Hoppe-Seylcr's Z. Physiol. Chem. Bd. 363, S. 1117-1131, September 1982 Analysis of the Primary Structure of the Strongly Hydrophobie Brain Myelin Proteolipid Apoprotein (Lipophilin) Isolation and Amino Acid Sequence Determination of Proteolytic Fragments Wilhelm STOFFEL , Werner SCHRÖDER , Heinz HILLEN and Rainer DEUTZMANN Institut für Physiologische Chemie der Universität zu Köln (Received 24 May 1982) Summary: Proteolipid apoprotein (lipophilin) and DM-20 protein from bovine brain white mat- ter proved to be identical in polyacrylamide gel electrophoresis and automated Edman degrada- tion of the TV-terminal end over 20 cycles. Lipophilin can be hydrolysed by trypsin, ther- molysin, chymotrypsin and subtilisin. We describe here a new, effective and rapid high- performance liquid Chromatographie separation method for hydrophilic and hydrophobic poly- peptides according to molecular mass on an analytical and preparative scale. Three large and several small peptides have been isolated from the tryptic and thermolysinolytic hydrolysate and purified by combined molecular sieve and high-performance Chromatographie separation and purification for automated Edman degradation. 40 amino acid residues of the large tryptic fragment and sequences of 43 and 22 amino acids of two thermolysinolytic fragments have been determined. These three polypeptides are partial structures of the 14 kDa large trypto- phan fragment I or the cyanogen bromide frag- ment I (18-19 kDa). Thermolysin also releases a polypeptide from incompletely reductively carboxymethylated lipophilin which is cleaved into the large ther- molysin fragment mentioned, 22 residues of which were analysed, and a 14 amino acids long se- quence of tryptophan fragment IV, described in the previous paper. Reductively carboxymethylated lipophilin, the lysine side chains of which were blocked with maleic anhydride, can be cleaved at arginine specific sites. Bio-Gel P-150 and high-performance Chromatographie purification yielded a polypep- tide, which upon performic acid oxidation was split into a 15 kDa and a 7.8 kDa polypeptide. The 15 kDa polypeptide resembles the TV-terminal end as proven by 31 cycles in Edman degradation. The 7.8 kDa polypeptide corresponds to the 72 amino acid C-terminal sequence, which equals cyanogen bromide fragments II, III and IV and embraces tryptophan fragment IV. Enzymes: Trypsin (EC 3.4.21.4); Thermolysin (EC 3.4.24.4); Chymotrypsin (EC 3.4.21.1); Subtilisin (EC 3.4.21.14). Abbreviations: DM-20 protein, myelin protein with molecular mass 20 kDa; Lipophilin, proteolipid apoprotein from brain white matter; Qiudrol, A^,7V,A^',A r '-tetrakis(2-hydroxypropyl)ethylenediamine; TosPheCI^Cl, tosyl-L-phenylalanin chloromethylketone. 0018/4888/82/0363-1117S02.00 © Copyright by Walter de Gruyter & Co · Berlin · New York Brought to you by | Purdue Universit Authenticated Download Date | 6/2/15 3:38