Purification and characterization of a neurite extension factor from bovine brain.
The extension of neurites by chicken embryo cerebral cortical neurons can be measured quantitatively at low cell density in serum-free, defined medium. An acidic, heat-stable protein fraction from bovine brain has been shown to have neurite extension activity in this assay. We report the use of reversed-phase HPLC to purify a neurite extension factor from this fraction to apparent homogeneity. The protein was characterized by NaDodSO4/PAGE. In the presence of reducing agents, the protein migrated as a single band, with an apparent molecular weight of 6500. In the absence of reducing agents, the protein showed bands at apparent molecular weights of 6500, 21,000-22,000, 30,000, and 40,000. Reduction and S-carboxymethylation of the protein abolished all biological activity and resulted in a shift of the apparent molecular weight to 11,000. The amino acid composition of the purified neurite-extension factor was nearly identical to that of bovine brain S100 beta. The amino acid sequences of peptides derived from trypsin or cyanogen bromide digests of the protein were identical to those found in S100 beta and accounted for 71 of 91 amino acids in the protein. However, three peptides obtained from cyanogen bromide digestion of the nonreduced protein appeared to be disulfide-linked dimers. Our results indicate that a biological activity, neurite extension, which is critical for the development of the nervous system, is associated with a disulfide form of S100 beta.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D002540:Cerebral Cortex / Q000187:drug effects; D002642:Chick Embryo; D003488:Cyanogen Bromide; D004220:Disulfides; D008970:Molecular Weight; D009414:Nerve Growth Factors / Q000302:isolation & purification* / Q000494:pharmacology; D010446:Peptide Fragments / Q000032:analysis; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins / Q000302:isolation & purification* / Q000494:pharmacology; D014357:Trypsin
D000596:Amino Acids; D004220:Disulfides; D009414:Nerve Growth Factors; D010446:Peptide Fragments; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; D014357:Trypsin; D003488:Cyanogen Bromide