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.
Chao
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D
Endocrine and Cell Regulation Research Groups, Faculty of Medicine, University of Calgary, Canada.
Severson
D L
DL
Zwiers
H
H
Hollenberg
M D
MD
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D002417:Cattle; D002846:Chromatography, Affinity; D015180:Electrophoresis, Gel, Two-Dimensional; D006031:Glycosylation; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins; D007526:Isoelectric Point; D008565:Membrane Proteins; D008970:Molecular Weight; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*
D002147:Calmodulin; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D010761:Phosphorus Radioisotopes; D011506:Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D000246:Adenosine Diphosphate Ribose; D011493:Protein Kinase C