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).
D000251:Adenosine Triphosphatases / Q000302:isolation & purification* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002499:Centrifugation, Density Gradient; D004734:Energy Metabolism; D066298:In Vitro Techniques; D008244:Lysophosphatidylcholines; D008929:Mitochondria, Heart / Q000201:enzymology*; D006180:Proton-Translocating ATPases
D008244:Lysophosphatidylcholines; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases