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ADP‐Ribosylation of Human Myelin Basic Protein
Author(s) -
Boulias Chris,
Moscarello Mario A.
Publication year - 1994
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1994.63010351.x
Subject(s) - adp ribosylation , chemistry , cholera toxin , myelin basic protein , nad+ kinase , polyacrylamide gel electrophoresis , gel electrophoresis , biochemistry , myelin , biology , enzyme , neuroscience , microbiology and biotechnology , central nervous system
When isolated myelin membranes were ADP‐ribosylated by [ 32 P]NAD + either in the absence of toxin (by the membrane ADP‐ribosyltransferase) or in the presence of cholera toxin, the same proteins were ADP‐ribosylated in both cases and myelin basic protein (MBP) was the major radioactive product. Therefore, cholera toxin was considered a good model for ADP‐ribosylation of myelin proteins. Although purified human MBP migrates as a single band on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis with a molecular mass of 20 kDa, the microheterogeneity that is masked under these conditions can be clearly demonstrated on alkaline‐urea gels at pH 10.6. At this pH, MBP is resolved into several components that differ one from the other by a single charge (charge isomers). These charge isomers can be resolved on CM52 columns at pH 10.6, and several can be ADP‐ribosylated. Component 1 (C‐1), the most cationic charge isomer, incorporated 1.79 mol of ADP‐ribose/mol of protein. C‐2 and C‐3 (which differ from C‐1 by the loss of one and two positive charges, respectively) incorporated slightly less at 1.67 and 1.63 mol of ADP‐ribose/mol of protein, respectively, whereas C‐8, the least cationic, incorporated less than 0.11 mol/mol of protein. In the presence of neutral hydroxylamine, the ADP‐ribosyl bond was shown to have a half‐life of about 80 min, suggesting an N ‐glycosidic linkage between ADP‐ribose and an arginyl residue of the protein. As MBP contains several components that are ADP‐ribosylated to different specific activities, the use of MBP, ADP‐ribosylated in the natural membrane, to identify the sites involved would yield a mixture of peptides difficult to resolve. Therefore, to identify the sites ADP‐ribosylated, an endoproteinase Lys‐C digest of C‐1 ADP‐ribosylated by cholera toxin was prepared. Two radioactive peptides were isolated by reversed‐phase HPLC. Amino acid and sequence analyses identified the radioactive peptides as residues 5–13 and 54–58 of the human sequence (sp. act., 0.89 and 0.62 nmol of ADP‐ribose/nmol of peptide, respectively). The ADP‐ribosylated residues were identified as Arg 9 and Arg 54 by automated and manual Edman sequencing. Taken together with our previous observation that MBP binds GTP at a single site, these data suggest that MBP functions as part of a signal transduction system in myelin.