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Kinetic studies of protein-protein interactions between the beef heart mitochondrial F(1)-ATPase and its natural inhibitor protein utilizing synthetic peptides
Abstract
Peptide analogs corresponding to the conserved region of the natural ATPase inhibitor protein from beef heart, Candida utilis and Saccharomyces cerevisiae mitochondria were synthesized by solid-phase methodologies and tested for ATPase inhibitory activity. These peptides were found to be noncompetitive inhibitors of the F$\sb1$-ATPase in acidic reaction mixtures. These results closely match those obtained for the naturally occurring inhibitor proteins. The amphiphilic alpha helical nature of these, as well as that of the intact inhibitor protein suggests what may be an important and general motif in some protein-protein interfaces. Additional peptides were synthesized that correspond to the region of the beef heart beta subunit that bears a substantial homology with the conserved region of the inhibitor protein. This region of the $\beta$ subunit appears to be near the binding site of the beef heart inhibitor protein. The close relationship of these peptides to those previously found to inhibit the F$\sb1$-ATPase catalyzed hydrolysis of ATP lead to the studies of these peptide as possible inhibitors of the ATPase reaction. Several of the peptides were found to be inhibitors of the ATPase activity. The best inhibitor was the peptide resembling the beef heart beta subunit from amino acids 394-413. This peptide is closely related to the peptides that are derived from the conserved region of the inhibitor protein. The results of these experiments are discussed with respect to the predicted secondary structure of the peptides and their observed inhibitory activity. Those peptides having the greatest amount of predicted alpha helical content and the presence of two charge clusters of amino acids appeared to be the better inhibitors. The peptide analog which had the two charged clusters separated by an insertion of five glycine residues showed different kinetic characteristics than those seen for the inhibitor peptide analogs. These results are discussed in relation to the inhibitor protein-F$\sb1$-ATPase interactions at the molecular level and its effects upon the catalytic activity of the enzyme.
Subject Area
Biochemistry|Agricultural chemicals
Recommended Citation
Stout, Jay Stewart, "Kinetic studies of protein-protein interactions between the beef heart mitochondrial F(1)-ATPase and its natural inhibitor protein utilizing synthetic peptides" (1991). ETD collection for University of Nebraska-Lincoln. AAI9133317.
https://digitalcommons.unl.edu/dissertations/AAI9133317