84] Soluble ATPase (F1) from a thermophilic bacterium: Purification, dissociation into subunits, and reconstitution from individual subunits

Abstract

This chapter discusses the purification, dissociation, and reconstitution of soluble ATPase (F 1 ) from a thermophilic bacterium. The activity to prevent leakage of protons from vesicles reconstituted with phospholipids and TF 0 is measured by means of a fluorescent dye or by direct pH measurement in a pH meter. The sensitivity to dicyclohexylcarbodiimide (DCCD) is measured in the presence of TF 0 and phospholipids. A unit of ATPase activity is that amount of enzyme, which catalyzes the turnover of one μmol of ATP per minute under the special assay condition. Specific activity is expressed as units per milligram of protein. The thermophilic bacterium PS3 is cultured with vigorous aeration in a medium containing a 0.8% polypeptone, 0.4% yeast extract, and 0.3% NaCl, pH 7.0, in the temperature range 65 ° –74 ° C for 12 hours. Large-scale cultures for 6 kg of cells per ton of medium are usually performed. The procedure involves three steps: extraction of TF 1 from the membranes, diethylaminoethyl-cellulose column chromatography, and Sepharose 6B column chromatography.