Photoaffinity labeling of mitochondrial adenosinetriphosphatase by 2-azidoadenosine 5'-(alpha-32P)diphosphate
2-Azidoadenosine 5'-diphosphate (2-azido-ADP) labeled with 32P in the alpha-position was prepared and used to photolabel the nucleotide binding sites of beef heart mitochondrial F1-ATPase. The native F1 prepared by the procedure of Knowles and Penefsky (Knowles, A. F., and Penefsky, H. S. (1972)) contained an average of 2.9 mol of tightly bound ADP plus ATP per mole of enzyme. Short-term incubation of F1 with micromolar concentrations of (alpha-32P)-2-azido-ADP in the dark in a Mg2+-supplemented medium resulted in the rapid supplementary binding of 3 mol of label/mol of F1, consistent with the presence of six nucleotide binding sites per F1. The Kd relative to the reversible binding of (alpha-32P)-2-azido-ADP to mitochondrial F1 in the dark was 5 microM in the presence of MgCl2 and 30 microM in the presence of ethylenediaminetetraacetic acid. A linear relationship between the percentage of inactivation of F1 and the extent of covalent photolabeling by (alpha-32P)-2-azido-ADP was observed for percentages of inactivation up to 90%, extrapolating to 2 mol of covalently bound (alpha-32P)-2-azido-ADP/mol of F1. Under these conditions, only the beta subunit was photolabeled. Covalent binding of one photolabel per beta subunit was ascertained by electrophoretic separation of labeled and unlabeled beta subunits based on charge differences and by mapping studies showing one major radioactive peptide segment per photolabeled beta subunit.
- Research Organization:
- Centre d'Etudes Nucleaires de Grenoble, France
- OSTI ID:
- 5636874
- Journal Information:
- Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 25; ISSN BICHA
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
ACID ANHYDRASES
ADP
ANIMALS
ATP
ATP-ASE
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CATTLE
CELL CONSTITUENTS
CHEMICAL BONDS
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
DOMESTIC ANIMALS
ENZYMES
HYDROLASES
ISOTOPES
LABELLING
LIGHT NUCLEI
MAMMALS
MITOCHONDRIA
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANOIDS
PHOSPHOHYDROLASES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOTOCHEMICAL REACTIONS
RADIOISOTOPES
RUMINANTS
VERTEBRATES