Purification from a yeast mutant of mitochondrial F1 with modified beta-subunit. High affinity for nucleotides and high negative cooperativity of ATPase activity.
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.
Falson
P
P
Laboratoire de Biologie et Technologie des Membranes du CNRS, Université Claude Bernard de Lyon, Villeurbanne, France.
Di Pietro
A
A
Jault
J M
JM
Gautheron
D C
DC
Boutry
M
M
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D002850:Chromatography, Gel; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D009154:Mutation; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004718:Saccharomycetales / Q000201:enzymology*; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000235:genetics; D013367:Submitochondrial Particles / Q000201:enzymology*
D046911:Macromolecular Substances; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D008274:Magnesium