Abstract
This study describes the influence of apolipoproteins on the hepatic lipase (HL)-mediated hydrolysis of phospholipids and triacylglycerol in high density lipoproteins (HDL). HL-mediated hydrolysis was assessed in well characterized, homogeneous preparations of spherical reconstituted high density lipoproteins (rHDL). The rHDL were comparable in size and lipid composition and contained either apoA-I ((A-I)rHDL) or apoA-II ((A-II)rHDL) as their sole apolipoprotein constituent. Preparations of rHDL containing only cholesteryl esters (CE) in their core, (A-I/CE)rHDL and (A-II/CE)rHDL, were used to assess phospholipid hydrolysis. Preparations of rHDL that contained triacylglycerol as their predominant core lipid, (A-I/TG)rHDL and (A-II/TG)rHDL, were used to assess both triacylglycerol and phospholipid hydrolysis. The rHDL contained trace amounts of either radiolabeled phospholipid or radiolabeled triacylglycerol. Hydrolysis was measured as the release of radiolabeled nonesterified fatty acids (NEFA) from the rHDL. Kinetic analysis showed that HL had a greater affinity for the phospholipids in (A-II/CE)rHDL (Km(app) = 0.2 mM) than in (A-I/CE)rHDL (Km(app) = 3.1 mM). This was also evident when hydrolysis was measured directly by quantitating NEFA mass. HL also had a greater affinity for the phospholipids and triacylglycerol in (A-II/TG)rHDL than in (A-I/TG)rHDL. The Vmax for phospholipid hydrolysis was, by contrast, greater for (A-I/CE)rHDL than for (A-II/CE)rHDL: 309.3 versus 49.1 nmol of NEFA formed/ml of HL/h. Comparable Vmax values were obtained for the hydrolysis of the phospholipids in (A-II/TG)rHDL and (A-I/TG)rHDL. In the case of triacylglycerol hydrolysis, the respective Vmax values for (A-I/TG)rHDL and (A-II/TG)rHDL were 1154.8 and 240.2 nmol of NEFA formed/ml of HL/h. These results show that apolipoproteins have a major influence on the kinetics of HL-mediated phospholipid and triacylglycerol hydrolysis in rHDL.
Highlights
Unlike lipoprotein lipase (LPL), which requires apolipoprotein C-II for maximal activity, there is no known protein cofactor for hepatic lipase (HL)
Since crosslinking studies have shown that spherical (A-I)reconstituted high density lipoproteins (HDL) (rHDL) and (AII)rHDL of comparable size and composition contain three and six apolipoprotein molecules/particle, respectively (16), the molar ratios in Table I are expressed relative to the number of apolipoprotein molecules
The (A-I)rHDL and (A-II)rHDL used in this study were comparable in size and lipid composition and differed only in their apolipoprotein content
Summary
Purification of ApoA-I and ApoA-II—ApoA-I and apoA-II were prepared from pooled human plasma donated by the Transfusion Service, Royal Adelaide Hospital. PLTP activity was quantitated as the transfer of L-3–1,2-di[1-14C]palmitoylphosphatidylcholine ([14C]DPPC) (112 mCi/mmol) (Amersham Pharmacia Biotech) from [14C]DPPC-labeled phospholipid vesicles to ultracentrifugally isolated HDL during a 2-h incubation at 37 °C (21). The two substrates (a and b), which were labeled with [14C]DPPC and contained CE in their core, were used to monitor phospholipid hydrolysis in the absence of triacylglycerol hydrolysis. Substrates c and d were labeled with [14C]DPPC and contained triacylglycerol as their predominant core lipid They are designated (A-I/TG)rHDL and (A-II/TG)rHDL and were used to study phospholipid hydrolysis against a background of triacylglycerol hydrolysis. The resulting [3H]triolein-labeled (A-I/TG)rHDL were isolated by sequential ultracentrifugation in the density range 1.063 Ͻ d Ͻ 1.21 g/ml using a TLA100.4 rotor (Beckman Instruments) as described elsewhere (17). Statistical Methods—The one-tailed, Student’s t test for two samples with equal variance was used to determine whether differences between values were significant
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