ANALYSIS OF MYELIN PROTEOLIPID PROTEIN AND F-0 ATPASE SUBUNIT-9 IN NORMAL AND JIMPY CNS

Membrane fractions and chloroform-methanol (C-M) extracts of jimpy (jp) and normal CNS at 17-20 days were examined by immunoblot and sequence analysis to determine whether myelin proteolipid protein (PLP) or DM-20 could be detected in jp CNS. No reactivity was detected in jp samples with several PLP antibodies (Abs) except with one Ab to amino acids 109-128 of normal PLP. Proteins in the immunoreactive bands similar to 26 Mr comigrating with PLP were sequenced for the first 10-12 residues. A sequence corresponding to PLP was found in normal CNS, as expected, but not in the band from jp CNS. Our results provide no evidence for an aberrant form of PLP in jp CNS at 17-20 days. This and other studies suggest that the abnormalities in jp brain are not due to toxicity of the mutant jp PLP/DM-20 proteins. Interestingly, a sequence identical to the amino terminus of the mature proton channel subunit 9 of mitochondrial F-0 ATPase was detected in the immunoreactive bands similar to 26 Mr in both normal and jp samples. This identification was supported by reactivity with an Ab to the F-0 subunit and by labeling with dicyclohexylcarbodiimide (DCCD). In contrast to PLP isolated from whole CNS, PLP isolated from myelin was devoid of F-0 subunit 9 based on sequence analysis and lack of reactivity with an Ab to the F-0 subunit, yet still reacted with DCCD. This finding rules out the possibility that contaminating F-0 ATPase gives rise to the DCCD binding exhibited by PLP and confirms the possibility that PLP has proton channel activity, as suggested by Lin and Lees (1,2).