Molecular properties of the growth-associated protein GAP-43 (B-50)

Abstract

The protein that has been identified in different contexts as growth-associated protein (GAP)-43, GAP-48, protein 4, B-50, F-1 gamma 5, and pp46, has been implicated in neural development, axonal regeneration, and the modulation of synaptic function. The present study investigated various properties of this protein (designated here as GAP/B-50), including its correct molecular weight and possible polymeric structure. GAP/B-50 was purified to greater than 90% homogeneity using an alkaline extraction procedure followed by a two-stage separation on a size-exclusion HPLC column. The equivalence of the purified protein to the B-50 phosphoprotein was confirmed by peptide digests, comigration, immunostaining, and amino acid composition. On a series of sodium dodecyl sulfate-polyacrylamide gels the apparent molecular weight of the protein was seen to vary inversely with the concentration of acrylamide in the gels. Using these data in the method of Ferguson, the molecular weight of GAP/B-50 was calculated to be 32.8 kilodaltons (kD), considerably lower than the previously reported values of 43-67 kD. The low molecular weight of the protein in the presence of detergent was confirmed by density centrifugation. In the absence of detergent, however, the protein was found to be part of a polymeric structure whose retention time by size-exclusion chromatography indicated a size of 124 kD; this property was also confirmed by density centrifugation under nondetergent conditions. These data suggest the possibility that the native form of GAP/B-50 in the presynaptic membrane may be a tetramer of four identical subunits.