PAF-acetylhydrolase activity on Lp(a) before and during Cu2+-induced oxidative modification in vitro

被引:52
作者
Karabina, SAP
Elisaf, MC
Goudevenos, J
Siamopoulos, KC
Sideris, D
Tselepis, AD
机构
[1] UNIV IOANNINA,DEPT CHEM,BIOCHEM LAB,GR-45110 IOANNINA,GREECE
[2] UNIV IOANNINA,SCH MED,DEPT INTERNAL MED,GR-45110 IOANNINA,GREECE
[3] UNIV IOANNINA,SCH MED,DEPT CARDIOL,GR-45110 IOANNINA,GREECE
关键词
PAF-acetylhydrolase; lipoprotein (a); Apo(a) isoform; lysophosphatidylcholine; oxidation;
D O I
10.1016/0021-9150(96)05872-8
中图分类号
R5 [内科学];
学科分类号
1002 ; 100201 ;
摘要
In human plasma with no detectable lipoprotein (a) (Lp(a)) levels, platelet-activating factor acetylhydrolase (PAF-AH) is associated with low density lipoprotein (LDL) and high density lipoprotein (HDL) with a distribution of 70 and 30%, respectively. We used a density gradient ultracentrifugation procedure to study the distribution of PAF-AH among lipoproteins in plasma containing Lp(a). Lp(a) was migrated as a broad band in the density region of d = 1.050-1.100 g/ml, independently of its isoform size. In plasma with Lp(a) levels 30-40 mg/dl or 80-100 mg/dl the PAF-AH activity migrated in this density region was 4 or 9% higher as compared to plasma having Lp(a) levels < 8 mg/dl (P < 0.05 or P < 0.02, respectively). Enrichment of plasma with the dense LDL(5) subfraction, significantly increased the enzyme activity distributed in this density region. The physicochemical properties of the Lp(a)-associated PAF-AH activity were similar to those reported for the LDL-associated enzyme. However, the kinetic constants in small Lp(a) isoforms were significantly higher compared to large ones. Isoform F had apparent K-m = 117 +/- 9 mu mol/l and V-max = 94 +/- 5 nmol/mg protein per min, and isoform S2/S3 had apparent K-m = 36 +/- 9 mu mol/l and V-max = 25 +/- 5 nmol/mg protein per min. Removal of apolipoprotein (a) (apo(a)) from Lp(a) by reductive cleavage with dithiothreitol, slightly affected the amount of PAF-AH existing on Lp(a) since, only 15 +/- 5% of the total enzyme activity dissociated from its particle after density gradient ultracentrifugation. During Cu2+-induced Lp(a) oxidation, the PAF-AH activity decreased from 10.90 +/- 2.30 nmol/mg per min to 2.57 +/- 0.56 nmol/mg per min 4 h after the initiation of the oxidation (P < 0.001). The apparent K-m of the enzyme remained essentially unchanged during oxidation, whereas V-max was significantly decreased from 58.6 +/- 7.8 nmol/mg protein per min to 38.2 +/- 8.7 nmol/mg protein per min (P < 0.03). An extensive hydrolysis of the endogenous phosphatidylcholine (PC) to lysophosphatidylcholine (Lyse-PC) was observed during Lp(a) oxidation, since the Lyso-PC/sphingomyelin molar ratio at the end of oxidation (0.55 +/- 0.09) was significantly higher than that beforeoxidation (0.19 +/- 0.01, P < 0.001). Our results show that the existence of Lp(a) in plasma alters the distribution of PAT-AH among the other lipoproteins. Apo(a) seems to affect the association of the enzyme with Lp(a) but does not bind itself to PAF-AH. During Lp(a) oxidation, the PAF-AH activity decreases whereas an extensive hydrolysis of the endogenous PC lu Lyse-PC is observed which is possibly due lo the PAF-AH activity.
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页码:121 / 134
页数:14
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