Purification and characterization of p68/70, regeneration-associated proteins from goldfish brain.
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 heterodimer. Subcellular localization and membrane extraction experiments indicated p68/70 to be a component of the plasma membrane associated primarily through hydrophobic interactions. p68/70 demonstrated biphasic behavior in phase partition experiments using Triton X-114. Analysis of hydrolytic products indicated p68/70 to be a glycoprotein, containing 11% carbohydrate.
Leski
M L
ML
Department of Biological Chemistry, University of Michigan, Ann Arbor 48104-1687.
Agranoff
B W
BW
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001921:Brain / Q000378:metabolism*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D053487:DEAD-box RNA Helicases; D006054:Goldfish / Q000378:metabolism*; D007525:Isoelectric Focusing; D009416:Nerve Regeneration / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011494:Protein Kinases; D020365:RNA Helicases; D013347:Subcellular Fractions / Q000378:metabolism; D014909:Wheat Germ Agglutinins / Q000378:metabolism
D000596:Amino Acids; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D014909:Wheat Germ Agglutinins; D011494:Protein Kinases; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases