Isolation of an active-site peptide of lipoprotein lipase from bovine milk and determination of its amino acid sequence.

Lipoprotein lipase from bovine milk reacted stoichiometrically with diisopropylphosphorofluoridate (DFP), an inactivator of serine esterases, resulting in the loss of enzymatic activity against triacylglycerols.The reaction obeyed first-order kinetics with a rate constant of 0.69 h-'.In order to isolate the peptide containing the diisopropylphosphoryl moiety (DIP), partially purified lipoprotein lipase was covalently labeled with 3HDFP, and the labeled protein was reduced, carboxymethylated, and further purified to about 90% homogeneity.Cyanogen bromide cleavage followed by gel filtration yielded a radioactive peptide of 6-8 kDa.This peptide was succinylated and then digested with Staphylococcus aureus Vs proteinase.From this digest, a peptide containing 0.95 mol of rSH] DIP/mol of peptide was isolated by gel-permeation chromatography followed by reverse-phase high performance liquid chromatography.Automated Edman degradation provided the following sequence: Ala-Ile-Gly-Ile-His-Trp-Gly-Gly-(DIP)Ser-Pro-Asn-Gln-Lys-Asn-Gly-Ala-Val-Phe-Ile-Asn-(Ser,Leu)-Glu.Analysis of the sequence for secondary structure suggests that the reactive serine of lipoprotein lipase is in a &turn, a structure similar to those of the active sites of most other serine proteinases.Lipoprotein lipase appears to share this secondary structure with other serine hydrolases despite significant differences in the primary structure of this domain.Lipoprotein lipase (EC 3.1.1.34)plays a central role in the regulation of lipid metabolism (1,2).Although the physiological functions of lipoprotein lipase are well established, its amino acid sequence, molecular properties, and catalytic mechanism are still not well defined.Earlier work using lipoprotein lipase from bovine milk (3) and rat heart (4) indicated that this enzyme can be inactivated by phenylmethanesulfonyl fluoride, diisopropylphosphorofluoridate (DFP'), and diethyl p-nitrophenyl phosphate (paraoxon).