P. Holvoet et al., PHOSPHOLIPID-BINDING AND LECITHIN-CHOLESTEROL ACYLTRANSFERASE ACTIVATION PROPERTIES OF APOLIPOPROTEIN-A-I MUTANTS, Biochemistry, 34(41), 1995, pp. 13334-13342
Recombinant human apolipoprotein A-I (ape A-I) and three deletion muta
nts: apo A-I(Delta Leu(44)-Leu(126)), apo A-I(Delta Glu(139)-Leu(170))
, and apo A-I(Delta Ala(190)-Gln(243)), purified from the periplasmic
space of Escherichia coli, were studied. The rate of turbidity decreas
e following mixing of apo A-I(Delta Ala(190)-Gln(243)) with dimyristoy
lphosphatidylcholine (DMPC) vesicles at 23 degrees C was 10-fold lower
than that of the other apo A-I proteins, confirming that the carboxy-
terminal region of apo A-I plays a role in rapid lipid binding. The St
okes radii of reconstituted high-density lipoproteins (rHDL), containi
ng dipalmitoylphosphatidylcholine and cholesterol, were larger for the
three apo A-I mutants [6.3 nm for apo A-I(Delta Leu(44)-Leu(126)), 6.
1 nm for apo A-I(Delta Glu(139)-Leu(170)), and 6.5 nm for apo A-I(Delt
a Ala(190)-Gln(243))] than for intact apo A-I (5.0 nm). The mutant rHD
L all contained 4 apo A-I molecules per particle as compared to 2 for
intact apo A-I. Circular dichroism measurements revealed 8 alpha-helic
es per apo A-I molecule, 5 per apo A-I(Delta Leu(44)-Leu(126)), 6 per
apo A-I(Delta Glu(139)-Leu(170)), and 4 per apo A-I(Delta Ala(190)-Gln
(243)) molecule as compared to predicted values of 8, 5, 6, and 6 alph
a-helices, respectively. The catalytic efficiencies for activation of
lecithin-cholesterol acyltransferase (LCAT) were 2-fold lower for apo
A-I(Delta Leu(44)-Leu(126)), 11-fold lower for apo A-I(Delta Glu(139)-
Leu(170)), and 8-fold lower for apo A-I(Delta Ala(190)-Gln(243)) than
for intact apo A-I, as a result of a significant decrease of V-max but
not of K-m values. In aggregate, these data suggest that deletion of
the amino-terminal or of the central domains of apo A-I has little eff
ect on lipid binding, whereas deletion of the carboxy-terminal domain
reduces the rate but not the extent of lipid binding. Furthermore, del
etion of the central domain [in apo A-I(Delta Glu(139)-Leu(170))] or c
onformational modifications in the central domain [lin apo A-I(Delta A
la(190)-Glu(243))] result in a decrease of the rate of LCAT activity t
hat is independent of the binding of LCAT to rHDL.