title: Association of phosphatidic acid with the bovine mitochondrial ADP/ATP carrier.
abstract: The beef heart adenine nucleotide carrier protein (Anc) of the inner mitochondrial membrane can be purified in a form stabilized by binding the inhibitor carboxyatractyloside. The protein is copurified with bound lipid. We show for the first time that phosphatidic acid, although a minor component, is one of the lipids bound to Anc. The short spin-lattice relaxation time found by (31)P magic angle spinning nuclear magnetic resonance (MAS/NMR) for phosphatidic acid indicates that it is tightly bound to the protein. However, this lipid also has a comparatively small chemical shift anisotropy, suggesting that it can undergo rapid reorientation in space. In contrast, most of the lipid bound to Anc shows anisotropic motion typical of a bilayer arrangement. The phosphatidic acid that is detected in the purified preparation of Anc is also shown to be present initially in the unfractionated mitochondria, prior to the isolation of Anc. In Triton-solubilized mitochondria, phosphatidic acid, cardiolipin, phosphatidylethanolamine, and phosphatidylcholine exhibit resonance lines in the static (31)P NMR spectra, but in the purified Anc, only the phosphatidylethanolamine and phosphatidylcholine can be detected by this method, even though the other lipids are still present. This demonstrates that the phosphatidic acid and cardiolipin are interacting with the Anc. The thermal denaturation of the Anc was determined by differential scanning calorimetry. The protein denatures at 74 degrees C both before and after the NMR studies with the same characteristics.
authors.affiliation: Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, OntarioL8N 3Z5, Canada. epand@mcmaster.ca
authors.forename: Richard M
authors.initials: RM
authors.lastname: Epand
authors.forename: Raquel F
authors.initials: RF
authors.lastname: Epand
authors.forename: Bob
authors.initials: B
authors.lastname: Berno
authors.forename: Ludovic
authors.initials: L
authors.lastname: Pelosi
authors.forename: Gérard
authors.initials: G
authors.lastname: Brandolin
mesh_terms: D000818:Animals; D002308:Cardiolipins / Q000737:chemistry; D002417:Cattle; D000226:Mitochondrial ADP, ATP Translocases / Q000737:chemistry* / Q000302:isolation & purification; D008968:Molecular Conformation; D019906:Nuclear Magnetic Resonance, Biomolecular; D010712:Phosphatidic Acids / Q000737:chemistry*; D011485:Protein Binding
chemical_list: D002308:Cardiolipins; D010712:Phosphatidic Acids; D000226:Mitochondrial ADP, ATP Translocases