Isolation and partial characterization of the inactive and active forms of human plasma phospholipid transfer protein (PLTP).
Plasma phospholipid transfer protein (PLTP) plays an important role in lipoprotein metabolism. Two forms of PLTP exist in human plasma, one catalytically active (high activity form, HA-PLTP) and the other inactive (low activity form, LA-PLTP) (Oka, T., Kujiraoka, T., Ito, M., Egashira, T., Takahashi, S., Nanjee, N. M., Miller, N. E., Metso, J., Olkkonen, V. M., Ehnholm, C., Jauhiainen, M., and Hattori, H. (2000) J. Lipid Res. 41, 1651-1657). The two forms are associated with macromolecular complexes of different size. The apparent size of LA-PLTP is 520 kDa and that of HA-PLTP is 160 kDa. Of the circulating PLTP mass only a minor portion is in the HA-PLTP form in normolipidemic subjects. In the present study we have isolated and partially characterized the LA and HA forms of PLTP. Both LA- and HA-PLTP bind to heparin-Sepharose and can be separated by elution with 0-0.5 m NaCl gradient, with HA-PLTP displaying higher affinity for the matrix. LA-PLTP was further purified using hydrophobic butyl-Sepharose and anti-PLTP immunoaffinity chromatography steps. HA-PLTP was subjected to a second heparin-Sepharose step and hydroxylapatite chromatography. Analysis of the two forms of PLTP by SDS-PAGE, Western blotting, immunoprecipitation, and gel filtration demonstrates that LA-PLTP is complexed with apoA-I whereas HA-PLTP is not. Instead, HA-PLTP copurified with apoE. Based on these findings we suggest a model in which nascent PLTP enters the circulation as a high specific activity form not associated with apoA-I. During or after the transfer of lipolytic surface remnants to HDL, PLTP is transferred to apoA-I-containing HDL particles and thereby becomes part of the low activity complex.
Kärkkäinen
Minna
M
Department of Molecular Medicine, National Public Health Institute, Biomedicum, P. O. Box 104, Helsinki FIN-00251, Finland.
Oka
Tomoichiro
T
Olkkonen
Vesa M
VM
Metso
Jari
J
Hattori
Hiroaki
H
Jauhiainen
Matti
M
Ehnholm
Christian
C
D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008055:Lipids / Q000097:blood; D008074:Lipoproteins / Q000097:blood; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids / Q000378:metabolism; D010956:Plasmapheresis
D002352:Carrier Proteins; D008055:Lipids; D008074:Lipoproteins; D008565:Membrane Proteins; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids