Purification, properties, sequencing, and cloning of a lipoprotein-associated, serine-dependent phospholipase involved in the oxidative modification of low-density lipoproteins.
A novel LDL-associated phospholipase A2 (LDL-PLA2) has been purified to homogeneity from human LDL obtained from plasma apheresis. This enzyme has activity toward both oxidized phosphatidylcholine and platelet activating factor (PAF). A simple purification procedure involving detergent solubilization and affinity and ion exchange chromatography has been devised. Vmax and Km for the purified enzyme are 170 micromol.min-1.mg-1 and 12 micromol/L, respectively. Extensive peptide sequence from LDL-PLA2 facilitated identification of an expressed sequence tag partial cDNA. This has led to cloning and expression of active protein in baculovirus. A lipase motif is also evident from sequence information, indicating that the enzyme is serine dependent. Inhibition by diethyl p-nitrophenyl phosphate and 3,4-dichloroisocoumarin and insensitivity to EDTA, Ca2+, and sulfhydryl reagents confirm that the enzyme is indeed a serine-dependent hydrolase. The protein is extensively glycosylated, and the glycosylation site has been identified. Antibodies to this LDL-PLA2 have been raised and used to show that this enzyme is responsible for >95% of the phospholipase activity associated with LDL. Inhibition of LDL-PLA2 before oxidation of LDL reduces both lysophosphatidylcholine content and monocyte chemoattractant ability of the resulting oxidized LDL. Lysophosphatidylcholine production and monocyte chemoattractant ability can be restored by addition of physiological quantities of pure LDL-PLA2.
Tew
D G
DG
Department of Mechanistic Enzymology, Welwyn, Hertfordshire, UK.
Southan
C
C
Rice
S Q
SQ
Lawrence
M P
MP
Li
H
H
Boyd
H F
HF
Moores
K
K
Gloger
I S
IS
Macphee
C H
CH
D000595:Amino Acid Sequence; D001024:Aortic Valve Stenosis / Q000235:genetics; D016367:Baculoviridae / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D006801:Humans; D008074:Lipoproteins / Q000378:metabolism*; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010741:Phospholipases A / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D054467:Phospholipases A2; D012694:Serine / Q000378:metabolism*
D008074:Lipoproteins; D008077:Lipoproteins, LDL; D012694:Serine; D010741:Phospholipases A; D054467:Phospholipases A2