Isolation of a highly active H+-ATPase from beef heart mitochondria

The lysolecithin extraction procedure originally described by Sadleret al. (1974) has been modified to yield a H⁺-ATPase with high levels of P_i-ATP exchange activity (400–600 nmol × min^–1 × mg^–1). This activity is further enhanced (1400–1600 nmol × min^–1 × mg^–1) following sucrose density gradient centrifugation in the presence of asolectin. This enhancement results in part from a lipid-dependent activation and in part from removal of inactive complexes. The H⁺ translocating activity of the complex has been determined spectrophotometrically using binding of oxonol VI as an indicator of membrane potential. P_i-ATP exchange, ATP hydrolysis, and oxonol binding are sensitive to energy-transfer inhibitors (oligomycin, rutamycin) and/or uncouplers (DNP, FCCP).