Isolation and identification of proteolipid proteins injimpy mouse brain

Proteolipids were isolated from 20 day old normal andjimpy mouse brain by extraction into chloroform-methanol (21, w/v), delipidated by size-exclusion HPLC, and analyzed by SDS-PAGE, Western blots, amino acid analyses, and N-terminal sequencing. SDS-PAGE showed that a major proteolipid fromjimpy mouse brain had an apparent molecular weight of 23 kDa, intermediate to that of PLP and DM-20 from normal mouse brain. Western blots with 3 different antibodies which recognize residues 200–224, 116–150, and 270–276 respectively recognized immunoreactive material in normal andjimpy PLP. Since antibody reactive with 270–276 did not recognizejimpy PLP, an altered C-terminus of thejimpy protein is suggested. These results demonstrated that a PLP can be partially purified fromjimpy mouse brain. Amino acid analyses failed to show the predicted increase in cysteinyl residues (predicted from cDNA) injimpy PLP. However, whenjimpy brain proteolipids were subjected to N-terminal sequencing, Gly, Leu, Leu, Gly the first four amino acids of PLP were detected. Thus, the partial purification of a proteolipid fromjimpy mouse brain, whose characteristics (apparent molecular weight, immunoreactivity, N-terminal sequence and relative net charge) strongly suggested that PLP of altered size is present injimpy mouse brain.Abbreviations BCIP 5-bromo-4-chloro-3-indolyl phosphate toluidine salt - MBP myelin basic protein - NBT -nitro blue tetrazolium chloride - PITC phenylisothiocyanate - PLP myelin proteolipid protein - PVDF polyvinylidene difluoride - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresisSpecial issue dedicated to Dr. Marjorie B. Lees.