Sap. Karabina et al., PAF-ACETYLHYDROLASE ACTIVITY ON LP(A) BEFORE AND DURING CU2-INDUCED OXIDATIVE MODIFICATION IN-VITRO(), Atherosclerosis, 125(1), 1996, pp. 121-134
In human plasma with no detectable lipoprotein (a) (Lp(a)) levels, pla
telet-activating factor acetylhydrolase (PAF-AH) is associated with lo
w density lipoprotein (LDL) and high density lipoprotein (HDL) with a
distribution of 70 and 30%, respectively. We used a density gradient u
ltracentrifugation procedure to study the distribution of PAF-AH among
lipoproteins in plasma containing Lp(a). Lp(a) was migrated as a broa
d 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 m
g/dl the PAF-AH activity migrated in this density region was 4 or 9% h
igher 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) s
ubfraction, significantly increased the enzyme activity distributed in
this density region. The physicochemical properties of the Lp(a)-asso
ciated PAF-AH activity were similar to those reported for the LDL-asso
ciated enzyme. However, the kinetic constants in small Lp(a) isoforms
were significantly higher compared to large ones. Isoform F had appare
nt K-m = 117 +/- 9 mu mol/l and V-max = 94 +/- 5 nmol/mg protein per m
in, 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 aff
ected 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 unchang
ed 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 phosphatidy
lcholine (PC) to lysophosphatidylcholine (Lyse-PC) was observed during
Lp(a) oxidation, since the Lyso-PC/sphingomyelin molar ratio at the e
nd of oxidation (0.55 +/- 0.09) was significantly higher than that bef
oreoxidation (0.19 +/- 0.01, P < 0.001). Our results show that the exi
stence of Lp(a) in plasma alters the distribution of PAT-AH among the
other lipoproteins. Apo(a) seems to affect the association of the enzy
me with Lp(a) but does not bind itself to PAF-AH. During Lp(a) oxidati
on, the PAF-AH activity decreases whereas an extensive hydrolysis of t
he endogenous PC lu Lyse-PC is observed which is possibly due lo the P
AF-AH activity.