MITOCHONDRIAL ATP SYNTHASE - FE2-CATALYZED FRAGMENTATION OF THE SOLUBLE F1-ATPASE()

The treatment of the soluble F-1-ATPase with the Fe2+-ascorbate oxidat ive system has resulted in the inactivation and fragmentation of the e nzyme. Up to 10 polypeptide fragments could be readily observed on the SDS-PAGE. Addition of free Mg2+ or EDTA effectively prevented inactiv ation and fragmentation. Both alpha and beta subunits of the F-1-ATPas e were cleaved, with predominant cleavage sites being identified on al pha. Oxidative fragmentation of the F-1-ATPase showed nucleotide depen dence. Removal of nucleotides from the F-1-ATPase as well as their exc ess in the medium dramatically affected the fragmentation pattern. On the basis of the M(r) of the fragments, their immunorecognition with t he antibodies against subunits of the F-1-ATPase, and the results of t he mild proteolysis of the F-1-ATPase with trypsin, cleavage sites are suggested to be located in the nucleotide-binding domain of both alph a and beta subunits. Finally, it is hypothesized that similar structur al damage of the F-1-ATPase may occur in mitochondrion in vivo under o xidative stress conditions. (C) 1996 Academic Press, Inc.