Properties and localization of the goldfish proteinp68/70.

Previous attempts to characterize the molecular events that support successful regeneration of axotomized goldfish retinal ganglion cells (RGC's) have led to the discovery of an acidic protein doublet (p68/70) which displays an increased incorporation of $\sp{35}$S-methionine following axotomy. This protein is approximately 68-70 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and is axonally transported within the optic nerve. The experiments reported here further characterize the properties and localization of this protein. The apparent molecular weight of p68/70 in SDS-PAGE was found to vary inversely with acrylamide concentration, suggesting that the previously assigned molecular weight of 68-70 kDa is an overestimate. Corrected molecular weights of 53.4 and 54.9 kDa were calculated for the two components of p68/70 by analyzing their migration on gels of differing acrylamide concentrations. In addition, the relationship of p68/70 to the following well-defined proteins was investigated: kinesin light chain, the low molecular weight neurofilament subunit and the inducible 70 kDa heat-shock protein. None of these proteins co-enriched with preparations of p68/70, indicating that the doublet is not closely related to any of them. An affinity-purified antibody to p68/70 (anti-p68/70) was developed and used to assess the species, tissue and cellular distribution of p68/70. The antibody cross-reacted with brain homogenates of goldfish and closely related fish species. All goldfish tissues expressed p68/70, although the levels varied over a 30-fold range. Immunolabeling of goldfish retina revealed prominent staining of RGC somata, dendrites and axons. Intense immunolabeling was also observed in the germinal epithelial cells and rod precursors and in all retinal layers near the peripheral margin, where recently differentiated neurons are found. In the tectum, the germinal zone was also highly labeled. During regeneration the immunoreactivity of the RGC somata and axons increased dramatically. The elevated expression of p68/70 in each of these manifestations of neuronal growth within the goldfish visual system suggests that p68/70 plays a role in growth and possibly in cellular development as well.PhDNeuroscienceUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/103514/1/9319657.pdfDescription of 9319657.pdf : Restricted to UM users only