Interaction of phospholipids with the detergent-solubilized ADP/ATP carrier protein as studied by spin-label electron spin resonance.
The interaction of spin-labeled phospholipids with the detergent-solubilized ADP/ATP carrier protein from the inner mitochondrial membrane has been investigated by electron spin resonance spectroscopy. The equilibrium binding of cardiolipin and phosphatidic acid was studied by titration of the protein with spin-labeled phospholipid analogues using a spectral subtraction protocol for the evaluation of the mobile and immobilized lipid portions. This analysis revealed the immobilization of two molecules of spin-labeled cardiolipin per protein dimer. Phosphatidic acid has a similar affinity for the protein surface as cardiolipin. The lipid-protein interaction was less pronounced with the neutral phospholipids and with phosphatidylglycerol. The importance of the electrostatic contribution to the phospholipid-protein interaction shows up with a strong dependence of the lipid binding on salt concentration. Cleavage by phospholipase A2 and spin reduction by ascorbate of the spin-labeled acidic phospholipids in contact with the protein surface suggest that these lipids are located on the outer perimeter of the protein. At reduced detergent concentration, the protein aggregated upon addition of small amounts of cardiolipin but remained solubilized when more cardiolipin was added. This result is discussed with respect to the aggregation state of the protein in the mitochondrial membrane. It is also tentatively concluded that binding of spin-labeled cardiolipin does not displace the tightly bound cardiolipin of mitochondrial origin, which was detected previously by 31P nuclear magnetic resonance spectroscopy.
Drees
M
M
Institut für Physikalische Biochemie, Universität München, Federal Republic of Germany.
Beyer
K
K
D001667:Binding, Competitive; D003902:Detergents; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D007700:Kinetics; D000226:Mitochondrial ADP, ATP Translocases / Q000302:isolation & purification / Q000378:metabolism*; D009713:Nucleotidyltransferases / Q000378:metabolism*; D010741:Phospholipases A; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism*; D012995:Solubility; D013113:Spin Labels; D013816:Thermodynamics
D003902:Detergents; D010743:Phospholipids; D013113:Spin Labels; D000226:Mitochondrial ADP, ATP Translocases; D009713:Nucleotidyltransferases; D010741:Phospholipases A; D054467:Phospholipases A2