FIGURE 5.

S34C forms ectopic disulfide bonds. A, myelin from sciatic nerves of adult mice was purified, separated using SDS-PAGE, under reducing and nonreducing conditions, and Western-blotted. P0 was detected using a rabbit polyclonal anti-P0 antibody (see “Experimental Procedures”). In the absence of DTT, P0 ran at ∼27-kDa (*). In all the S63C transgenics, an additional band of around 50 kDa was detected (arrowhead). This band disappeared after treatment with DTT, which caused P0 to run at around 30 kDa (arrow). B, no modifications due to DTT were evident in MBP (detected using anti-MBP antibody), used as a loading control. C, Western blot and Coomassie gel of WT and S63C(+/+) purified myelin run under nonreducing conditions. In this Western blot, P0 was detected using a mouse monoclonal anti-P0 antibody. The two bands corresponding to the putative monomer and dimer (arrow and arrowhead, respectively) were excised for MALDI-TOF MS. Note that MpzS63C is a transgene randomly inserted away from the Mpz locus. Therefore, S63C+/− is, for example, an abbreviation of MpzS63C//Mpz+/− and contains the MpzS63C transgene in addition to one wild type Mpz and one Mpz null allele. D, tables showing the peptides containing the Cys³⁴ residue identified via MALDI-TOF MS in the 27- and 50-kDa bands. MH+, monoisotopic measured mass of the peptide; #Mod, number of modifications; ppm, part per million mass difference (measured mass-computed mass). Note that in the tables the amino acids are numbered according to the preprotein, including the 29-amino acid signal peptide (therefore, Cys³⁴ is at position 63).