Porcine Pancreatic Lipase

Abstract

The single polypeptide chain of about 460 amino acids of porcine pancreatic lipase (EC 3.1.1.3) was fragmented into 5 peptides by cyanogen bromide cleavage. The sequence of the first 3 cyanogen bromide peptides (CNI, CNII, CNIII), including a total of 234 amino acids, was fully elucidated. Automatic or manual Edman degradation was performed on the different peptides. Fragmentations of the CN peptides were accomplished by digestions with trypsin [EC 3.4.2.1.4] (after citraconylation of 1,2-cyclohexanedione treatment), chymotrypsin [EC 3.4.21.1] and Staphylococcus aureus external protease [EC 3.4.99.-]. Hydrolysis of unreduced material by pepsin [EC 3.4.23.1] and thermolysin [EC 3.4.24.4], performed in order to determine the S-S bridge positions, provided useful overlapping peptides. The glycan moiety of lipase is bound to Asn-166. The non-essential tyrosine specifically blocked by diisopropylphosphorofluoridate is Tyr-49 in a cluster of asparagine and glutamine residues. The existence of a highly hydrophobic sequence (206-217) at the C terminus of the CNII fragment is noteworthy.