Use of trypsin to monitor conformational changes of mitochondrial adenosinetriphosphatase induced by nucleotides and phosphate.
Upon incubation with trypsin, the adenosine-5'-triphosphatase (ATPase) activity of the nucleotide-depleted F1 is first rapidly and slightly activated and then slowly inactivated. The first phase is simultaneous with the conversion of the alpha subunit into an alpha' fragment which migrates between alpha and beta on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The second phase is related to the proteolysis of the three main subunits, alpha', beta, and gamma. Preincubation of the enzyme with low concentrations of adenosine 5'-diphosphate (ADP) or adenosine 5'-triphosphate (ATP) does not modify the slight increase of activity but efficiently prevents the inactivation induced by trypsin. The alpha leads to alpha' conversion is not affected whereas the further proteolysis of alpha', beta, and gamma does not occur. On the contrary, even high concentrations of GDP only slightly lower the trypsin-induced inactivation. The presence of endogenous tightly bound nucleotides also partially lowers the sensitivity to trypsin since F1 is less rapidly inactivated and proteolyzed than the nucleotide-depleted F1. Phosphate, at high concentrations, both slows down the first phase of activation and simultaneous alpha leads to alpha' conversion and prevents the second phase of inactivation and proteolysis of the main subunits. Pretreatment of the nucleotide-depleted F1 with trypsin under conditions where the ATPase activity is largely inhibited only slightly modifies, however, the hysteretic behavior of the enzyme: the ADP binding and the concomitant hysteretic inhibition of the residual activity are not markedly diminished. The purified ATPase-ATP synthase complex binds very few ADP's and is not hysteretically inhibited. Its ATPase activity is rapidly activated but not further inhibited by trypsin. Preincubation of the complex with ADP does not modify the effects of trypsin.
D000818:Animals; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology*; D010710:Phosphates / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D012265:Ribonucleotides / Q000494:pharmacology*; D013552:Swine; D014357:Trypsin / Q000378:metabolism*
D010710:Phosphates; D012265:Ribonucleotides; D014357:Trypsin; D006180:Proton-Translocating ATPases