Trypsin cleavage of the alpha-subunit of beef heart F1-ATPase abolishes ATP synthesis and ATP-driven energy-transduction capabilities.
Previous work has shown that mild trypsin treatment eliminates energy-transduction capability and tight (non-exchangeable)nucleotide binding in beef heart mitochondrial F1-ATPase (Leimgruber, R.M. and Senior, A.E. (1976) J. Biol. Chem. 251, 7103-7109). The structural change brought about by trypsin was, however, too subtle to be identified by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis, and was not defined. In this work we have applied two-dimensional electrophoresis (isoelectric focussing then sodium dodecyl sulfate polyacrylamide gradient electrophoresis) to the problem, and have determined that the alpha-subunit of F1 is altered by the mild trypsin treatment, whereas no change was detected in beta-, gamma-, delta- or epsilon-subunits. Binding of ADP to the trypsin-treated F1 was compared to binding to control enzyme over a range of 0-40 muM ADP in a 30 min incubation period. There was no difference between the two enzymes, KADPd in Mg2+ -containing buffer was about 2 muM in each. Since the tight (nonexchangeable)sites are abolished in trypsin-treated F1, this shows that tight exchangeable ADP-binding sites are different from the tight nonexchangeable ADP-binding sites. There was no effect of trypsin cleavage of the alpha-subunit on beta-subunit conformation as judged by aurovertin fluorescence studies. The cleavage of the alpha-subunit which occurred was judged to occur very close to the C- or N-terminus of the subunit and constitutes therefore a small and specific chemical modification which abolishes overall function in F1 but leaves partial functions intact.
D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000096:biosynthesis*; D000818:Animals; D001313:Aurovertins / Q000378:metabolism; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D004735:Energy Transfer; D007525:Isoelectric Focusing; D046911:Macromolecular Substances; D009206:Myocardium / Q000201:enzymology*; D006180:Proton-Translocating ATPases; D014357:Trypsin / Q000378:metabolism*
D001313:Aurovertins; D046911:Macromolecular Substances; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D014357:Trypsin; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases