Phospholipid binding and lecithin-cholesterol acyltransferase activation properties of apolipoprotein A-I mutants.
Recombinant human apolipoprotein A-I (apo A-I) and three deletion mutants: apo A-I(delta Leu44-Leu126), apo A-I(delta Glu139-Leu170), and apo A-I(delta Ala190-Gln243), purified from the periplasmic space of Escherichia coli, were studied. The rate of turbidity decrease following mixing of apo A-I(delta Ala190-Gln243) with dimyristoylphosphatidylcholine (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 Stokes radii of reconstituted high-density lipoproteins (rHDL), containing dipalmitoylphosphatidylcholine and cholesterol, were larger for the three apo A-I mutants [6.3 nm for apo A-I(delta Leu44-Leu126), 6.1 nm for apo A-I(delta Glu139-Leu170), and 6.5 nm for apo A-I(delta Ala190-Gln243)] than for intact apo A-I (5.0 nm). The mutant rHDL all contained 4 apo A-I molecules per particle as compared to 2 for intact apo A-I. Circular dichroism measurements revealed 8 alpha-helices per apo A-I molecule, 5 per apo A-I(delta Leu44-Leu126), 6 per apo A-I(delta Glu139-Leu170), and 4 per apo A-I(delta Ala190-Gln243) molecule as compared to predicted values of 8, 5, 6, and 6 alpha-helices, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Holvoet
P
P
Center for Molecular and Vascular Biology, University of Leuven, Belgium.
Zhao
Z
Z
Vanloo
B
B
Vos
R
R
Deridder
E
E
Dhoest
A
A
Taveirne
J
J
Brouwers
E
E
Demarsin
E
E
Engelborghs
Y
Y
D000595:Amino Acid Sequence; D016632:Apolipoprotein A-I / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D001483:Base Sequence; D003001:Cloning, Molecular; D004789:Enzyme Activation; D006801:Humans; D008075:Lipoproteins, HDL / Q000737:chemistry*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000737:chemistry; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D010957:Plasmids; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D015183:Restriction Mapping; D017384:Sequence Deletion; D013050:Spectrometry, Fluorescence
D016632:Apolipoprotein A-I; D008075:Lipoproteins, HDL; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D010743:Phospholipids; D011994:Recombinant Proteins; D007862:Phosphatidylcholine-Sterol O-Acyltransferase