Apolipoprotein A-I-cell membrane interaction: extracellular assembly of heterogeneous nascent HDL particles.
The ability of lipid-free human apoA-I expressed by transfected Chinese hamster ovary (CHO) cells to form apoA-I-lipid complexes extracellularly when incubated with CHO cell monolayers was investigated. Lipid-free apoA-I was incubated with nontransfected CHO-C19 cells for 24 h and extracellular assembly products were isolated at d < or = 1.235 g/ml; approximately 12% of the incubated apoA-I floated at d < or = 1.235 g/ml when apoA-I was added at 10 micrograms/ml. The composition of the particles was 51.3% protein, 20.3% phospholipid, and 28.3% cholesterol. Electron microscopy of the apoA-I-lipid complexes revealed that discoidal particles 15.4 +/- 4.1 nm diameter predominated but some vesicular particles 34.7 +/- 16.8 nm diameter were also in evidence. Nondenaturing gradient gel electrophoresis of the extracellular assembly products formed after 24 h incubation with 10 micrograms/ml apoA-I showed particle size heterogeneity with major bands at 11.2 and 9.0 nm; additional minor components banded at 7.3, 17.7, and 19.5 nm. This size distribution, as well as composition and electron microscopic structure, is similar to that of complexes isolated from the medium of CHO cells transfected with the human apoA-I gene. The formation of extracellular assembly complexes was concentration-dependent such that at 2 micrograms apoA-I/ml for 24 h, primarily 7.3 nm complexes were formed; at 4 micrograms/ml the distribution was more heterogeneous and the major band peaked at 9.2 nm, while at 8 micrograms/ml the 7.3 nm component was greatly diminished and the 11.2 nm component was the major one.(ABSTRACT TRUNCATED AT 250 WORDS)
Forte
T M
TM
Molecular and Nuclear Medicine Department, Lawrence Berkeley Laboratory, Berkeley, CA 94720.
Goth-Goldstein
R
R
Nordhausen
R W
RW
McCall
M R
MR
D000818:Animals; D016632:Apolipoprotein A-I / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008075:Lipoproteins, HDL / Q000378:metabolism*; D008563:Membrane Lipids / Q000378:metabolism*; D008854:Microscopy, Electron; D010316:Particle Size; D010743:Phospholipids / Q000378:metabolism; D014162:Transfection
D016632:Apolipoprotein A-I; D008075:Lipoproteins, HDL; D008563:Membrane Lipids; D010743:Phospholipids