoprotein. These PNS cell lines should prove useful for further studies of the control of Schwann cell differentiation.     

Bony Fish Myelin: Evidence for Common Major Structural Glycoproteins in Central and Peripheral Myelin of Trout

Peripheral nervous system (PNS) myelin from the rainbow trout (Salmo gairdneri) banded at a density of 0.38 M sucrose. The main myelin proteins consisted of (1) two basic proteins, BPa and BPb (11,500 and 13,000 MW, similar to those of trout central nervous system (CNS) myelin proteins BP1 and BP2), and (2) two glycosylated components, IPb (24,400 MW) and IPc (26,200 MW). IPc comigrated with trout CNS myelin protein IP2 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas trout CNS myelin protein IP1 had a lower molecular weight (23,000). Following two-dimensional separation, however, both IPb and IPc from PNS showed two components; the more acidic component of IPc comigrated with IP2 from CNS. PNS tissue autolysis led to the formation of IPa (20,000 MW), consisting of two components in isoelectric focusing of which again the more acidic one comigrated with the CNS autolysis product IP0. Limited enzymatic digestion of isolated IP proteins from PNS and CNS led to closely similar degradation patterns, being most pronounced in the case of IP2 and IPc. Immunoblotting revealed that all IP components from trout PNS and CNS myelins reacted with antibodies to trout IP1 (CNS) and bovine P0 protein (PNS) whereas antibodies to rat PLP (CNS) were entirely unreactive. All BP components from trout PNS and CNS myelins bound to antibodies against human myelin basic protein. On the basis of these studies trout PNS and CNS myelins contain at least one common IP glycoprotein, whereas other members of the IP myelin protein family appear closely related. In the CNS myelin of trout the IP components appear to replace PLP.(ABSTRACT TRUNCATED AT 250 WORDS)