Protein kinase C substrates from bovine brain. Purification and characterization of neuromodulin, a neuron-specific calmodulin-binding protein.
Although such solubility is uncommon among proteins generally, several bovine brain proteins were found to be soluble in 2.5% perchloric acid, and many of them were in vitro substrates for protein kinase C (Ca2+/phospholipid-dependent enzyme). Two of the perchloric acid-soluble brain proteins were purified, p43 and p17. P43 and p17 could be phosphorylated by protein kinase C only in the presence of Ca2+ and phospholipids and neither was a substrate for protein kinase II. P43 was subsequently identified as the neurospecific, calmodulin-binding protein, neuromodulin (also designated P-57, GAP43, B50, or F1) (Alexander, K. H., Wakim, B. T., Doyle, G. S., Walsh, K. A., and Storm, D. R. (1988) J. Biol. Chem. 263, 7544-7549). A rapid purification method for neuromodulin was developed taking advantage of its newly discovered property, solubility in 2.5% perchloric acid, and of its previously recognized calmodulin-binding property. Evidence was obtained that neuromodulin isolated from cytosolic extract exists as a mixture of molecular forms and that the Ca2+-binding S100 protein-beta discriminates among the different neuromodulin isoforms in forming covalent complexes via disulfide bridges; this discrimination may be explained by analogous differences observed between the NH2-terminal amino acid sequences of p57 and F1. Solubility in 2.5% perchloric acid was demonstrated for another rat brain protein kinase C substrate, p87. We suggest that perchloric acid solubility might be a common property of protein kinase C substrates.
Baudier
J
J
Laboratoire de Physique, Faculte de Pharmacie, Universite Louis Pasteur, Strasbourg, France.
Bronner
C
C
Kligman
D
D
Cole
R D
RD
D000818:Animals; D001923:Brain Chemistry; D002118:Calcium / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D019922:GAP-43 Protein; D008970:Molecular Weight; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D012995:Solubility
D002148:Calmodulin-Binding Proteins; D019922:GAP-43 Protein; D011493:Protein Kinase C; D002118:Calcium