In 1969 V. Bocchini and Pietro U. Angeletti at the Laboratory of Cell Biology in Rome devised a method for purifying NGF from mouse salivary glands. With the large quantities of NGF made available by their technique Ruth Hogue Angeletti and Ralph A. Bradshaw of the Washington University School of Medicine were able to determine the sequence of amino acids in the protein. Biologically active NGF is a dimer, or complex of two identical polypeptide chains, each of which has a molecular weight of 13.250 daltons. The NGF molecule has a marked predominance of positively charged amino acids and has a net positive charge at neutral pH. In addition the folded polypeptide chain is stabilized by three covalent sulfur-sulfur bridges between units of the amino acid cysteine at different positions along the chain. Such disulfide bonds are common in proteins that are actively secreted from cells, such as insulin and antibodies. The restraint these molecular bridges impose on the conformation of the polypeptide chain is apparently necessary to prevent its denaturation and inactivation in an environment that is much more subject to adverse conditions than the interior of the cell.
A comparison of the sequence of amino acids in NGF with that of several other polypeptides by William A. Frazier of Washington University revealed that NGF and insulin have certain sequences in common. This observation led to the hypothesis that the gene for NGF evolved from an ancestral gene for proinsulin, the large precursor polypeptide that is cleaved to yield the active molecule of insulin. Frazier suggested that the ancestral proinsulin gene had duplicated itself and the two copies had subsequently evolved divergently, giving rise to proinsulin and a postulated precursor polypeptide for NGF. Although this intriguing possibility remains theoretical, the similarities in the amino acid sequences of NGF and insulin are not great enough to result in any similarity of function: the two molecules have completely different target cells and biological activities.
In 1967 Silvio S. Varon and Eric M. Shooter of the Stanford University School of Medicine isolated NGF by a different procedure and found that the NGF dimer was present as a stable complex with two copies each of two other proteins, which they designated the alpha and gamma subunits. This finding was puzzling, since it was known that the NGF dimer by itself was biologically active. Further investigation revealed that the gamma subunit is a specific enzyme that cleaves polypeptide chains only at a point adjacent to the amino acid arginine. The alpha subunit, on the other hand, has no detectable biological activity. This odd association of three disparate proteins demanded an explanation.
Shooter hypothesized that the alpha and gamma subunits serve to activate, store and protect the NGF molecule. According to his scheme the initial gene product in the manufacture of NGF is a large precursor polypeptide called proNGF, two copies of which form a dimer. Two gamma subunits then associate with the proNGF dimer and cleave the precursor chains to generate the active NGF dimer. Unlike the typical complex of an enzyme and its product, which rapidly dissociates, the 'gamma subunits remain bound to the NGF dimer after the cleavage of the proNGF chains. (The two alpha subunits may bind either to the proNGF dimer before cleavage or to the complex of the gamma subunit and the NGF dimer after cleavage.) The association of the gamma and alpha subunits with the NGF dimer apparently serves to protect it from further degradation by other protein-cleaving enzymes in the body fluids. Shooter's hypothesis recently received support when he and Edward A. Berger isolated the proNGF molecule and demonstrated that incubation of this precursor with the gamma subunit resulted in its complete conversion into active NGF.
The biological activity of a protein resides in its three-dimensional structure, which consists of not only its amino acid sequence but also the precise folding pattern of the polypeptide chain (the secondary structure) and the suprafolding of two or more such chains to form a unique globular entity (the tertiary structure). The only reliable way to determine the secondary and tertiary structure of a protein molecule is to crystallize it and mathematically analyze the crystal's X-ray-diffraction pattern. The recent crystallization of NGF by A. Wlodawer. Keith O. Hodgson and Shooter is therefore an important first step in determining the three-dimensional structure of the molecule by x-ray crystallography.