| Autor: |
von Ballmoos, Christoph, Appoldt, Yvonne, Brunner, Josef, Granier, Thierry, Vasella, Andrea, Dimroth, Peter |
| Zdroj: |
Journal of Biological Chemistry; February 2002, Vol. 277 Issue: 5 p3504-3510, 7p |
| Abstrakt: |
A carbodiimide with a photoactivatable diazirine substituent was synthesized and incubated with the Na+-translocating F1F0ATP synthase from both Propionigenium modestumandIlyobacter tartaricus. This caused severe inhibition of ATP hydrolysis activity in the absence of Na+ions but not in its presence, indicating the specific reaction with the Na+binding c-Glu65residue. Photocross-linking was investigated with the substituted ATP synthase from both bacteria in reconstituted 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC)-containing proteoliposomes. A subunit c/POPC conjugate was found in the illuminated samples but no a-c cross-links were observed, not even after ATP-induced rotation of the c-ring. Our substituted diazirine moiety on c-Glu65was therefore in close contact with phospholipid but does not contact subunit a. Na+in/22Na+outexchange activity of the ATP synthase was not affected by modifying the c-Glu65sites with the carbodiimide, but upon photoinduced cross-linking, this activity was abolished. Cross-linking the rotor to lipids apparently arrested rotational mobility required for moving Na+ions back and forth across the membrane. The site of cross-linking was analyzed by digestions of the substituted POPC using phospolipases C and A2and by mass spectroscopy. The substitutions were found exclusively at the fatty acid side chains, which indicates that c-Glu65is located within the core of the membrane. |
| Databáze: |
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