Biomedical subjects
Publications and source records attributed to G E Fagg.
The relative proportions of four myelin basic proteins (preL, L, preS,S) were determined in myelin subfractions prepared from the forebrains of quaking and littermate control mice. The distribution pattern of each protein was similar in both mutant and control fractions. The S component was the only basic protein present in low amounts in myelin from the mutant.
Myelin was purified from adult rabbit sciatic nerve by two procedures: discontinuous gradient centrifugation and continuous gradient zonal centrifugation. Two fractions were obtained from the discontinuous gradient. The fraction floating on 0.32 M sucrose and the fraction recovered from the 0.32/0.85 M sucrose interface showed typical myelin membranes by electron microscopy and typical myelin proteins by gel electrophoresis. The specific activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) decreased from the top to the bottom of the discontinuous gradient. The myelin separated by zonal centrifugation on a continuous sucrose gradient showed three distinct peaks (on monitoring optical density) at 0.10, 0.30 and 0.57 M sucrose. The latter peak yielded 92% of the material applied. The two minor peaks of low density exhibited high CNP and acetylcholinesterase (AChE) activities but the specific activity of both enzymes increased markedly at the heavy end of the gradient. The zonal fractions showed typical myelin proteins in all fractions by polyacrylamide gel electrophoresis but with important quantitative differences. These results indicate that PNS myelin shows significant heterogeneity.
Biochemical characterization of the development of myelin in vitro was extended to an analysis of myelin protein composition in cultures of explanted foetal rat spinal cord. Myelin fractions were isolated from pooled explants after 12-30 days in vitro and, for comparison, from the spinal cords of rats of equivalent developmental ages. Electron microscopic examination of the culture myelin fractions revealed the presence of multilamellar myelin fragments and some single membranes. All fractions were analyzed using a micro-polyacrylamide gel electrophoresis technique. Qualitatively similar protein profiles were observed for myelin isolated from either cultures or from spinal cords. Fractions from cultures contained a greater proportion of high molecular weight proteins than those from spinal cords, although with respect to the 'major' myelin proteins, a quantitatively similar developmental pattern was observed both in vivo and in vitro.
The carbonic anhydrase activity of myelin fractions isolated from the optic nerves of adult and immature (20-day-old) rats was examined. The specific activity in both total homogenate and myelin fractions was about 2-fold higher in adult than in immature animals and at both ages, the activity in the homogenate was higher than in myelin. After subfractionation by zonal gradient centrifugation, it was shown that carbonic anhydrase activity was greatest in the heaviest myelin particles at both ages. These data are consistent with the hypothesis that a small proportion of the total enzyme activity is localised in myelin.
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Myelin of considerable purity may be isolated from small (minimum 1 mg wet weight) samples of central nervous tissue, using a 4-step centrifugation procedure. The separation of myelin proteins by micro-linear gradient polyacrylamide gel electrophoresis yields similar results to those obtained by macro-scale (homogeneous) gel systems. These techniques have been employed for a preliminary study of the regional composition of myelin fractions from the Quaking mouse.