PURIFICATION AND CHARACTERIZATION OF P68 70, REGENERATION-ASSOCIATED PROTEINS FROM GOLDFISH BRAIN/

Two acidic proteins (p68/70) previously shown to be associated with re generation of the goldfish optic nerve were purified 887-fold from bra in homogenates of Carassius auratus. Purification to homogeneity was a chieved by sequential chromatography of a 100,000 g brain supernatant fraction on DEAE-Sephacel, Cu2+- charged iminodiacetic acid agarose, a nd gel filtration. The Stokes radius of the doublet was determined to be 5.8 nm, and the sedimentation coefficient calculated to be 5.2. Fro m these values a molecular mass of 128 kDa and a frictional coefficien t 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 hi ghly elongated and associate as homodimers or as a heterodimer. Subcel lular localization and membrane extraction experiments indicated p68/7 0 to be a component of the plasma membrane associated primarily throug h hydrophobic interactions. p68/70 demonstrated biphasic behavior in p hase partition experiments using Triton X-114. Analysis of hydrolytic products indicated p68/70 to be a glycoprotein, containing 11% carbohy drate.