Radiation inactivation of proteins: temperature-dependent inter-protomeric energy transfer in ox liver catalase.
The radiation-inactivation method is widely used to determine the oligomeric structure of enzymes without need for solubilization or purification. We have used purified ox liver catalase, a tetrameric enzyme in solution, to study energy transfer between associated promoters responsible for oligomer inactivation. However, after freeze-drying the tetramer dissociates into an asymmetric dimer. In the present paper we compare both the radiation-inactivation size (obtained by following the activity decay) and the target size (obtained by measuring the amount of remaining protein by SDS/PAGE) of catalase under various states of aggregation and temperature. At -78 degrees C, only one promoter was fragmented after being hit by a gamma-ray and, as expected, this protomer was also inactivated. This result was obtained when either catalase was in tetrameric or in dimeric forms. However, at 38 degrees C, even though a single monomer was fragmented as at -78 degrees C, the whole dimer was inactivated. This result suggests that, at the higher temperature, there is a transfer of energy from the fragmented protomer to the other associated protomer, causing inactivation of the whole dimer. The inactivation of oligomeric enzymes is a two-step mechanism involving: (1) fragmentation of the hit monomer, followed by (2) temperature-dependent energy transfer from the fragmented towards the associated protomer. Thus we conclude that the radiation-inactivation size reflects the transfer of absorbed energy inside the oligomer which causes inactivation of one or several monomers.
Potier
M
M
Hôpital Sainte-Justine, Département de Pédiatrie, Université de Montréal, Québec, Canada.
Villemure
J F
JF
Thauvette
L
L
D000818:Animals; D002374:Catalase / Q000737:chemistry* / Q000528:radiation effects*; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D004735:Energy Transfer; D004789:Enzyme Activation; D005612:Freeze Drying; D005615:Freezing; D005720:Gamma Rays; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008970:Molecular Weight; D013696:Temperature
D046911:Macromolecular Substances; D002374:Catalase