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 hydrolysis 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 interactions involving B‐50 could have common structural determinants. Chymotryptic digestion of B‐50 in the presence or absence of a nonionic detergent and ACTH demonstrated that hydrolysis is potentiated by a lipid‐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 appears to occur only in the presence of a nonionic detergent or lipid and the absence of calcium. A structure‐activity study for ACTH‐mediated inhibition of B‐50 proteolysis by an endogenous protease that copurifies with B‐50 in a detergent extract of synaptosomal plasma membranes showed that ACTH 1–24 , ACTH 5–24 , ACTH 5–16 , dynorphin, and corticostatin inhibited the conversion of rat B‐50 to B‐50 41–226 . In contrast, ACTH 7–16 , Org 2766 , and neurotensin had no detectable effect on B‐50 proteolysis at concentrations of 10 and 50 µ M . The results indicate that in common with effects in other B‐50‐containing systems, inhibition of proteolysis is related to the presence of a basic amphiphilic helix in those ACTH fragments and analogues that were inhibitory and, moreover, the presence of this motif in other peptides appears to 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 regulation by posttranslational enzymes and binding proteins as a consequence of its ability to adapt an amphiphilic helix conformation.