Apolipoproteins regulate the kinetics of endothelial lipase-mediated hydrolysis of phospholipids in reconstituted high-density lipoproteins.
Endothelial lipase (EL) is a newly identified member of the triglyceride lipase gene family that hydrolyzes high-density lipoprotein (HDL) phospholipids. This study investigates the ability of the major apolipoproteins of rHDL to regulate the kinetics of EL-mediated phospholipid hydrolysis in well-characterized, homogeneous preparations of spherical rHDL. The rHDL contained either apoA-I as the only apolipoprotein, (A-I)rHDL, apoA-II as the only apolipoprotein, (A-II)rHDL, or apoA-I as well as apoA-II, (A-I/A-II)rHDL. The rHDL were comparable in terms of size and lipid composition and contained cholesteryl esters (CE) as their sole core lipid. Phospholipid hydrolysis was quantitated as the mass of nonesterified fatty acids (NEFA) released from the rHDL during incubation with EL. The V(max) of phospholipid hydrolysis for (A-I/A-II)rHDL [391.9 +/- 12.9 nmol of NEFA formed (mL of EL)(-1) h(-1)] was greater than (A-I)rHDL [152.8 +/- 4.7 nmol of NEFA formed (mL of EL)(-1) h(-1)]. The energy of activation (E(a)) for the hydrolysis reactions was calculated to be 52.1 and 34.8 kJ mol(-1) for (A-I)rHDL and (A-I/A-II)rHDL, respectively. Minimal phospholipid hydrolysis was observed for the (A-II)rHDL. Kinetic analysis showed that EL has a higher affinity for the phospholipids in (A-I)rHDL [K(m)(app) = 0.10 +/- 0.01 mM] than in (A-I/A-II)rHDL [K(m)(app) = 0.27 +/- 0.03 mM]. Furthermore, (A-I)rHDL is a competitive inhibitor of the EL-mediated phospholipid hydrolysis of (A-I/A-II)rHDL. These results establish that apolipoproteins are major determinants of the kinetics of EL-mediated phospholipid hydrolysis in rHDL.
Caiazza
Daniela
D
Lipid Research Group, The Heart Research Institute, Camperdown, Sydney, NSW 2050, Australia. d.caiazza@hri.org.au
Jahangiri
Anisa
A
Rader
Daniel J
DJ
Marchadier
Dawn
D
Rye
Kerry-Anne
KA
D015060:1,2-Dipalmitoylphosphatidylcholine / Q000378:metabolism*; D000818:Animals; D016632:Apolipoprotein A-I / Q000378:metabolism*; D016633:Apolipoprotein A-II / Q000378:metabolism*; D019556:COS Cells; D002522:Chlorocebus aethiops; D006801:Humans; D008049:Lipase / Q000378:metabolism*; D008075:Lipoproteins, HDL / Q000378:metabolism*
D016632:Apolipoprotein A-I; D016633:Apolipoprotein A-II; D008075:Lipoproteins, HDL; D015060:1,2-Dipalmitoylphosphatidylcholine; C410382:LIPG protein, human; D008049:Lipase