Study of the nucleotide binding site of the yeast Schizosaccharomyces pombe plasma membrane H+-ATPase using formycin triphosphate-terbium complex

Journal Article · Thu Mar 05 04:00:00 EST 1987 · J. Biol. Chem.; (United States)

OSTI ID:6594070

Ronjat, M; Lacapere, J J; Dufour, J P; Dupont, Y

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), Journal Name: J. Biol. Chem.; (United States) Vol. 7; ISSN JBCHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACID ANHYDRASES
ALKALINE EARTH METAL COMPOUNDS
ATP
ATP-ASE
BARYONS
BIOCHEMICAL REACTION KINETICS
CELL CONSTITUENTS
CELL MEMBRANES
DEUTERIUM
ELEMENTARY PARTICLES
ENZYME ACTIVITY
ENZYMES
FERMIONS
FLUORESCENCE
FUNGI
HADRONS
HYDROGEN ISOTOPES
HYDROLASES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LIGHT NUCLEI
LUMINESCENCE
MAGNESIUM COMPOUNDS
MEMBRANE PROTEINS
MEMBRANE TRANSPORT
MEMBRANES
METABOLISM
MICROORGANISMS
NUCLEI
NUCLEONS
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PHOSPHOHYDROLASES
PLANTS
PROTEINS
PROTONS
RARE EARTH COMPOUNDS
REACTION KINETICS
RECEPTORS
SACCHAROMYCES
STABLE ISOTOPES
TERBIUM COMPOUNDS
TRACER TECHNIQUES
YEASTS