Purification from a yeast mutant of mitochondrial F1 with modified beta-subunit. High affinity for nucleotides and high negative cooperativity of ATPase activity.

Falson, P;Di Pietro, A;Jault, J M;Gautheron, D C;Boutry, Marc
(1989) Biochimica et Biophysica Acta : international journal of biochemistry and biophysics — Vol. 975, n° 1, p. 119-126 (1989)

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  • Falson, P
    Author
  • Di Pietro, A
    Author
  • Jault, J M
    Author
  • Gautheron, D C
    Author
  • Boutry, MarcUCLouvain
    Author
Abstract
Mitochondrial F1 containing genetically modified beta-subunit was purified for the first time from a mutant of the yeast Schizosaccharomyces pombe. Precipitation by poly(ethylene glycol) allowed us to obtain a very stable and pure enzyme from either mutant or wild-type strain. In the presence of EDTA, purified F1 retained high amounts of endogenous nucleotides: 4.6 mol/mol and 3.7 mol/mol for mutant and wild-type F1, respectively. The additional nucleotide in mutant F1 was ATP; it was lost in the presence of Mg2+, which led to a total of 3.4 mol of nucleotides/mol whereas wild-type F1 retained all its nucleotides. Mutant F1 bound more exogenous ADP than wild-type F1 and the same total nucleotide amount was reached with both enzymes. Kinetics of ATPase activity revealed a much higher negative cooperativity for mutant than for wild-type F1. Bicarbonate abolished this negative cooperativity, but higher concentrations were required for mutant F1. The mutant enzyme was more sensitive than the wild-type one to azide inhibition and ADP competitive inhibition; this indicated stronger interactions between nucleotide and F1 in the mutant enzyme. The latter also showed increased sensitivity to N,N'-dicyclohexylcarbodiimide irreversible inhibition.
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Falson, P., Di Pietro, A., Jault, J. M., Gautheron, D. C., & Boutry, M. (1989). Purification from a yeast mutant of mitochondrial F1 with modified beta-subunit. High affinity for nucleotides and high negative cooperativity of ATPase activity. Biochimica et Biophysica Acta : international journal of biochemistry and biophysics, 975(1), 119-126. https://hdl.handle.net/2078.5/208970 (Original work published 1989)