Purification and Characterization of p68/70, Regeneration-Associated Proteins from Goldfish Brain

Abstract: Two acidic proteins (p68/70) previously shown to be associated with regeneration of the goldfish optic nerve were purified 887-fold from brain homogenates of Carassius auratus. Purification to homogeneity was achieved by sequential chromatography of a 100,000 g brain supernatant fraction on DEAE-Sephacel, Cu²⁺-charged iminodiacetic acid agarose, and gel filtration. The Stokes radius of the doublet was determined to be 5.8 nm, and the sedimentation coefficient calculated to be 5 2. From these values a molecular mass of 128 kDa and a frictional coefficient ratio of 1.6 were calculated. Chromatofocusing on a high-resolution DEAE column resolved the protein doublet into three dimeric species of p68, p68/70, and p70. These results indicate that the proteins are highly elongated and associate as homodimers or as a hetero-dimer. Subcellular localization and membrane extraction experiments indicated p68/70 to be a component of the plasma membrane associated primarily through hydro-phobic interactions. p68/70 demonstrated biphasic behavior in phase partition experiments using Triton 114. Analysis of hydrolytic products indicated p68/70 to be a glyco-protein, containing 11% carbohydrate.