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

The lysolecithin extraction procedure originally described by Sadler et al. (1974) has been modified to yield a H+-ATPase with high levels of Pi-ATP exchange activity (400-600 nmol x min-1 x mg-1). This activity is further enhanced (1400-1600 nmol x min-1 x 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. Pi-ATP exchange, ATP hydrolysis, and oxonol binding are sensitive to energy-transfer inhibitors (oligomycin, rutamycin) and/or uncouplers (DNP, FCCP).