Aurovertin, a fluorescent probe of conformational change in beef heart mitochondrial adenosine triphosphatase

T Chang, H S Penefsky
1973 Journal of Biological Chemistry  
Formation of a complex between aurovertin and soluble mitochondrial adenosine triphosphatase (FJ was accompanied by a 55-fold enhancement of fluorescence, an increase in the polarization of fluorescence from 0.278 for the free form to 0.375 for the bound form and a decrease in absorption at 366 nm. The fluorescence intensity of the complex was partially quenched by addition of ATP or Mg++ and enhanced by ADP. Two binding sites for aurovertin were found on F1 in the presence of ATP and one site
more » ... f ATP and one site in the presence of either ADP, Mg++, or dilute buffer. The dissociation constants of the fluorescent complex were 0.52, 0.07, 0.013, and 0.04 PM in the presence of ATP, ADP, Mg++, or in buffer, respectively. It is proposed that of the two binding sites for aurovertin on F1, only one site participates in inhibition of ATPase activity. Addition of succinate to a fluorescent complex of aurovertin and submitochondrial particles gave rise to an enhancement of fluorescence which depended on maintenance of an energized state. The changes in fluorescence of bound aurovertin were interpreted in terms of changes in the conformational state of the ATPase. The antibiotic aurovertin was first introduced as a tool for the study of oxidative phosphorylation by Lardy et al. (1) who established that this compound was a potent inhibitor of osidative phosphorylation, the 3'Pi-ATP eschange reaction, and the eschange of I*0 between the oxygens of Pi and ATP catalyzed by rat liver mitochondria. In addition, aurovertin inhibited the hydrolysis of ATP stimulated by some uncouplers (1). Subsequcntly, it was found by Lenaz (2) and Lee and Ernster (3) that the forward reaction of ATP synthesis in oxidative phosphorylation was much more sensitive to aurovertin inhibition than the reverse energy transfer reactions (supported by either ATP or succinate oxidation) such as the reduction of DPN+ by succinate, the energy-linked reduction of TPN+ by DPNH, and the translocation of Ca++. The adenosine triphosphatase activ-*
pmid:4266808 fatcat:qgqpqhtjjff5jntuaotxeofdvy