Purification and characterization of a neurite extension factor from bovine brain.

Abstract

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, heatstable 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 (3. The amino acid sequences of peptides derived from trypsin or cyanogen bromide digests of the protein were identical to those found in S100 P 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 (3.The purification and characterization of proteins that influence neuronal development are essential steps in understanding the development of the nervous system. Such purifications rely on in vitro bioassays to identify molecules that have biological activities (1). These molecules can be divided into two broad classes: (i0 substrate-attached materials, such as laminin (2), that stimulate neurite outgrowth, presumably by promoting adhesion to the substratum, and (ii) soluble molecules, such as nerve growth factor, that promote neuronal survival and/or neuronal differentiation (3). Tissue extracts or conditioned media have been used as starting materials for the purification of neurotrophic molecules, but these starting materials may contain multiple growth-promoting activities (1). Thus, the success of purifying any one of these substances depends on the specificity of the bioassay in addition to its reproducibility and sensitivity.We have studied molecules that influence the development of neurons in the central nervous system. One of us (D.K.) has described a well-defined bioassay for neurotrophic substances active on central neurons (4). This assay quantitatively measures neurite extension from chicken embryo cerebral cortical neurons grown at low cell density in a serum-free, defined medium. By using this assay, an acidic, heat-stable protein fraction was isolated from bovine brain (4).In this paper, we report the purification of this neurite extension factor (NEF) to apparent homogeneity by use of reversed-phase HPLC. The amino acid sequences ofpeptides d...

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