Purification and Characterization of a Neurite Extension Factor from Bovine Brain

• Published in: Proceedings of the National Academy of Sciences - PNAS 1985-10, Vol.82 (20), p.7136-7139
• Main Authors: Kligman, Douglas, Marshak, Daniel R.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Amino Acids - analysis; Animals; Biological and medical sciences; Brain; Brain Chemistry; Bromides; Cattle; Cell differentiation, maturation, development, hematopoiesis; Cell physiology; Cerebral Cortex - drug effects; Chick Embryo; Cyanogen Bromide; Dimers; Disulfides; Fundamental and applied biological sciences. Psychology; Molecular and cellular biology; Molecular Weight; Nerve Growth Factors - isolation & purification; Nerve Growth Factors - pharmacology; Nervous system; Neurites; Neurons; Peptide Fragments - analysis; S100 Calcium Binding Protein beta Subunit; S100 Proteins - isolation & purification; S100 Proteins - pharmacology; Trypsin; Ungulates
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.82.20.7136

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 β . The amino acid sequences of peptides derived from trypsin or cyanogen bromide digests of the protein were identical to those found in S100 β 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 β .