DETERGENTS AND PEPTIDES ALTER PROTEOLYSIS AND CALMODULIN-BINDING OF B-50 GAP-43 IN-VITRO

The neuronal growth-associated protein B-50/GAP-43 is a substrate for protein kinase C, binds to calmodulin in a calcium-independent manner, and in vitro is subject to an endogenous and chymotrypsin-mediated hy drolysis in the vicinity of the single kinase C phosphorylation site. All of these processes can be influenced by corticotrophin (ACTH). In the present study we have investigated whether these biochemical inter actions involving B-50 could have common structural determinants. Chym otryptic digestion of B-50 in the presence or absence of a nonionic de tergent and ACTH demonstrated that hydrolysis is potentiated by a lipi d-like environment that primarily affects the protein rather than the protease or the peptide. Furthermore, this lipid dependency appears to extend to the binding of dephosphorylated B-50 to calmodulin, which a ppears to occur only in the presence of a nonionic detergent or lipid and the absence of calcium. A structure-activity study for ACTH-mediat ed inhibition of B-50 proteolysis by an endogenous protease that copur ifies with B-50 in a detergent extract of synaptosomal plasma membrane s showed that ACTH(1-24), ACTH(5-24), ACTH(5-16), dynorphin, and corti costatin inhibited the conversion of rat B-50 to B-40(41-226). In cont rast, ACTH(7-16), Org(2766), and neurotensin had no detectable effect on B-50 proteolysis at concentrations of 10 and 50 mu M. The results i ndicate that in common with effects in other B-50-containing systems, inhibition of proteolysis is related to the presence of a basic amphip hilic helix in those ACTH fragments and analogues that were inhibitory and, moreover, the presence of this motif in other peptides appears t o confer inhibitory activity. The results are discussed with reference to the putative secondary structure of B-50 and changes that may take place in the presence of membrane lipids or nonionic detergents. The conclusions of this study suggest that in vitro B-50 is subject to reg ulation by posttranslational enzymes and binding proteins as a consequ ence of its ability to adapt an amphiphilic helix conformation.