The use of hybrid molecules in a study of the equilibrium between nerve growth factor monomers and dimers.
The major protein in beta nerve growth factor preparations, beta1NGF, is a dimer in which both peptide chains have COOH-terminal arginine residues. Digestion of beta1NGF with carboxypeptidase B produced a dimer, beta3NGF, in which both chains lack these terminal arginine residues. Exposure of mixtures of beta1 and beta3NGF dimers to 8 M urea to produce monomers, followed by removal of urea to allow recombination, resulted in the formation of the hybrid beta2NGF, comprising one arginine-containing and one arginine-less chain, as well as the parent dimers. The amount of the three dimers formed was close to that expected from random association of monomers. Hybrid beta2NGF was also formed from mixtures of beta1 and beta3NGF where incubated at pH 2.6 to 4.5. The formation of beta2NGF has a half-time of 6 h at pH 4.0 and 4 degrees C. Its rate of formation decreased above pH 4.5, becoming minimal between pH 9.5 and pH 10.5, and increased with increasing temperature. The amount of beta2NGF formed was determined by the lowest pH to which the parent mixture was exposed, irrespective of its prior history. These data suggest that the hybrid is formed by the same mechanism in the absence and presence of the urea step. An approximate value for Kd, the equilibrium dissociation constant of the dimer equilibrium monomer equilibrium was derived. Its value was 3 - 10(-10) M at pH 4.0 and 4 degrees C. The alpha-subunit of 7S NGF decreased the rate of formation of beta2NGF not only at pHs where an alphabeta complex is stable, but also at an acid pH where no complex formation is observed by sedimentation analysis, suggesting that the present methodology offers a more sensitive probe of subunit interactions. In contrast, the gamma subunit and a number of indifferent proteins had little or no effect on the appearance of beta2NGF at the pHs studied.
D000818:Animals; D001120:Arginine; D055598:Chemical Phenomena; D002621:Chemistry; D006207:Half-Life; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D051379:Mice; D009414:Nerve Growth Factors / Q000138:chemical synthesis; D010443:Peptide Chain Termination, Translational; D013696:Temperature; D013997:Time Factors; D014508:Urea
D009414:Nerve Growth Factors; D014508:Urea; D001120:Arginine