Structure of bovine milk lipoprotein lipase.
The primary structure of bovine milk lipoprotein lipase (bLPL) was determined by alignment of peptides produced by tryptic digestion, Staphylococcus aureus V8 protease digestion, and cyanogen bromide cleavage. bLPL consists of 450 amino acid residues. Most tryptic peptides were isolated and analyzed, except for the dipeptide, Glu-Lys (position 423-424), and the 2 Lys at positions 416 and 488. Peptides resulting from digestion by S. aureus V8 protease and cyanogen bromide cleavage filled the missing part and completed the primary sequence of bLPL. The NH2 terminus of bLPL was determined to be Asp by sequencing the intact protein with a gas phase sequencer for up to 30 residues, whereas the COOH terminus was identified as Gly through, carboxyl peptidase Y cleavage. The enzyme contains 10 cysteine residues, all of which exist in disulfide linkages. They are formed between Cys29 and Cys42, Cys218 and Cys241, Cys266 and Cys285, Cys277, and Cys280, and Cys420 and Cys440. The sites of N-glycosylation were identified at Asn44 and Asn361. In accordance with a common structural homology of serine-type esterases, -G-X-S-X-G- (Yang, C. Y., Manoogian, D., Pao, Q., Lee, F., Knapp, R. D., Gotto, A. M., Jr., and Pownall, H. J. (1987) J. Biol. Chem., 262, 3086-3191), the active site serine of bLPL was assigned to the serine at position 134. The chymotrypsin nick of bLPL was determined to be between residues 390 and 391. A model of the enzyme is proposed on the basis of our data and available chemical data.
Yang
C Y
CY
Division of Atherosclerosis and Lipoprotein Research, Baylor College of Medicine, Houston, Texas 77030.
Gu
Z W
ZW
Yang
H X
HX
Rohde
M F
MF
Gotto
A M
AM
Pownall
H J
HJ
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D005260:Female; D008071:Lipoprotein Lipase; D008892:Milk / Q000201:enzymology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D010447:Peptide Hydrolases; D011487:Protein Conformation; D012689:Sequence Homology, Nucleic Acid; D012697:Serine Endopeptidases
D010446:Peptide Fragments; D008071:Lipoprotein Lipase; D010447:Peptide Hydrolases; D012697:Serine Endopeptidases; D003488:Cyanogen Bromide