Inactivation mechanism of tetrameric .beta.-galactosidase by gamma-rays involves both fragmentation and temperature-dependent denaturation of protomers

Abstract

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)

The Active Streptococcal Hyaluronan Synthases (HASs) Contain a Single HAS Monomer and Multiple Cardiolipin Molecules

“…For example, Boyer and Kempner (42) found no rearrangement of glutathione Stransferase after irradiation. Other studies have also shown that a variety of irradiated proteins do not dissociate, precluding any exchange of subunits (43,44). Similar reasoning would apply to a HAS protein whose activity required the noncovalent association of other molecules (cofactors).…”

The role of phenylalanine in structure–function relationships of phenylalanine hydroxylase revealed by radiation target analysis

“…The tetrameric structure of the enzyme ␤-galactosidase is retained after a single radiation hit, as assessed by sizeexclusion chromatography (33); multiple radiation events are required to alter the tetramer elution pattern. These data imply that radiation-damaged subunits are still able to maintain the subunit interactions involved in a tetrameric association.…”

Radiation inactivation of galactose oxidase, a monomeric enzyme with a stable free radical

“…This finding is consistent with all reports that irradiated protein molecules do not dissociate even though there are scissions of the polypeptide backbone. 10,11 …”

“…10,11 Even though there are scissions of the polypeptide backbone, the radiation-fragmented protein molecules do not dissociate except under denaturing conditions. Only a single peak eluted from these size-exclusion columns; it contained essentially all the surviving protein and enzymatic activity.…”