Amino-Acid Sequence of Human and Bovine Brain Myelin Proteolipid Protein (Lipophilin) is Completely Conserved

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Abstract
Proteolipid protein (PLP) was isolated from white matter of human brain by chloroform/methanol extraction and further purified by chromatography. Performic acid oxidation yielded a product homogenous in NaDodSO4-polyacrylamide electrophoresis with a molecular mass of 30 kDa [Kdalton]. The carboxymethylated PLP was chemically cleaved with cyanogen bromide into 4 fragments: CNBr I 22-24 kDa, CNBr II 5 kDa, CNBr III 1.4 kDa and CNBr IV 0.7 kDa. HBr/dimethylsulfoxide cleavage at tryptophan residues released 4 fragments: Trp I 14-16 kDa, Trp II 2.0 kDa, Trp III 5 kDa and Trp IV 7 kDa. Hydrophilic fragments were enriched in 50% formic acid (CNBr II, III, IV and Trp II and III); hydrophobic peptides precipitated from this solvent were CNBr I, Trp I and IV. The fragments were separated by gel filtration with 90% formic acid as solvent and finally purified by gel permeation HPLC [high performance liquid chromatography] (Si 60 and Si 100) for automated liquid and solid-phase Edman degradation. Large fragments were further cleaved with different proteinases (trypsin, V8-proteinase, endoproteinase Lys-C and thermolysin). An improved strategy was used in the sequencing of the human proteolipid protein compared with the approach to the structural elucidation of bovine brain PLP. The amino-acid sequence of human PLP contains 276 residues, the same as found in bovine proteolipid protein. The 2 sequences proved to be identical. The possible importance of the conservative structure of this integral membrane protein is discussed.