Nerve growth factor (NGF) is a signaling protein [4] and growth factor implicated in a wide range of development and maintenance functions. NGF was discovered through a series of experiments in the 1950s on the development of the chick [5] nervous system. Since its discovery, NGF has been found to act in a variety of tissues throughout development and adulthood. It has been implicated in immune function, stress response, nerve maintenance, and in neurodegenerative diseases. It is named for its effect on the critical role it plays in the growth and organization [6] of the nervous system during embryonic development.

Abstract.-The nerve growth factor protein was purified over 100-fold from adult mouse salivary glands. The first step was a gel filtration on Sephadex G-100 at pH 7.5 of the aqueous gland extract. After gel filtration, most of the NGF activity was eluted in the 80,000-90,000-molecular-weight region (G-100 pool). The G-100 pool was dialyzed at pH 5.0 and fractionated by CM52 cellulose chromatography at pH 5.0. Recovery from CM52 cellulose columns was quantitative for protein and ranged 80-100 per cent for the nerve growth factor activity; the latter was almost completely carried by a protein which did not show any heterogeneity when examined by several analytical tests. The purified nerve growth factor showed an S20,w, = 2.43, a D20 = 7.30 and a 30,000 molecular weight. The over-all recovery was about 45 per cent.The nerve growth factor is a protein which selectively stimulates the growth and differentiation of sympathetic and embryonic sensory nerve cells." 2 It was first observed in certain mouse sarcomas and later found at much higher levels in other sources, such as snake venom (820 = 2.2)3-5 and submaxillary glands of adult mice (S20 = 4.33).6-8The isolation from mouse submaxillary glands of a nerve growth factor form of 140,000 mol wt was recently reported.9' 10 This high-moleculer-weight nerve growth factor was mostly inactivated upon exposure to pH below 5.0 and above 8.0; inactivation was accompanied by dissociation into acidic, basic, and neutral components, called a-, ,B-, and 'y-subunits, respectively. Once separated by chromatography on carboxymethyl (CM) cellulose, only the B subunit displayed nerve growth factor activity, which, however, accounted for only 25 per cent of the amount loaded on the column. Recovery of the total nerve growth factor activity and reconstitution of the 140,000-molecular-weight protein were obtained by mixing the three components at neutral pH."' 12Studies from this laboratory did not support these findings.2' 1 A procedure is here described for the purification of the nerve growth factor in which pH was kept between 7.5 and 5.0, i.e., within values reported"' 12 as not affecting either the activity or the molecular size of the nerve growth factor. The purified nerve growth factor showed a 30,000 mol wt, accounted for full recovery of biological activity, and did not require any other protein component for maximal response.Materials and Methods.-Submaxillary glands were excised from adult albino mice (25-30 gm body weight), freed of fat and connective tissue, and stored frozen at -20°u ntil used. The usual amount was 10 gm of glands (wet weight) from 100 mice.Sephadex G-100 (Uppsala, Sweden) columns were calibrated as indicated by Andrews.'4 DE52 cellulose and CM52 cellulose (Whatman, London) were used as anion exchanger and cation exchanger, respectively. Fractions from gel filtration or ion exchange chro-787

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