Nerve growth factor from Crotalus adamenteus snake venom

Nerve growth factor (NGF) from the venom of the Crotalus adamenteus snake can be isolated as a basic protein (β) or as a higher molecular weight protein complex (γβ) by two different purification schemes. The β form is a basic polypeptide dimer which sediments at 2.5 S in a neutral pH sucrose gradient. Electrophoretic analysis of β in the presence of sodium dodecyl sulfate yields a single protein band migrating as a 14,000- to 14,450-dalton polypeptide which is consistent with the molecule having 8 to 12 more amino acid residues than the mouse β polypeptide. Analysis of the Crotalus β-NGF by isoelectric focusing on polyacrylamide disc gels shows two protein bands focusing at a pI of 9.4 and 9.5. The less basic band co-migrates with mouse β-NGF and both molecular species have indistinguishable specific biological activities. The specific biological activity of the Crotalus β ranges between 8 and 16 ng/biological unit, comparing well with the 3 to 16 ng/biological unit range of mouse β-NGF obtained in these experiments. The amino acid compositions of mouse β, Crotalus β, and Naja naja NGF compare strikingly. Isolation of the Crotalus γβ complex yields a high molecular weight component of 40,000 to 54,000. Dissocciation of the complex under acidic conditions yields an acidic protein with arginyl esterase activity, Crotalus γ, and no biological activity, and a basic protein devoid of arginyl esterase activity, Crotalus β. This basic subunit (β) has a specific biological activity and electrophoretic properties similar to the basic protein directly purified from whole venom. Kinetic analysis of the Crotalus γβ, Crotalus γ, and Crotalus β, shows that the arginyl esterase specific activity of Crotalus γ is strongly stimulated when the Crotalus γ is isolated and assayed apart from Crotalus β. Treatment of Crotalus β-NGF with the mouse γ subunit generates a molecular species with electrophoretic properties similar to the Naja naja NGF species described by Hogue-Angeletti et al. (Hogue-Angeletti, R.A., Frazier, W.A., Jacobs, J.W., Niall, H.D., and Bradshaw, R. A. (1976) Biochemistry 15, 26-34). The similarities between mouse β-NGF and Crotalus adamenteus β-NGF and the isolation from a snake venom of a complexed form of NGF similar to the mouse NGF complex would agree with a strong evolutionary preservation of NGF molecular forms in vertebrates.