Studies on the beef heart mitochondrial F/sub 1/-ATPase with the photoaffinity label BzATP
Thesis/Dissertation
·
OSTI ID:7164350
The photoaffinity analog of ATP, 3'-O-(4-benzoyl) benzoyl ATP (BzATP), was used in kinetics and binding studies to investigate the mechanism of the beef heart mitochondrial F/sup 1/-ATPase. New methods were developed for the synthesis and purification of non-radioactive BzATP, /sup 3/H-BzATP, and ..gamma..-/sup 32/P-BzATP, and the molar absorption coefficient for BzATP was determined. Experimental conditions for photolysis and binding studies were defined in which the stability of both BzATP and F/sub 1/ was maintained. Initial experiments examined the kinetic interactions between F/sub 1/ and BzATP. In the absence of actinic illumination, BzATP was a slow substrate for the enzyme and behaved as a classical competitive inhibitor versus ATP. Under photolytic conditions, BzATP inactivated F/sub 1/ with pseudo first-order kinetics, and the photoinactivation reaction showed rate saturation suggesting specific, reversible binding of BzATP to F/sub 1/ prior to covalent bond formation. ATP protected against F/sub 1/ photoinactivation and F/sub 1/ preparations partially modified covalently yielded the same K/sub m/ for ATP as unmodified enzyme preparations. These results strongly suggested that BzATP was bound to catalytic sites on the enzyme.
- Research Organization:
- New York Univ., NY (USA)
- OSTI ID:
- 7164350
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACID ANHYDRASES
ANIMALS
ATP
ATP-ASE
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BODY
CARDIOVASCULAR SYSTEM
CATTLE
CELL CONSTITUENTS
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
DECOMPOSITION
DOMESTIC ANIMALS
ENZYME ACTIVITY
ENZYMES
FRACTIONATION
HEART
HYDROLASES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LABELLED COMPOUNDS
LIGHT NUCLEI
MAMMALS
MITOCHONDRIA
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANOIDS
ORGANS
PHOSPHOHYDROLASES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOTOCHEMICAL REACTIONS
PHOTOLYSIS
RADIOISOTOPES
REACTION KINETICS
RUMINANTS
SEPARATION PROCESSES
TRACER TECHNIQUES
TRITIUM COMPOUNDS
VERTEBRATES
59 BASIC BIOLOGICAL SCIENCES
ACID ANHYDRASES
ANIMALS
ATP
ATP-ASE
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BODY
CARDIOVASCULAR SYSTEM
CATTLE
CELL CONSTITUENTS
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
DECOMPOSITION
DOMESTIC ANIMALS
ENZYME ACTIVITY
ENZYMES
FRACTIONATION
HEART
HYDROLASES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LABELLED COMPOUNDS
LIGHT NUCLEI
MAMMALS
MITOCHONDRIA
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANOIDS
ORGANS
PHOSPHOHYDROLASES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOTOCHEMICAL REACTIONS
PHOTOLYSIS
RADIOISOTOPES
REACTION KINETICS
RUMINANTS
SEPARATION PROCESSES
TRACER TECHNIQUES
TRITIUM COMPOUNDS
VERTEBRATES