Mapping of nucleotide-depleted mitochondrial F1-ATPase with 2-azido-(alpha-/sup 32/P)adenosine diphosphate. Evidence for two nucleotide binding sites in the beta subunit
Photolabeling of nucleotide binding sites in nucleotide-depleted mitochondrial F1 has been explored with 2-azido (alpha-/sup 32/P)adenosine diphosphate (2-N3(alpha-/sup 32/P) ADP). Control experiments carried out in the absence of photoirradiation in a Mg2+-supplemented medium indicated the presence of one high affinity binding site and five lower affinity binding sites per F1. Similar titration curves were obtained with (/sup 3/H)ADP and the photoprobe 3'-arylazido-(/sup 3/H)butyryl ADP (( /sup 3/H)NAP4-ADP). Photolabeling of nucleotide-depleted F1 with 2-N3(alpha-/sup 32/P)ADP resulted in ATPase inactivation, half inactivation corresponding to 0.6-0.7 mol of photoprobe covalently bound per mol F1. Only the beta subunit was photolabeled, even under conditions of high loading with 2-N3(alpha-/sup 32/P)ADP. The identification of the sequences labeled with the photoprobe was achieved by chemical cleavage with cyanogen bromide and enzymatic cleavage by trypsin. Under conditions of low loading with 2-N3(alpha-/sup 32/P)ADP, resulting in photolabeling of only one vacant site in F1, covalently bound radioactivity was located in a peptide fragment of the beta subunit spanning Pro-320-Met-358 identical to the fragment photolabeled in native F1. With a heavier load of photoprobe, leading to nearly 4 mol of photoprobe covalently bound per mol F1, an additional region of the beta subunit was specifically labeled, corresponding to a sequence extending from Gly-72 to Arg-83. The isolated beta subunit also displayed two binding sites for 2-N3-(alpha-/sup 32/P)ADP.
- Research Organization:
- Centre d'Etudes Nucleaires, Grenoble, France
- OSTI ID:
- 5784696
- Journal Information:
- J. Biol. Chem.; (United States), Journal Name: J. Biol. Chem.; (United States) Vol. 262:31; ISSN JBCHA
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
ACID ANHYDRASES
ADP
AMINO ACID SEQUENCE
ANIMALS
ATP-ASE
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CATTLE
DAYS LIVING RADIOISOTOPES
DOMESTIC ANIMALS
ENZYME ACTIVITY
ENZYMES
HYDROLASES
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
MAMMALS
MEMBRANE PROTEINS
MOLECULAR STRUCTURE
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PHOSPHOHYDROLASES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PROTEINS
RADIOISOTOPES
RECEPTORS
RUMINANTS
TRACER TECHNIQUES
VERTEBRATES