Radiolabelling 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 of a protein in a crude bovine brain homogenate, which upon two-dimensional gel electrophoresis migrated with an acidic pI (< 4.5) and an apparent 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 in brain homogenates, we first purified bovine brain MARCKS using calmodulin-Sepharose affinity chromatography and we then supplemented the crude ADP-ribosylation reaction mixture with this purified MARCKS fraction. 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, established the identity of this constituent as MARCKS. The radiolabelling of MARCKS was lower in comparison with the ADP-ribosylation of the related neuronal protein B-50/GAP-43 under identical reaction conditions. The potential functional consequences of the ADP-ribosylation of MARCKS are discussed and the possibility is raised that other members of the MARCKS family, such as the F52/MacMARCKS/MRP protein, may also be subject to ADP-ribosylation.