Abstract
The bovine heart mitochondrial F1-ATPase (MF1) is reversibly inhibited in the dark by 4-amino-1-octylquinaldinium (AOQ) with an I0.5 value of 48 μM. When irradiated in the presence of AOQ, MF1 is photoinactivated with an apparent Kd of 12 μM. About 1.1 mol of [3H]AOQ were incorporated per mol of MF1 on complete photoinactivation. Fractionation of a cyanogen bromide digest of MF1 photolabeled with [3H]AOQ followed by fractionation of peptic digests of partially purified cyanogen bromide fragments led to isolation of two CNBr/peptic fragments labeled with3H. Sequence analysis of the labeled peptides revealed that one contained residues 423–441 of the β subunit. A gap in position 2 of the sequence indicates that βPhe424 is derivatized. The phenyl side-chain of this residue is part of a pocket that binds the adenine moiety of ATP or ADP at catalytic sites. The other peptide, which was labeled to a greater extent, contained residues 342–358 of the β subunit, but in this case, no gap was found in the sequence indicating that the derivatized amino-acid side-chain might not have survived the conditions of automatic Edman degradation. This peptide contains βTyr345, the side-chain of which is also a component of the pocket that binds the adenine moiety of ATP or ADP to catalytic sites. However, for the reason stated, there is no direct evidence that βTyr345 is labeled in this peptide.
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Bullough, D. A., Ceccarelli, E. A., Roise, D., and Allison, W. S. (1989) Inhibition of the bovine heart F1-ATPase by cationic dyes and amphipathic peptides.Biochim. Biophys. Acta 975, 377–383.
Zhuo, S. and Allison, W. S. (1988) Inhibition and photoinactivation of the bovine heart mitochondrial F1-ATPase by the cytotoxic agent, dequalinium. Biochem.Biophys. Res. Comm. 152, 968–972.
Zhuo, S., Paik, S. R., Register, J. A., and Allison, W. S. (1993) Photoinactivation of the bovine heart mitochondrial F1-ATPase by [14C]dequalinium cross-links phenylalanine-403 or phenylalanine-406 of an α subunit to a site or sites contained within residues 440–459 of a β subunit.Biochemistry 32, 2219–2227.
Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Structure at 2.8 Å resolution of F1-ATPase from bovine heart mitochondria.Nature 370, 621–628.
Weber, J. and Senior, A. E. (1997) Catalytic mechanism of F1-ATPase.Biochim. Biophys. Acta 1319, 19–58.
Esch, F. S. and Allison, W. S. (1978) Identification of a tyrosine residue at a nucleotide binding site in the β subunit of mitochondrial ATPase with p-fluorosulfony[14C]benzoyl-5′-adenosine.J. Biol. Chem. 253, 6100–6106.
Penefsky, H. S. (1977) Reversible binding of Pi by beef heart mitochondrial adenosine triphosphatase.J. Biol. Chem. 252, 2891–2899.
Austin, W. C., Potter, M. D., and Taylor, E. P. (1958) Potential trypanocides. The action of polymethylene dihalides on 4-aminoquinaldine.J. Chem. Soc. (London), 1489–1498.
Bradford, M. M. (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 72, 248–254.
Jault, J.-M. and Allison, W. S. (1994) Hysteretic inhibition of the bovine heart mitochondrial F1-ATPase is due to saturation of noncatalytic sites with ADP which blocks activation of the enzyme by ATP.J. Biol. Chem. 269, 319–325.
Penin, F., Godinot, C., and Gautheron, D. (1984) Two-dimensional gel electrophoresis of membrane proteins using anionic and cationic detergents.Biochim. Biophys. Acta 775, 239–245.
Schägger, H. and von Jagow, G. (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range of 1 to 100 kDa.Anal. Biochem. 166, 368–379.
Garin, J., Boulay, F., Issartel, J. P., Lunardi, J., and Vignais, P. V. (1986) Identification of amino acid residues photolabeled by 2-azido[α-32P]adenosine diphosphate in the β subunit of beef heart mitochondrial F1-ATPase.Biochemistry 25, 4431–4437.
Bullough, D. A. and Allison, W. S. (1986) Inactivation of the bovine heart mitochondrial F1-ATPase by 5′-p-fluorosulfonylbenzoyl[3H]inosine is accompanied by modification of tyrosine-345 in a single β subunit.J. Biol. Chem. 261, 14171–14177.
Paik, S. R., Jault, J.-M., and Allison, W. S. (1994) Inhibition and inactivation of the F1 adenosinetriphosphatase fromBacillus PS3 by dequalinium and activation of the enzyme by lauryl dimethylamine oxide.Biochemistry 33, 126–133.
Ren, H.-M. and Allison, W. S. (1997) Photoinactivation of the F1-ATPase from spinach chloroplasts by dequalinium is accompanied by derivatization of methionine β183.J. Biol. Chem. 272, 32294–32300.
Kagawa, Y. and Nukiwa, N. (1981) Conversion of a stable ATPase to labile ATPase by acetylation, and the αβ and αγ subunit complexes during its reconstitution.Biochem. Biophys. Res. Comm. 100, 1370–1376.
Van Raaij, M. J., Abrahams, J. P., Leslie, A. G. W., and Walker, J. E. (1996) The structure of bovine F1-ATPase complexed with the antibiotic inhibitor aurovertin B.Proc. Natl. Acad. Sci. USA 93, 6913–6917.
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Grodsky, N.B., Allison, W.S. The adenine pocket of a single catalytic site is derivatized when the bovine heart mitochondrial F1-ATPase is photoinactivated with 4-amino-1-octylquinaldinium. Cell Biochem Biophys 31, 285–294 (1999). https://doi.org/10.1007/BF02738243
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DOI: https://doi.org/10.1007/BF02738243