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.
Aloyo
V J
VJ
Department of Pharmacology, Medical College of Pennsylvania, Philadelphia 19129.
Zwiers
H
H
De Graan
P N
PN
Gispen
W H
WH
D000818:Animals; D003366:Cosyntropin / Q000494:pharmacology*; D019922:GAP-43 Protein; D007700:Kinetics; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009474:Neurons / Q000201:enzymology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013574:Synaptosomes / Q000201:enzymology
D019922:GAP-43 Protein; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D003366:Cosyntropin; D011493:Protein Kinase C