Inactivation mechanism of tetrameric beta-galactosidase by gamma-rays involves both fragmentation and temperature-dependent denaturation of protomers.
The radiation inactivation method is widely used to estimate the molecular size of membrane-bound enzymes, receptors, and transport systems in situ. The method is based on the principle that exposure of frozen solutions or lyophilized protein preparations to increasing doses of ionizing radiations results in a first-order decay of biological activity proportional to radiation inactivation size of the protein. This parameter is believed to reflect the "functional unit" of the protein defined as the minimal assembly of structure (protomers) required for expression of a given biological activity. We tested the functional unit as a concept to interpret radiation inactivation data of proteins with Escherichia coli beta-galactosidase, where the protomers are active only when associated in a tetramer. Gamma-Irradiation of beta-galactosidase at both -78 and 38 degrees C followed by quantitation of the residual unfragmented promoter band by SDS-polyacrylamide gel electrophoresis yielded the protomer size, indicating that only one protomer is fragmented by each radiation hit. By following the enzyme activity as a function of dose it was found that only the protomer that has been directly hit and fragmented at -78 degrees C was effectively inactivated. In contrast, at 38 degrees C, it was the whole tetramer that was inactivated. beta-Galactosidase cannot have two different functional units depending on temperature. The inactivation of the whole beta-galactosidase tetramer at 38 degrees C is in fact related to protomer fragmentation but also to the production of stable denatured protomers (detected by gel-filtration HPLC and differential UV spectroscopy) due to energy transfer from fragmented protomers toward unhit protomers.(ABSTRACT TRUNCATED AT 250 WORDS)
Potier
M
M
Service de Génétique Médicale, Hôpital Sainte-Justine, Université de Montréal, Québec, Canada.
Thauvette
L
L
Michaud
L
L
Giroux
S
S
Beauregard
G
G
D004789:Enzyme Activation / Q000187:drug effects / Q000528:radiation effects; D004926:Escherichia coli / Q000201:enzymology; D005720:Gamma Rays; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D008958:Models, Molecular; D011487:Protein Conformation / Q000528:radiation effects; D011489:Protein Denaturation; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature; D001616:beta-Galactosidase / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000528:radiation effects*
D006146:Guanidines; D001616:beta-Galactosidase; D019791:Guanidine