Cross antigenicity of proteins released from peripheral nerves of four classes of vertebrates: a fluorescent immunohistochemical study
Garwood, Margaret Mary
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The efflux protein from frog sciatic nerve was purified, quantitated, and its electrophoretic properties were determined. The 8th and 9th dorsal root ganglia and attached sciatic nerves from 6 frogs were excised and placed in a two-compartment chamber. The ganglia were incubated in ('3)H-leucine in Ringer (2000 (mu)Ci/ml) and oxygenated for the duration of the run (22 hours). The axons were bathed in oxygenated frog Ringer with cycloheximide to inhibit the incorporation of isotope into protein by non-neuronal cells. At the end of the run, the nerve trunk preparations were removed and placed into 10% trichloroacetic acid to fix the nerve for axonal transport studies and remove any unincorporated radioactivity. The efflux-containing solution in the outer compartment (containing the nerve trunks) was collected and purified of plasma proteins ("treated") by "batch" immunoaffinity chromatography. Protein quantitation revealed that the amount of protein released from the nerve trunks was approximately 5 (mu)g protein per 100 mg tissue per 24 hours. Electrophoretic characterization of the efflux protein was accomplished by SDS and Tris polyacrylamide gel electrophoresis. The efflux protein, in both electrophoretic systems, was separable into three peaks. On SDS polyacrylamide gels, the three peaks were shown to have molecular weights of 110,000, 65,000, and 55,000 daltons. For the immunological and histological studies of the efflux protein, antibodies to purified efflux protein were produced for use with the indirect fluorescent antibody technique. By this technique, the released efflux protein was immunologically cross-reactive across a range of vertebrate classes; also, the three-dimensional distribution of the efflux protein in tissue sections appeared tube-like and suggests a possible association with axolemmal or myelin membranes.