PMID:7727499
| Citation |
Miyauchi, M, Tozawa, K and Yoshida, M (1995) F1-ATPase alpha-subunit made up from two fragments (1-395, 396-503) is stabilized by ATP and complexes containing it obey altered kinetics. Biochim. Biophys. Acta 1229:225-32 |
|---|---|
| Abstract |
Inferred from the crystal structure of mitochondrial F1-ATPase (Abrahams, J.P. et al. (1994) Nature 370, 621-628), the proteinase-sensitive region around Phe-395 of thermophilic F1-ATPase alpha-subunit corresponds to the loop which connects main part of the carboxyl-terminal helical bundle domain with the ATP binding domain. This loop is in contact with the gamma- and adjacent beta-subunits. Two polypeptides corresponding to the sequence 1-395 and 396-503 of the alpha-subunit were expressed in Escherichia coli cells and they were copurified as an apparently functional alpha-subunit (alpha(395/396)) made up of two polypeptides. The isolated alpha(395/396) was stabilized by ATP-Mg, but not by ADP-Mg, although it bound both ATP-Mg and ADP-Mg with similar affinities (Kd, 11 microM and 14 microM, respectively). The alpha(395/396) was reconstitutable into alpha(395/396)3 beta 3 and alpha(395/396)3 beta 3 gamma complexes. Different from the intact the ATP-Mg-induced dissociation into alpha 1 beta 1 heterodimers. ATP hydrolysis by the alpha(395/396)3 beta 3 gamma complex underwent a slow initial phase, whereas the intact alpha 3 beta 3 gamma complex exhibited an accelerated initial phase. Steady-state ATPase activity at various ATP concentrations showed negative cooperativity for the intact alpha 3 beta 3 gamma complex but apparently positive cooperativity for the alpha(395/396)3 beta 3 gamma complex. The ATPase activities at a saturating ATP concentration of the complexes containing the alpha(395/396) were 180% of those containing intact alpha-subunits. These results indicate that a loop around Phe-395 is involved in intersubunit interaction in F1-ATPase. |
| Links | |
| Keywords |
Adenosine Diphosphate/metabolism; Adenosine Triphosphate/metabolism; Base Sequence; Cloning, Molecular; Enzyme Stability; Escherichia coli/genetics; Kinetics; Molecular Sequence Data; Oligodeoxyribonucleotides; Peptide Fragments/chemistry; Peptide Fragments/genetics; Peptide Fragments/metabolism; Protein Binding; Proton-Translocating ATPases/chemistry; Proton-Translocating ATPases/genetics; Proton-Translocating ATPases/metabolism; Recombinant Proteins |
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