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Localisation of adenine nucleotide-binding sites on beef-heart mitochondrial ATPase by photolabelling with 8-azido-ADP and 8-azido-ATP

Wagenvoord, R.J.; Van Der Kraan, I.; Kemp, A.

Biochimica et Biophysica Acta 548(1): 85-95

1979

ISSN/ISBN: 0006-3002
PMID: 158387
Accession: 005822611
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In addition to the previosuly studied 8-azido-ATP, 8-azido-ADP is a suitable photoaffinity label for beef-heart mitochondrial ATPase (F1). Photolysis at 350 nm of 8-azido-ADP in the presence of isolated F1 leads to inactivation of ATPase activity. Both ATP and ADP (but not AMP) protect against the inactivation. In the absence of Mg2+, 8-azido-ADP binds almost equally to the .alpha. and .beta. subunits of F1, whereas in the presence of Mg2+ the .alpha. subunits are predominantly labeled. The ATPase activity is completely inhibited when 2 molecules of 8-azido-ADP are bound/molecule F1. 8-Azido-ATP and ATP are competitive substrates for F1, indicating that in the presence of Mg2+ 8-azido-ATP binds to the same site as ATP. The amount of tightly bound nucleotides in F1 is not significantly changed upon incubation with 8-azido-ATP either in the light or the dark. 8-Azido-ATP is also a suitable photoaffinity label for F1 in the isolated ATPase complex and submitochondrial particles, photolabeling leading to inactivation of ATPase activity. As is the case for isolated F1 and .alpha. and/or .beta. subunits of F1 in the ATPase complex or particles are specifically labeled with 8-azido-ATP. Oxidative phosphorylation and the ATP-driven reduction of NAD+ by succinate are also inhibited by photolabeling Mg-ATP particles with 8-azido-ATP. In contrast to the uncoupled ATPase activity, where the 2 ATP-binding sites do not interact, cooperation between the 2 sites is required for ATP hydrolysis coupled to reduction of NAD+ by succinate.

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Related References

Wagenvoord, R.J.; Kemp, A.; Slater, E.C. 1980: The number and localisation of adenine nucleotide-binding sites in beef-heart mitochondrial ATPase (F1) determined by photolabelling with 8-azido-ATP and 8-azido-ADP Biochimica et Biophysica Acta 593(2): 204-211
Wagenvoord, R.J.; Van der Kraan, I.; Kemp, A. 1977: Specific photolabelling of beef-heart mitochondrial ATPase by 8-azido-ATP Biochimica et Biophysica Acta 460(1): 17-24
Martins, I.S.; Penefsky, H.S. 1994: Covalent modification of catalytic sites on membrane-bound beef heart mitochondrial ATPase by 2-azido-adenine nucleotides European Journal of Biochemistry 224(3): 1057-1065
Xue, Z.X.; Zhou, J.M.; Melese, T.; Cross, R.L.; Boyer, P.D. 1987: Chloroplast F1 ATPase has more than three nucleotide binding sites, and 2-azido-ADP or 2-azido-ATP at both catalytic and noncatalytic sites labels the beta subunit Biochemistry 26(13): 3749-3753
Zhixiong, X.U.E.; Junmei, Z.H.O.U.; Melese, T.; Cross, R.L.; Boyer, P.D. 1987: Chloroplast F1 ATPase has more than three nucleotide binding sites, and 2-azido-ADP or 2-azido-ATP at both catalytic and noncatalytic sites labels the β subunit Biochemistry (Easton) 26(13): 3749-3753
Xue, Z.; Zhou, J.M.; Melese, T. 1987: Chloroplast F1 ATPase has more than three nucleotide binding sites, and 2-azido-ADP or 2-azido-ATP at both catalytic and noncatalytic sites labels the b subunit Biochemistry (American Chemical Society) 26: 49-53
Wagenvoord, R.J.; Verschoor, G.J.; Kemp, A. 1981: Photolabelling with 8-azido-adenine nucleotides of adenine nucleotide-binding sites in isolated spinach chloroplast ATPase (CF1) Biochimica et Biophysica Acta 634(2): 229-236
Cross, R.L.; Cunningham, D.; Ben Aram, C.; Zhou, Z.M.; Xue, Z.; Boyer, P.D. 1987: Photoaffinity labeling of noncatalytic and catalytic nucleotide sites on mitochondrial f 1 atpase with 2 azido atp Federation Proceedings 46(6): 1967
Mueller, D.; Hudson, R.A.; Lee, C.P. 1979: Photo affinity labeling of beef heart sub mitochondrial membranes with an azido derivative of quinacrine Federation Proceedings 38(3 Part 1): 574
Vogel, P.D.; Nett, J.H.; Sauer, H.E.; Schmadel, K.; Cross, R.L.; Trommer, W.E. 1992: Nucleotide binding sites on mitochondrial F1-ATPase. Electron spin resonance spectroscopy and photolabeling by azido-spin-labeled adenine nucleotides support an adenylate kinase-like orientation Journal of Biological Chemistry 267(17): 11982-11986
Lunardi, J.; Garin, J.; Issartel, J.P.ul 1987: Mapping of nucleotide-depleted mitochondrial F1-ATPase with 2-azido-(a-32P)adenosine diphosphate. Evidence for two nucleotide binding sites in the b subunit The Journal of Biological Chemistry 262: 172-81
Lunardi, J.; Garin, J.; Issartel, J.P.; Vignais, P.V. 1987: Mapping of nucleotide-depleted mitochondrial F1-ATPase with 2-azido-[α32P]adenosine diphosphate: evidence for two nucleotide binding sites in the β subunit The Journal of Biological Chemistry 262(31): 15172-15181
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Selman, B.R. 1985: Localization of adenine nucleotide tight binding sites on thylakoid bound chloroplast coupling factor 1 by the use of 2 azido photoaffinity analogs Schaefer, G. Icsu (international Council Of Scientific Unions) Short Reports, Vol. 3. Third European Bioenergetics Conference; Hannover, West Germany, Sept. 2-7, . Xlviii 745p. Cambridge University Press: New York, N.y., Usa; Cambridge, England. Illus. 290-291
Garin, J.; Vincon, M.; Gagnon, J. 1994: Photolabeling of mitochondrial F1-H+ATPase by 2-azido(3H)ADP and 8-azido(3H)ADP entrapped as fluorometal complexes into catalytic sites of the enzyme Biochemistry (American Chemical Society) 33: 72-7