Labeling of bovine heart cytochrome c oxidase with analogues of phospholipids. Synthesis and reactivity of a new cardiolipin benzaldehyde probe.
The syntheses of two new radioactive probes derived from cardiolipin and phosphatidylcholine are reported. These probes are derivatives of natural lipids and contain an amine-specific benzaldehyde in the head-group region. This functional group allows a choice of timing of the reaction (e.g., after equilibration and detergent removal) because an irreversible covalent bond is formed only upon the addition of reducing agent. These probes, as well as a benzaldehyde analogue of phosphatidic acid, and a water-soluble benzaldehyde reagent were covalently attached to bovine heart cytochrome c oxidase. After reconstitution into vesicles, the lipid-benzaldehyde probes selectively incorporated into the smaller polypeptides of the enzyme, while the remaining subunits (I-IV) exhibited little incorporation of label. The accessibility of amine groups labeled under the conditions used here was independent of the structural and charge differences between the benzaldehyde probes. This suggests that all three lipid probes react with polypeptides of the cytochrome c oxidase complex at general contact sites for membrane phospholipids. A water-soluble benzaldehyde reagent predominantly labeled subunits IV, Va, and Vb and polypeptides of VII-VIII. A comparison of these results facilitates a more refined view of the disposition of polypeptides of cytochrome c oxidase in respect to the lipid and aqueous phases.
Kuppe
A
A
Institute of Molecular Biology, University of Oregon, Eugene 97403.
Mrsny
R J
RJ
Shimizu
M
M
Firsan
S J
SJ
Keana
J F
JF
Griffith
O H
OH
D000818:Animals; D001547:Benzaldehydes / Q000378:metabolism*; D002308:Cardiolipins / Q000378:metabolism; D002417:Cattle; D003576:Electron Transport Complex IV / Q000378:metabolism*; D007202:Indicators and Reagents; D009682:Magnetic Resonance Spectroscopy; D008929:Mitochondria, Heart / Q000201:enzymology*; D010743:Phospholipids / Q000138:chemical synthesis / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013367:Submitochondrial Particles / Q000201:enzymology*
D001547:Benzaldehydes; D002308:Cardiolipins; D007202:Indicators and Reagents; D010743:Phospholipids; D003576:Electron Transport Complex IV; C032175:benzaldehyde