Tightly associated cardiolipin in the bovine heart mitochondrial ATP synthase as analyzed by 31P nuclear magnetic resonance spectroscopy.
The bovine heart F0F1-ATPase preparation (Serrano, R., Kanner, B., and Racker, E. (1976) J. Biol. Chem. 251, 2453-2461) has been further delipidated. The lipid-deficient preparation contained 2.5 mol of cardiolipin, 1 mol of phosphatidylcholine (PC), and 1 mol of phosphatidylethanolamine (PE) per mol of F0F1. When reconstituted with asolectin the delipidated preparation exhibited an activity of 13 mumol of ATP hydrolyzed/min/mg of protein which was 88% oligomycin-sensitive. The phospholipids in this preparation were analyzed by 31P NMR spectroscopy to determine if they were immobilized by the enzyme (rendered NMR-invisible). The PC and PE were below the limits of detection under the conditions utilized and the cardiolipin was NMR-invisible until the enzyme was denatured by addition of either 1% sodium dodecyl sulfate or 8 M urea. Addition of cardiolipin to the delipidated preparation and subsequent analysis by NMR spectroscopy revealed that approximately 4 mol of cardiolipin were immobilized per mol of F0F1 ATPase. The enzyme appears to have high affinity for cardiolipin exclusively, since PC (a prominent inner membrane lipid), phosphatidyl serine (an acidic phospholipid), and phosphatidyl glycerol (the precursor to cardiolipin) were not immobilized (rendered NMR-invisible) when added to the delipidated preparation.
Eble
K S
KS
Department of Biochemistry, Wake Forest University Medical Center, Winston-Salem, North Carolina 27103.
Coleman
W B
WB
Hantgan
R R
RR
Cunningham
C C
CC
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002308:Cardiolipins / Q000378:metabolism*; D002417:Cattle; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008929:Mitochondria, Heart / Q000201:enzymology*; D010713:Phosphatidylcholines / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000378:metabolism; D011489:Protein Denaturation; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D014508:Urea / Q000494:pharmacology
D002308:Cardiolipins; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D012967:Sodium Dodecyl Sulfate; D000255:Adenosine Triphosphate; D014508:Urea; D006180:Proton-Translocating ATPases