Study of the nucleotide binding site of the yeast Schizosaccharomyces pombe plasma membrane H+-ATPase using formycin triphosphate-terbium complex
The plasma membrane of yeasts contains an H+-ATPase similar to the other cation transport ATPases of eukaryotic organisms. This enzyme has been purified and shows H+ transport in reconstituted vesicles. In the presence of Mg2+, formycin triphosphate (FTP) is hydrolyzed by the H+-ATPase and supports H+ transport. When combined with terbium ion, FTP (Tb-FTP) and ATP (Tb-ATP) are no longer hydrolyzed. Competition between Mg-ATP and Tb-FTP for ATP hydrolysis indicates that terbium-associated nucleotides bind to the catalytic site of the H+-ATPase. The fluorescent properties of the Tb-FTP complex were used to study the active site of the H+-ATPase. Fluorescence of Tb-FTP is greatly enhanced upon binding into the nucleotide site of H+-ATPase with a dissociation constant of 1 microM. Tb-ATP, Tb-ADP, and Tb-ITP are competitive inhibitors of Tb-FTP binding with Ki = 4.5, 5.0, and 6.0 microM, respectively. Binding of Tb-FTP is observed only in the presence of an excess of Tb3+ with an activation constant Ka = 25 microM for Tb3+. Analysis of the data reveals that the sites for Tb-FTP and Tb3+ binding are independent entities. In standard conditions these sites would be occupied by Mg-ATP and Mg2+, respectively. These findings suggest an important regulatory role of divalent cations on the activity of H+-ATPase. Replacement of H/sub 2/O by D/sub 2/O in the medium suggests the existence of two types of nucleotide binding sites differing by the hydration state of the Tb3+ ion in the bound Tb-FTP complex.
- Research Organization:
- Centre d'Etudes Nucleaires de Grenoble, France
- OSTI ID:
- 6594070
- Journal Information:
- J. Biol. Chem.; (United States), Vol. 7
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
ATP
BIOCHEMICAL REACTION KINETICS
METABOLISM
RECEPTORS
ATP-ASE
ENZYME ACTIVITY
NUCLEOTIDES
PROTONS
MEMBRANE TRANSPORT
CELL MEMBRANES
DEUTERIUM
FLUORESCENCE
MAGNESIUM COMPOUNDS
SACCHAROMYCES
TERBIUM COMPOUNDS
TRACER TECHNIQUES
ACID ANHYDRASES
ALKALINE EARTH METAL COMPOUNDS
BARYONS
CELL CONSTITUENTS
ELEMENTARY PARTICLES
ENZYMES
FERMIONS
FUNGI
HADRONS
HYDROGEN ISOTOPES
HYDROLASES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LIGHT NUCLEI
LUMINESCENCE
MEMBRANE PROTEINS
MEMBRANES
MICROORGANISMS
NUCLEI
NUCLEONS
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PHOSPHOHYDROLASES
PLANTS
PROTEINS
RARE EARTH COMPOUNDS
REACTION KINETICS
STABLE ISOTOPES
YEASTS
550201* - Biochemistry- Tracer Techniques