Effects of pronase and neuraminidase treatment on a myelin-associated glycoprotein in developing brain.

Rats (14 days old) were injected with [14c]fucose and young adult rats with [3H]fucose in order to label the myelin-associated glycoproteins. As previously reported, the major [14C]fucose-labelled glycoprotein in the immature myelin had a higher apparent molecular weight on sodium dodecyl sulphate/polyacrylamide gels that the [3H]fucose-labelled glycoprotein in mature myelin. This predominant doubly labelled glycoprotein component was partially purified by preparative gel electrophoresis and converted to glycopeptides by extensive Pronase digestion. Gel filtration on Sephadex G-50 separated the glycopeptides into several clases, which were designted A,B, C AND D, from high to low molecular weight. The 14C-labelled glycopeptides from immature myeline were enriched in the highest-molecular-weight class A relative to the 3H-labelled glycopeptides from mature myelin. Neuraminidase treatment of the glycoprotein before Pronase digestion greatly decreased the proportion of glycopeptides fractionating in the higher-molecular-weight classes and largely eliminated the developmental differences that were apparent by gel filtration. However, neuraminidase treatment did not decrease the magnitude of the developmental difference revealed by electrophoresing the intact glycoprotein on sodium dodecyl sulphate gels, although it did decrease the apparent molecular weight of the glycoprotein from both the 15-day-old and adult rats by an amount comparable in magnitude to that developmental difference. The results from gel filtration of glycopeptides indicate that there is a higher content of large molecular weight, sialic acid-rich oligosaccharide units in the glycoprotein of immature myelin. However, the higher apparent molecular weight for the glycoprotein from 15-day-old rats on sodium dodcyl sulphate gels is not due primarily to its higher sialic acid content.     

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.     

Lectin-binding proteins in central-nervous-system myelin. Detection of glycoproteins of purified myelin on polyacrylamide gels by [3h]concanavalin A binding.

Concanavalin A strongly agglutinates purified fragments of immature and mature rat brain myelin, but only weakly agglutinates mature bovine and human myelin fragments. A sensitive method involving [3H]concanavalin binding to sodium dodecyl sulphate/polyacrylamide gels was used to detect the concanavalin A-binding proteins in purified myelin. When applied to mature rat brain myelin proteins that had been labelled in vivo with [14C]fucose, the distribution of the [3H]concanavalin A on the gel was very similar to that of [14C]fucose with the major peak corresponding to the major myelin-associated glycoprotein. The technique revealed that the immature form of the myelin-associated glycoprotein with a slightly larger apparent molecular weight also bound concanavalin A, and that in purified immature rat myelin the quantitative importance of some of the other glycoproteins in binding concanavalin A was increased relative to the myelin-associated glycoprotein. The separated proteins of bovine and human myelin bound more [3H]-concanavalin A than those of rat myelin. In these species, the myelin-associated glycoprotein was a major concanavalin A-binding protein, although two higher-molecular-weight glycoproteins also bound significant quantities of [3H]concanavalin A. The results indicate that there are receptors for concanavalin A on the surface of rat, bovine and human myelin membranes and suggest that the myelin-associated glycoprotein is one of the principal receptors.     

EVIDENCE THAT THE MAJOR PROTEIN IN RAT SCIATIC NERVE MYELIN IS A GLYCOPROTEIN

Evidence is presented that the major protein of rat sciatic nerve myelin is a glycoprotein. When myelin proteins were separated by polyacrylamide gel electrophoresis, the major band which was stained with amido black–Coomassie blue was also stained with periodic acid-Schiff reagents for carbohydrate. Radioactive labelling of myelin in vivo with [³H]leucine and [¹⁴C]fucose, followed by electrophoresis of the proteins, indicated that with both isotopes the major labelled peak corresponded to the major stained band. In addition, a second smaller peak of [¹⁴C]fucose migrated ahead of the major peak. Delipidated myelin contained galactose, mannose, fucose and sialic acid.     

[3 5S]Sulfate incorporation into myelin glycoproteins I. Central nervous system

The in vivo incorporation of [^{3 5}S]sulfate and [³H]fucose into rat brain myelin was investigated. Most of the ^{3 5}S in the myelin was in sulfatide, but about 4% was associated with the residual proteins after chloroform/methanol extraction. Polyacrylamide gel electrophoresis of these proteins indicated that the major ^{3 5}S-labeled component corresponded to the major fucose-labeled glycoprotein. The labeling of this predominant glycoprotein with sulfate was more selective than with fucose, since there was relatively little incorporation of sulfate into some of the minor fucose-labeled glycoproteins. There was little or no ^{3 5}S associated with proteolipid or basic protein on polyacrylamide gels. The fucose-labeled glycoproteins were converted to glycopeptides by pronase digestion and separated into two major classes by gel filtration on Sephadex-G50. Only the higher molecular weight class contained significant amounts of ^{3 5}S. The association of ^{3 5}S with the glycopeptides was not due to binding of sulfatide or free inorganic sulfate. The results indicate that the predominant myelin-associated glycoprotein in rat brain is sulfated.     

Lectin-binding proteins in central-nervous-system myelin. Binding of glycoproteins in purified myelin to immobilized lectins

The capacities of immature and mature rat brain myelin, bovine myelin and human myelin to be agglutinated by soya-bean agglutinin, Ricinus communis agglutinin, wheatgerm agglutinin, and Lotus tetragonolobus agglutinin were examined. The first two lectins, which are specific for galactose and N-acetylgalactosamine, strongly agglutinated immature and mature rat myelin, weakly agglutinated bovine myelin, but did not affect human myelin. The other myelin and lectin combinations resulted in very weak or no agglutination. [(3)H]Fucose-labelled glycoproteins of purified adult rat brain myelin were solubilized with sodium dodecyl sulphate and allowed to bind to concanavalin A-Sepharose and each of the other lectins mentioned above, which had been immobilized on agarose. About 60% of the radioactive fucose was in glycoproteins that bound to concanavalin A-Sepharose and these glycoproteins could be eluted with solutions containing methyl alpha-d-mannoside and sodium dodecyl sulphate. Periodate/Schiff staining or radioactive counting of analytical gels showed that most of the major myelin-associated glycoprotein (apparent mol.wt. approx. 100000) bound to the concanavalin A, whereas the glycoproteins that did not bind were mostly of lower molecular weight. Preparative polyacrylamide-gel electrophoresis of the glycoprotein fraction that was eluted with methyl alpha-d-mannoside yielded a relatively pure preparation of the myelin-associated glycoprotein. Similar results were obtained with each of the other lectins, i.e. the myelin-associated glycoprotein was in the fraction that bound to the immobilized lectin. Double-labelling experiments utilizing [(3)H]fucose-labelled glycoproteins from adult myelin and [(14)C]fucose-labelled glycoproteins from 14-day-old rat brain myelin did not reveal any difference in the binding of the mature and immature glycoproteins to any of the immobilized lectins. The results in this and the preceding paper [McIntyre, Quarles & Brady (1979) Biochem. J.183, 205-212] suggest that the myelin-associated glycoprotein is one of the principal receptors for concanavalin A and other lectins in myelin, and that this property can be utilized for the purification of this glycoprotein.     

[3 5S]Sulfate incorporation into myelin glycoproteins II. Peripheral nervous tissue

The in vivo incorporation of [^{3 5}S]sulfate, [³H]fucose and [³H]leucine into sciatic nerve myelin was investigated. Polyacrylamide gel electrophoresis of the proteins indicated that the ^{3 5}S-labeling of proteins occurred almost exclusively in the major myelin protein. A smaller myelin glycoprotein migrating just ahead of the major one was labeled with [³H]fucose but did not incorporate ^{3 5}S to a detectable extent. There was little or no ^{3 5}S associated with basic proteins on polyacrylamide gels when the proteins were extracted with chloroform/methanol. Fucose-labeled myelin glycoproteins were converted to glycopeptides by pronase digestion. The glycopeptides gave a single peak on Sephadex G-50 in which the ³H and ^{3 5}S coincided. The association of ^{3 5}S with glycopeptides was not caused by binding of sulfatide or free inorganic sulfate. This study shows that the major myelin protein in the sciatic nerve of the rat is glycosylated and sulfated.     

EVIDENCE FOR THE CLOSE ASSOCIATION OF A GLYCOPROTEIN WITH MYELIN IN RAT BRAIN

Abstract— Myelin was purified from rats which had been injected intracerebrally with radioactive fucose in order to label specifically the glycoproteins. Myelin contained a small amount of fucose-labelled glycoproteins in comparison to that in other subcellular fractions, but polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate revealed a unique pattern of radioactive glycoproteins dominated by a major peak. The same glycoprotein was not prominent in the other subcellular fractions which were examined. This major glycoprotein in the myelin fraction was also labelled after injection with [³H]glucosamine or N -[³H]acetylmannosamine. It was the most intensely staining myelin protein when gels were treated with periodic acid-Schiff reagents, an indication that, in terms of protein-bound carbohydrate, it is the major glycoprotein in the myelin fraction. The glycoprotein was present in myelin purified from rats ranging in age from 14 days to 14 months. Extensive recycling of the myelin through the purification procedures did not significantly reduce the amount of glycoprotein in the myelin. Double label experiments with [³H]fucose and [¹⁴C]fucose were used to compare glycoproteins in myelin purified from white and grey matter, respectively, and from mixed homogenates of myelinated and unmyelinated brain. The results obtained from these experiments suggested that the glycoprotein is closely associated with myelin and that it is not in an unrelated contaminating structure. Possible locations of the glycoprotein are discussed. They include the myelin membrane itself, the oligodendroglial plasma membrane, and the axolemma of myelinated axons.     

Synthesis of glycoproteins in a single identified neuron of Aplysia californica

Incorporation of L-[³H]fucose into glycoproteins was studied in R2, the giant neuron in the abdominal ganglion of Aplysia. [³H]fucose injected directly into the cell body of R2 was readily incorporated into glycoproteins which, as shown by autoradiography, were confined almost entirely to the injected neuron. Within 4 h after injection, 67% of the radioactivity in R2 had been incorporated into glycoproteins; at least 95% of these could be sedimented by centrifugation at 105,000 g, suggesting that they are associated with membranes. Extraction of the particulate fraction with sodium dodecyl sulfate (SDS), followed by gel filtration on Sephadex G-200 and polyacrylamide gel electrophoresis in SDS revealed the presence of only five major radioactive glycoprotein components which ranged in apparent molecular weight from 100,000 to 200,000 daltons. Similar results were obtained after intrasomatic injection of [³H]N-acetylgalactosamine. Mild acid hydrolysis of particulate fractions released all of the radioactivity in the form of fucose. When ganglia were incubated in the presence of [³H]fucose, radioactivity was preferentially incorporated into glial cells and connective tissue. In contrast to the relatively simple electrophoretic patterns obtained from cells injected with [³H]fucose, gel profiles of particulate fractions labeled with [¹⁴C]valine were much more complex.     

Schwann Cell Surface Proteins and Glycoproteins

Abstract: To identify surface sialoglycoproteins of rat Schwann cells and to compare molecular weights of these sialoglycoproteins with those present in rat peripheral nervous system myelin, we prepared Schwann cells from sciatic nerves of 1–3-day-old rats and cultured them in monolayer. Surface sialoglycoproteins of the cultured cells were tritium-labeled by the periodateborohydride procedure and compared with sialoglycoproteins of adult rat peripheral nervous system myelin by fluorography following polyacrylamide slab gel electrophoresis in sodium dodecyl sulfate. Three radioactive bands with apparent molecular weights of 114,000–132,000, 105,000–115,000, and 44,000–56,000 were observed in both the Schwann cell and myelin preparations. Bands of similar apparent molecular weights were noted in Schwann cells metabolically radiolabeled with d -[1,6-³H]glucosamine. A band co-migrating with myelin P₀ glycoprotein was the most intensely radiolabeled of all peptides in periodate-B³H₄^?treated myelin, but was present in only trace amounts in periodate-B³H₄^? or d -[1,6-³H]glucosamine radiolabeled Schwann cells. Many presumably non-myelin glycoproteins were identified in the cultured Schwann cells by the periodate-borohydride procedure and by incubation of the cells with d -[1,6-³H]glucosamine. An immunoprecipitation technique was used to detect radiolabeled peptides in a nonionic detergent extract of freshly prepared, surface-radioiodinated Schwann cells that were bound by a rabbit anti-Schwann cell serum preabsorbed with rat fibroblasts. Many radioactive peptides were detected in the immunoprecipitate, but the two most intensely radiolabeled had apparent molecular weights of 105,000–115,000 and 95,000–106,000. This study has identified a number of glycoproteins synthesized by cultured rat Schwann cells which resemble in apparent molecular weight the glycoproteins expressed in rat peripheral nervous system myelin and has defined Schwann cell surface proteins recognized by a specific anti-rat Schwann cell antiserum.     

Effects of Inhibitors of Oligosaccharide Processing on P0Protein Synthesis and Incorporation into PNS Myelin

Four inhibitors of oligosaccharide processing were used to investigate their effects on the transport of PNS myelin glycoproteins through the secretory pathway, as well as to gain further insight into the structure of the oligosaccharide chains of the P0 and 19-kDa glycoproteins. Several different inhibitors of oligosaccharide processing were incubated with chopped peripheral nerves from young rats (21-24 days of age) and the uptake of 14C-amino acid and [3H]fucose or [3H]mannose was measured in P0 and the 19-kDa glycoprotein after separation of homogenate and myelin proteins on polyacrylamide gels. [3H]Mannose was not found as suitable as [3H]fucose as an oligosaccharide precursor because glucose used as an energy source profoundly inhibited the uptake of [3H]mannose. The substitution of pyruvate as an energy source, however, resulted in incomplete glycosylation, poor amino acid uptake, and truncated oligosaccharide chains. Endoglycosidase H cleaved approximately 50% of the P0 labeled with [3H]fucose and 14C-amino acid. The lower molecular weight protein resulting from endoglycosidase H cleavage contained approximately one-half the [3H]fucose label on the protein, whereas one-half remained on the oligosaccharide chain of the undegraded P0, indicating that at least one-half the P0 has a hybrid structure. Deoxynojirimycin, deoxymannojirimycin, and castanospermine inhibited incorporation of [3H]fucose into the oligosaccharide chains of P0 and the 19-kDa glycoprotein as predicted from their action in blocking various stages of trimming of high mannose structures before the addition of fucose. P0 synthesized in the presence of these inhibitors was cleaved to a greater extent by endoglycosidase H than the normal protein, indicating increased vulnerability to this enzyme with arrest of normal processing. Similar results were obtained for the 19-kDa glycoprotein. Both the incompletely processed P0 and the 19-kDa glycoprotein formed in the presence of these inhibitors appeared to be transported normally into myelin.     

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.     

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.     

A comparison of [3H]galactose and [3H]fucose uptake with the morphological and histochemical changes observed in mucous secretion in chemically induced rat colonic carcinoma

Summary The alterations in carbohydrate metabolism which occur in the distal colon of rats during carcinogenesis induced by dimethylhydrazine were investigated using [³H]galactose and [³H]fucose as glycoprotein precursors.A statistically significant decrease in [³H]galactose uptake was observed in dysplastic epithelia. These findings are consistent with the alterations in mucin composition with predominance of sialomucins shown in these areas by histochemical methods. Furthermore, changes in the gradient of [³H]galactose incorporation along the crypt epithelium were also found in the histological and histochemically non-involved colonic mucosa of dimethylhydrazine-treated rats, as compared with controls.No significant variations were seen in [³H]fucose incorporation.These results correlate well with our previous histochemical observations and are further evidence of the profound alterations in glycoprotein synthesis affecting the whole colonic mucosa during carcinogenesis.     

Respiration of 14CO2 by intact animals of various species given l-[1-14C]fucose or d-[1-14C]arabinose

The production of ¹⁴CO₂ from l-[1-¹⁴C]fucose and d-[1-¹⁴C]arabinose has been studied in five mammalian species.Cats, guinea pigs, mice, and rabbits respired about 22% of the label of l[1-¹⁴C]fucose or of d-[1-¹⁴C]arabinose within 6 h after intraperitoneal injection of the sugar. Rats respired only 1.5% of the l-fucose label and 5% of the d-arabinose label in the same time period.Liver homogenates from cat, guinea pig, and rabbit produced significantly more ¹⁴CO₂ from l-[1-¹⁴C]fucose or d-[1-¹⁴C]arabinose than mouse or rat liver homogenates. Unlike those of the other species, guinea pig liver homogenates had very low l-fucose dehydrogenase activity.The results suggest that substantial catabolism of l-fucose and d-arabinose occurs in the tissues of some animal species. Investigators wishing to employ l-fucose as a tracer of glycoprotein metabolism must, therefore, ensure that the species that they employ does not metabolize l-fucose to products interfering with their studies.     

The degradation and turnover of fucosylated glycoproteins in the plasma membrane of a neuroblastoma-cell line

When monolayer cultures of neuroblastoma N2a cells were prelabelled with [³H]fucose to steady state, and then reincubated in complete medium in the presence of unlabelled 40mm-l-fucose, there was a rapid metabolism of fucosylated cellular macromolecules and the specific radioactivity of the acid-insoluble material decreased by 22% within 2h. After this period of time the remaining radioactive glycoproteins appeared to be more stable and the rate of loss of specific radioactivity markedly decreased. Since fucose is known to be associated predominantly with plasma-membrane components, the analysis of fucosylated glycoproteins was characterized in plasma-membrane fractions by polyacrylamide-gel electrophoresis. Two experimental approaches were used to measure glycoprotein degradation and turnover in the cell-surface membranes. In one set of experiments, with a similar incubation procedure to that used with intact cells, three membrane components were rapidly degraded (150000, 130000 and 48000 daltons), but another surface glycoprotein (68000 daltons) appeared to be more slowly metabolized than the mean rate of glycoprotein degradation. The relationship of the degradation of membrane glycoproteins to their turnover was analysed by dual-label experiments that used both [¹⁴C]fucose and [³H]fucose. Glycoproteins of the surface membrane of neuroblastoma cells were found to turn over at heterogeneous rates. The components mentioned above that exhibited significantly rapid rates of degradation, were also shown to turn over more rapidly than the average surface component. In addition to the membrane components detected by the use of only [³H]fucose, dual-label experiments illustrated that numerous surface glycoproteins were metabolized more rapidly or slowly than most of the cell-surface constituents.     

Biosynthesis and release of glycoproteins by human skin fibroblasts in culture

1. Confluent human skin fibroblasts maintained in a chemically defined medium incorporate l-[1-³H]fucose in a linear manner with time into non-diffusible macromolecules for up to 48h. Chromatographic analysis demonstrated that virtually all the macromolecule-associated ³H was present as [³H]fucose. 2. Equilibrium CsCl-density-gradient centrifugation established that [³H]fucose-labelled macromolecules released into the medium were predominantly glycoproteins. Confirmation of this finding was provided by molecular-size analyses of the [³H]fucose-labelled material before and after trypsin digestion. 3. The [³H]fucose-labelled glycoproteins released into fibroblast culture medium were analysed by gel-filtration chromatography and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. These techniques demonstrated that the major fucosylated glycoprotein had an apparent mol.wt. of 230000–250000; several minor labelled species were also detected. 4. Dual-labelling experiments with [³H]fucose and ¹⁴C-labelled amino acids indicated that the major fucosylated glycoprotein was synthesized de novo by cultured fibroblasts. The non-collagenous nature of this glycoprotein was established by three independent methods. 5. Gel-filtration analysis before and after reduction with dithiothreitol showed that the major glycoprotein occurs as a disulphide-bonded dimer when analysed under denaturing conditions. Further experiments demonstrated that this glycoprotein was the predominant labelled species released into the medium when fibroblasts were incubated with [³⁵S]cysteine. 6. The relationship between the major fucosylated glycoprotein and a glycoprotein, or group of glycoproteins, variously known as fibronectin, LETS protein, cell-surface protein etc., is discussed.     

Incorporation of glycoproteins into peripheral nerve myelin

Peripheral nerve myelin contains a dominant low molecular weight glycoprotein called Po. To study the metabolism of this glycoprotein, tritiated fucose was injected into the peripheral nerves of adult mice and developing rats, and the temporal distribution of label was examined by autoradiography and gel electrophoresis. Mice and rat pups, injected with fucose, were sacrificed from 1 h to 98 days later. Series of autoradiographs were prepared. At the shortest labeling periods, newly formed product was confined to juxtanuclear Schwann cell cytoplasm, in association with regions rich in Golgi apparatus. After longer labeling periods, silver grain levels in Schwann cell cytoplasm decreased; concomitantly, there was an increase of silver grains associated with myelin. In adult animals, label associated with myelin was concentrated over outer layers of thickly myelinated fibers. Even at the longest time intervals examined (72 and 98 days), this distribution of label was largely retained. In contrast, in developing animals, label became associated with inner layers of the thicker sheaths. At no time was label observed over axons. Gel electrophoresis revealed that tritiated fucose was a suitable precursor for the faster migrating peripheral nerve glycoprotein(s). At all times examined, there was a single major peak of radioactivity that co-migrated on sodium dodecyl sulfate (SDS) acrylamide gels with the Po protein. Sometimes, a faster migrating shoulder of radioactivity was noted. With increased labeling periods, there was an enrichment of radioactivity associated with Po, indicative of a relatively slow turnover rate.     

External labeling of galactose in surface membrane glycoproteins of the intact myelin sheath.

The molecular organization of surface galactose residues in glycoproteins of the intact myelin sheath was investigated using the enzymatic membrane probe, galactose oxidase. Rat spinal cords treated under physiological conditions with this nonpermanent probe were labeled specifically in galactose residues by reduction with tritiated sodium borohydride. The enzymatically modified proteins from isolated myelin were analyzed electrophoretically and their specific radioactivities determined. Results indicated tritium label associated with a surprising variety of high molecular weight proteins. The most extensively labeled peak corresponded to the major myelin glycoprotein as indicated by the coincidence of tritium label with that of [14C]fucose used as an internal marker for the glycoproteins. The radioactivity associated with this protein was 1.1 to 2.7 times higher after treatment with galactose oxidase when compared to reduction in the absence of the enzyme and 1.4 to 4.8 times higher when oxidized and reduced after prior treatment with neuraminidase. The results suggest a complex heterogeneity of minor glycoproteins associated with isolated myelin. It is concluded that from this complexity of glycoproteins, a major glycoprotein is at least partially localized on the external surface of either the intact myelin sheath or the closely associated oligodendroglial plasma membrane. Such a localization of this glycoprotein and the probable localization of the other glycoproteins enhances their potential role in specific interactions in the process of mpyelination or myelin maintenance.     

Receptors for Dolichos biflorus agglutinin: New cell surface markers on a spontaneous leukemia

$\text{Dolichos biflorus}$ agglutinin (DBA), which is specific for terminal α-N-acetylgalactosamine, bound to a spontaneous leukemia cell of $\text{GRS}\text{A}$ mice, but not to lymphoid cells of the host. The DBA receptors were isolated from the leukemia cell labeled with [³H]-galactose after detergent solubilization and affinity chromatography on DBA-agarose. The major component of the receptors migrated as a glycoprotein of apparent molecular weight 100,000 upon SDS gel electrophoresis. Alkaline treatment degraded the glycoproteins, releasing oligosaccharides of molecular weight around 1,000.