Oligomeric states of the detergent-solubilized human serum paraoxonase (PON1).
Human plasma paraoxonase (HuPON1) is a high density lipoprotein (HDL)-bound enzyme exhibiting antiatherogenic properties. The molecular basis for the binding specificity of HuPON1 to HDL has not been established. Isolation of HuPON1 from HDL requires the use of detergents. We have determined the activity, dispersity, and oligomeric states of HuPON1 in solutions containing mild detergents using nondenaturing electrophoresis, size exclusion chromatography, and cross-linking. HuPON1 was active whatever its oligomeric state. In nonmicellar solutions, HuPON1 was polydisperse. In contrast, HuPON1 exhibited apparent homogeneity in micellar solutions, except with CHAPS. The enzyme apparent hydrodynamic radius varied with the type of detergent and protein concentration. In C(12)E(8) micellar solutions, from sedimentation velocity, equilibrium analytical ultracentrifugation, and radioactive detergent binding, HuPON1 was described as monomers and dimers in equilibrium. A decrease of the detergent concentration shifted this equilibrium toward the formation of dimers. About 100 detergent molecules were associated per monomer and dimer. The assembly of amphiphilic molecules, phospholipids in vivo, in sufficiently large aggregates could be a prerequisite for anchoring of HuPON1 and then allowing stabilization of the enzyme activity. Changes of HDL size and shape could strongly affect the binding affinity and stability of HuPON1 and result in reduced antioxidative capacity of the lipoprotein.
Josse
Denis
D
Unité d'Enzymologie, Centre de Recherches du Service de Santé des Armées, 24 avenue des Maquis du Grésivaudan, BP 87, 38702 La Tronche Cedex, France. denis.josse@amedd.army.mil
Ebel
Christine
C
Stroebel
David
D
Fontaine
Alice
A
Borges
Frédéric
F
Echalier
Aude
A
Baud
Delphine
D
Renault
Frédérique
F
Le Maire
Marc
M
Chabrieres
Eric
E
Masson
Patrick
P
D000818:Animals; D043303:Aryldialkylphosphatase; D015153:Blotting, Western; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004950:Esterases / Q000097:blood / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D007700:Kinetics; D008051:Lipid Bilayers; D008075:Lipoproteins, HDL / Q000378:metabolism; D008823:Micelles; D011485:Protein Binding; D011817:Rabbits; D014461:Ultracentrifugation
D003432:Cross-Linking Reagents; D003902:Detergents; D008051:Lipid Bilayers; D008075:Lipoproteins, HDL; D008823:Micelles; D004950:Esterases; D043303:Aryldialkylphosphatase; C468406:PON1 protein, human