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

ABSTRACT: 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.

SUBMITTER: McIntyre LJ

PROVIDER: S-EPMC1161548 | biostudies-other | 1979 Nov

REPOSITORIES: biostudies-other

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Project description: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.

Project description:Hydrogels are important structural and operative components of microfluidic systems, finding diverse utility in biological sample preparation and interrogation. One inherent challenge for integrating hydrogels into microfluidic tools is thermodynamic molecular partitioning, which reduces the in-gel concentration of molecular solutes (e.g., biomolecular regents), as compared to the solute concentration in an applied solution. Consequently, biomolecular reagent access to in-gel scaffolded biological samples (e.g., encapsulated cells, microbial cultures, target analytes) is adversely impacted in hydrogels. Further, biomolecular reagents are typically introduced to the hydrogel via diffusion. This passive process requires long incubation periods compared to active biomolecular delivery techniques. Electrotransfer is an active technique used in Western blots and other gel-based immunoassays that overcomes limitations of size exclusion (increasing the total probe mass delivered into gel) and expedites probe delivery, even in millimeter-thick slab gels. While compatible with conventional slab gels, electrotransfer has not been adapted to thin gels (50-250 μm thick), which are of great interest as components of open microfluidic devices (vs enclosed microchannel-based devices). Mechanically delicate, thin gels are often mounted on rigid support substrates (glass, plastic) that are electrically insulating. Consequently, to adapt electrotransfer to thin-gel devices, we replace rigid insulating support substrates with novel, mechanically robust, yet electrically conductive nanoporous membranes. We describe grafting nanoporous membranes to thin-polyacrylamide-gel layers via silanization, characterize the electrical conductivity of silane-treated nanoporous membranes, and report the dependence of in-gel immunoprobe concentration on transfer duration for passive diffusion and active electrotransfer. Alternative microdevice component layers─including the mechanically robust, electrically conductive nanoporous membranes reported here─provide new functionality for integration into an increasing array of open microfluidic systems.

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

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