Chemical modification of thiol groups of mitochondrial F1-ATPase from the yeast Schizosaccharomyces pombe. Involvement of alpha- and gamma-subunits in the enzyme activity
Mitochondrial F1-ATPase from the yeast Schizosaccharomyces pombe has been prepared under a stable form and in relatively high amounts by an improved purification procedure. Specific chemical modification of the enzyme by the thiol reagent N-ethylmaleimide (NEM) at pH 6.8 leads to complete inactivation characterized by complex kinetics and pH dependence, indicating that several thiols are related to the enzyme activity. A complete protection against NEM effect is afforded by low concentrations of nucleotides in the presence of Mg/sup 2 +/, with ADP and ATP being more efficient than GTP. A total binding of 5 mol of (/sup 14/C)NEM/mol of F1-ATPase is obtained when the enzyme is 85% inactivated: 3 mol of the label are located on the alpha-subunits and 2 on the gamma-subunit. Two out of the 3 mol on the alpha-subunits bind very rapidly before any inactivation occurs. Complete protection by ATP against inactivation by NEM prevents the modification of three essential thiols out of the group of five thiols labeled in the absence of ATP: one is located on a alpha-subunit and two on the gamma-subunit. These two essential thiols of the gamma-subunit can be differentiated by modification with 6,6'-dithiodinicotinic acid (CPDS), another specific thiol reagent. A maximal binding of 4 mol of (/sup 14/C)CPDS/mol of enzyme is obtained, concomitant to a 25% inhibition. Sequential modification of the enzyme by CPDS and (/sup 14/C)NEM leads to the same final deep inactivation as that obtained with (/sup 14/C)NEM alone. One out of the two thiols of the gamma-subunit is no longer accessible to (/sup 14/C)NEM after CPDS treatment. When incubated at pH 6.8 with (/sup 3/H)ATP in the presence of Mg/sup 2 +/, F1-ATPase is able to bind 3, largely exchangeable, mol of nucleotide/mol of enzyme. Modification of the three essential thiols by NEM dramatically decreases the binding of /sup 3/H-nucleotide down to about 1 mol/mol of enzyme.
- Research Organization:
- Universite Claude Bernard de Lyon, Villeurbanne, France
- OSTI ID:
- 5268137
- Journal Information:
- J. Biol. Chem.; (United States), Vol. 16
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
ATP-ASE
INACTIVATION
THIOLS
BIOLOGICAL EFFECTS
CARBON 14 COMPOUNDS
IMIDES
MITOCHONDRIA
TRACER TECHNIQUES
YEASTS
ACID ANHYDRASES
CELL CONSTITUENTS
ENZYMES
FUNGI
HYDROLASES
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
MICROORGANISMS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
ORGANOIDS
PHOSPHOHYDROLASES
PLANTS
560302* - Chemicals Metabolism & Toxicology- Microorganisms- (-1987)