Photolabeling of mitochondrial F1-H+ATPase by 2-azido[3H]ADP and 8-azido[3H]ADP entrapped as fluorometal complexes into the catalytic sites of the enzyme.
In the presence of ADP and fluorometals, the ATPase activity of the catalytic sector, F1, of beef heart mitochondrial ATPase is strongly inhibited; this inhibition is dependent on the entrapment of ADP-fluoroaluminate complexes into the nucleotide binding sites of F1 [Lunardi, J., Dupuis, A., Garin, J., Issartel, J. P., Michel, L., Chabre, M., & Vignais, P. V. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8958-8962]. We described here the effect of fluoroaluminate on the binding of 2-azido[3H]ADP and 8-azido[3H]ADP to beef heart mitochondrial F1 in the absence and presence of light. When the incubation medium was supplemented with NaF and AlCl3, and maintained in the dark, both 2-azido[3H]ADP and 8-azido[3H]ADP were able to elicit inhibition of F1-ATPase activity, exactly like ADP did. Upon photoirradiation, 2-azido[3H]ADP and 8-azido[3H]ADP bound covalently to F1. Labeling was restricted to the beta subunit of F1, and the same tyrosine residue, beta-Tyr-345, was labeled by either of the photoprobes. This is in contrast with the previous findings that in the absence of fluoroaluminate both the alpha and beta subunits of F1 were photolabeled by 8-azido[3H]ADP, and that two different regions of the beta subunits were labeled, centered on beta-Tyr-345 in the case of 2-azido[3H]ADP [Garin, J., Boulay, F., Issartel, J.P., Lunardi, J., & Vignais, P. V. (1986) Biochemistry 25, 4431-4437] and beta-Tyr-311 that of 8-azido[3H]ADP [Hollemans, M., Runswick, M., Fearnley, I.H., & Walker, J.E. (1983) J. Biol. Chem. 258, 9307-9313].(ABSTRACT TRUNCATED AT 250 WORDS)
Garin
J
J
Département de Biologie Moléculaire et Structurale, CEA, Grenoble, France.
Vinçon
M
M
Gagnon
J
J
Vignais
P
P
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000345:Affinity Labels; D000535:Aluminum / Q000494:pharmacology; D000077410:Aluminum Chloride; D017607:Aluminum Compounds / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites; D002417:Cattle; D002712:Chlorides / Q000494:pharmacology; D003488:Cyanogen Bromide; D005461:Fluorine / Q000494:pharmacology; D008027:Light; D008929:Mitochondria, Heart / Q000201:enzymology*; D008969:Molecular Sequence Data; D010777:Photochemistry; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D012969:Sodium Fluoride / Q000494:pharmacology; D014316:Tritium; D014443:Tyrosine / Q000378:metabolism
D000345:Affinity Labels; D017607:Aluminum Compounds; D001386:Azides; D002712:Chlorides; D014316:Tritium; C053629:fluoroaluminum; D005461:Fluorine; D000077410:Aluminum Chloride; D014443:Tyrosine; D000244:Adenosine Diphosphate; C022295:2-azidoadenosine 3',5'-diphosphate; D012969:Sodium Fluoride; D000535:Aluminum; D006180:Proton-Translocating ATPases; C021622:8-azidoadenosine diphosphate; D003488:Cyanogen Bromide