Synthesis and properties of azidonitrophenyl pyrophosphate, a photoaffinity probe of the nucleotide binding sites of mitochondrial F1-ATPase
- Centre d'Etudes Nucleaires, Grenoble (France)
4-Azido-2-nitrophenyl pyrophosphate (azido-PPi) labeled with 32P in the alpha position was prepared and used to photolabel beef heart mitochondrial F1. Azido-PPi was hydrolyzed by yeast inorganic pyrophosphatase, but not by mitochondrial F1-ATPase. Incubation of F1 with (alpha-32P)azido-PPi in the dark under conditions of saturation resulted in the binding of the photoprobe to three sites, two of which exhibited a high affinity (Kd = 2 microM), the third one having a lower affinity (Kd = 300 microM). Mg2+ was required for binding. As with PPi, the binding of 3 mol of azido-PPi/mol of F1 resulted in the release of one tightly bound nucleotide. ADP, AMP-PNP, and PPi competed with azido-PPi for binding to F1, but Pi and the phosphate analogue azidonitrophenyl phosphate did not. The binding of (32P)Pi to F1 was enhanced at low concentrations of azido-PPi, as it was in the presence of low concentrations of PPi. Sulfite, which is thought to bind to an anion-binding site on F1, inhibited competitively the binding of both ADP and azido-PPi, suggesting that the postulated anion-binding site of F1 is related to the exchangeable nucleotide-binding sites. Upon photoirradiation of F1 in the presence of (alpha-32P)azido-PPi, the photoprobe became covalently bound with concomitant inactivation of F1. The plots relating the inactivation of F1 to the covalent binding of the probe were rectilinear up to 50% inactivation.
- OSTI ID:
- 7027333
- Journal Information:
- Biochemistry; (USA), Vol. 28:26; ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
ATP-ASE
RECEPTORS
PYROPHOSPHATES
BIOSYNTHESIS
AFFINITY
BIOCHEMICAL REACTION KINETICS
CATTLE
CHEMICAL PROPERTIES
ENZYME ACTIVITY
HEART
MITOCHONDRIA
NUCLEOTIDES
PHOSPHORUS 32
TRACER TECHNIQUES
ACID ANHYDRASES
ANIMALS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BODY
CARDIOVASCULAR SYSTEM
CELL CONSTITUENTS
DAYS LIVING RADIOISOTOPES
DOMESTIC ANIMALS
ENZYMES
HYDROLASES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LIGHT NUCLEI
MAMMALS
MEMBRANE PROTEINS
NUCLEI
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANOIDS
ORGANS
OXYGEN COMPOUNDS
PHOSPHOHYDROLASES
PHOSPHORUS COMPOUNDS
PHOSPHORUS ISOTOPES
PROTEINS
RADIOISOTOPES
REACTION KINETICS
RUMINANTS
SYNTHESIS
VERTEBRATES
550201* - Biochemistry- Tracer Techniques