Evidence for defective incorporation of proteins in myelin of the quaking mutant mouse

The defect in myelinogenesis present in the Quaking mutant mouse was investigated using a double radioisotope technique for comparing the incorporation of amino acid into myelin proteins of normal and mutant mice. Quaking mice and littermate controls recieved intracranial injections of 150 μCi ³H]glycine and 25 μCi of ¹⁴C]glycine respectively. After 2 h their brains were combined and jointly processed to obtain subcellular fractions. The ³H/¹⁴C ratio for the myelin subfraction was 1.88 as compared to a ³H/¹⁴C ratio of 3.0 for the other subfractions, indicating a 40% decrease in glycine incorporation into myelin of Quaking mice. Myelin proteins were separated by discontinuous gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) and the ³H/¹⁴C ratios determined in each gel slice. In contrast to the microsomal subfractions which gave a ³H/¹⁴C ratio of 2.6 across the gel, the ³H/¹⁴C ratio of myelin showed large variations with values ranging from 0.54 for proteolipid protein to 2.0 for some of the high molecular weight proteins. During development, the Quaking mutant exhibited a preferential depression in glycine incorporation into proteolipid protein in 18-day-old mice, while in older animals (32–54 days) the fast migrating basic protein, as well as the proteolipid protein, was labeled to a significantly lesser extent.