Premium
Purification and Characterization of p68/70, Regeneration‐Associated Proteins from Goldfish Brain
Author(s) -
Leski Michael L.,
Agranoff Bernard W.
Publication year - 1994
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1994.62031182.x
Subject(s) - chemistry , chromatography , periplasmic space , chromatofocusing , size exclusion chromatography , molecular mass , agarose , biochemistry , enzyme , escherichia coli , gene
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, Cu 2+ ‐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.