RADIOLABELING OF BOVINE MYRISTOYLATED ALANINE-RICH PROTEIN-KINASE-C SUBSTRATE (MARCKS) IN AN ADP-RIBOSYLATION REACTION

In an ADP-ribosylation reaction, we have observed the radiolabelling o f a protein in a crude bovine brain homogenate, which upon two-dimensi onal gel electrophoresis migrated with an acidic pi (<4.5) and an appa rent molecular mass (80-90 kDa) consistent with the properties of the myristoylated, alanine-rich, protein kinase C substrate protein termed MARCKS, To establish the identity of this radiolabelled constituent i n brain homogenates, we first purified bovine brain MARCKS using calmo dulin-Sepharose affinity chromatography and we then supplemented the c rude ADP-ribosylation reaction mixture with this purified MARCKS fract ion. Concordant increases in radiolabelling and silver staining of the same protein component from the MARCKS-supplemented ADP-ribosylation reaction, as compared with the ADP-ribosylated crude homogenate, estab lished the identity of this constituent as MARCKS. The radiolabelling of MARCKS was lower in comparison with the ADP-ribosylation of the rel ated neuronal protein B-50/GAP-43 under identical reaction conditions. The potential functional consequences of the ADP-ribosylation of MARC KS are discussed and the possibility is raised that other members of t he MARCKS family, such as the F52/MacMARCKS/MRP protein, may also be s ubject to ADP-ribosylation.