Characteristics of the non-exchangeable nucleotide binding sites of mitochondrial F1 revealed by dissociation and reconstitution with 2-azido-ATP.
The dissociation of mitochondrial F1-ATPase with 3 M LiCl at 0 degrees C, followed by reconstitution, has been analysed. FPLC over a gel filtration column in the dissociation buffer revealed the presence of two protein moieties, an alpha 3 gamma delta epsilon complex and single beta-subunits. When the dissociation and chromatography is performed at pH 6.2, the former protein moiety still contains some adenine nucleotides. Reconstitution of the dissociated complex is not possible any more after FPLC, probably due to the loss of residual adenine nucleotides. After a single column centrifugation step one nucleotide per F1 still remains bound. For reconstitution, additional ATP, or a suitable analog, is required. 2-Azido-ATP, but not 8-azido-ATP or ITP, can replace ATP during the reconstitution. F1, reconstituted in the presence of 2-azido-ATP, contains three tightly bound nucleotides, similar to freshly isolated F1, of which in this case one is an adenine nucleotide and two are azido-adenine nucleotides. One of the latter can be rapidly exchanged and is bound to a catalytic site. Covalent binding (at a beta-subunit) of the other tightly bound 2-azido-ATP by ultraviolet illumination does not result in inhibition of the enzyme. Digestion of F1 with trypsin, followed by HPLC, showed that the label is not bound to the fragment containing Tyr-368, nor to the fragment containing Tyr-345. This result was confirmed by CNBr digestion, followed by SDS-urea PAGE. We conclude that during dissociation of F1 one tightly bound nucleotide (ADP) remains bound at an alpha/beta interface site and that for reconstitution binding of ATP to a (non-catalytic) beta-site is required. The conformation of this site differs from that of the two catalytic beta-sites.
Hartog
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AF
E.C. Slater Institute for Biochemical Research, University of Amsterdam, Netherlands.
Edel
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CM
Lubbers
F B
FB
Berden
J A
JA
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000345:Affinity Labels; D000818:Animals; D001386:Azides / Q000378:metabolism*; D001665:Binding Sites; D002417:Cattle; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D008929:Mitochondria, Heart / Q000201:enzymology; D009711:Nucleotides / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000378:metabolism*
D000345:Affinity Labels; D001386:Azides; D009711:Nucleotides; C042231:2-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases