Synthesis and properties of azidonitrophenyl pyrophosphate, a photoaffinity probe of the nucleotide binding sites of mitochondrial F1-ATPase

Journal Article · Mon Dec 25 23:00:00 EST 1989 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00452a021· OSTI ID:7027333

Michel, L; Garin, J; Issartel, J P; Vignais, P V ^[1]

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1. 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), Journal Name: Biochemistry; (USA) Vol. 28:26; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACID ANHYDRASES
AFFINITY
ANIMALS
ATP-ASE
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BIOSYNTHESIS
BODY
CARDIOVASCULAR SYSTEM
CATTLE
CELL CONSTITUENTS
CHEMICAL PROPERTIES
DAYS LIVING RADIOISOTOPES
DOMESTIC ANIMALS
ENZYME ACTIVITY
ENZYMES
HEART
HYDROLASES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LIGHT NUCLEI
MAMMALS
MEMBRANE PROTEINS
MITOCHONDRIA
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANOIDS
ORGANS
OXYGEN COMPOUNDS
PHOSPHOHYDROLASES
PHOSPHORUS 32
PHOSPHORUS COMPOUNDS
PHOSPHORUS ISOTOPES
PROTEINS
PYROPHOSPHATES
RADIOISOTOPES
REACTION KINETICS
RECEPTORS
RUMINANTS
SYNTHESIS
TRACER TECHNIQUES
VERTEBRATES