Phosphorylation of the neuronal protein kinase C substrate B-50: in vitro assay conditions alter sensitivity to ACTH.

We have explored the hypothesis that changes in the in vitro assay conditions alter both the extent of endogenous phosphorylation of B-50 protein in synaptosomal plasma membrane (SPM) and also the ability of the neuropeptide, ACTH-(1-24) to inhibit the phosphorylation of this protein. B-50 phosphorylation is influenced by preincubation, pH and ionic strength. ACTH-(1-24)-induced inhibition of B-50 phosphorylation varies with ionic strength and SPM protein concentration. Reduction of the buffer ionic strength and the SPM protein concentration enhances the ability of ACTH-(1-24) to inhibit B-50 phosphorylation. Furthermore, loss of ACTH-(1-24) by adsorption to plastic pipettes and test tubes reduces the peptide concentration in the assay. Addition of a low concentration of bovine serum albumin (BSA) essentially eliminates this loss without affecting the extent of phosphate incorporation into B-50. These data provide an explanation for the relatively high (and variable) IC50 values for ACTH-(1-24)-induced inhibition of B-50 phosphorylation reported in the literature. Further, these data suggest that in vitro assay conditions must be carefully investigated before modulation of protein phosphorylation can adequately be studied.