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ADP-ribosylation of myelin basic proteins isolated from normal and mutant mouse brains
1Hospital for Sick Children, Department of Biochemistry, Toronto, Ontario, Canada.
Abstract:
Myelin basic proteins (MBPs) have been shown to be ADP-ribosylated in-vitro by cholera toxin in the presence of NAD. Since acid-soluble extracts of brain contain other proteins in the 14-32 kD range (such as histones) in addition to MBP's, the identification of the ADP-ribosylated proteins was uncertain. To determine that only the MBP's were ADP-ribosylated, the acid-soluble fractions from several murine mutants were prepared. Thus, in the Shiverer mutants, none of the proteins in the 14-32 kD range were ADP-ribosylated; in the Myelin-deficient mutant, some of ADP-ribosylation was observed but none in the Jimpy mutant, consistent with our demonstration that the least cationic isomer cannot be ADP-ribosylated.
Insights
Cholera toxin ADP-ribosylates myelin basic proteins (MBPs) in vitro. Using specific mouse mutants, researchers confirmed that only MBPs undergo this modification, not other brain proteins.
Area of Science:
- Neuroscience
- Biochemistry
- Molecular Biology
Background:
- Myelin basic proteins (MBPs) are crucial for myelin sheath formation and maintenance in the central nervous system.
- ADP-ribosylation is a post-translational modification involving the transfer of ADP-ribose from NAD+ to target proteins.
- Cholera toxin is a known ADP-ribosyltransferase enzyme.
Purpose of the Study:
- To confirm that Myelin basic proteins (MBPs) are specifically ADP-ribosylated by cholera toxin in vitro.
- To differentiate MBP ADP-ribosylation from potential modifications of other acid-soluble brain proteins.
- To investigate the role of specific MBP isoforms in ADP-ribosylation using murine mutants.
Main Methods:
- In vitro ADP-ribosylation assays using cholera toxin and NAD+ on acid-soluble brain extracts.
- Analysis of ADP-ribosylated proteins in wild-type and genetically distinct murine mutants (Shiverer, Myelin-deficient, Jimpy).
- Protein identification based on molecular weight (14-32 kD range) and electrophoretic properties.
Main Results:
- In Shiverer mutants, no proteins in the 14-32 kD range were ADP-ribosylated, indicating the absence of specific MBP targets.
- Myelin-deficient mutants showed partial ADP-ribosylation, suggesting the involvement of specific MBP isoforms.
- The Jimpy mutant exhibited no detectable ADP-ribosylation, supporting the finding that the least cationic MBP isomer is not modified.
Conclusions:
- Myelin basic proteins (MBPs) are the exclusive targets of cholera toxin-mediated ADP-ribosylation within the studied acid-soluble brain protein fraction.
- Specific isoforms of Myelin basic proteins are differentially susceptible to ADP-ribosylation.
- The study validates the use of specific murine mutants to identify protein targets of enzymatic modifications in complex biological samples.