Open AccessAggregation of pyrene‐labeled microsomal glutathione S ‐transferase
Author(s) -
PIEMONTE Fiorella,
CACCURI Anna M.,
MORGENSTERN Ralf,
ROSATO Nicola,
FEDERICI Giorgio
Publication year - 1993
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1993.tb18290.x
Subject(s) - pyrene , chemistry , maleimide , glutathione , microsome , fluorescence , thiol , transferase , enzyme , microsoma , stereochemistry , biochemistry , organic chemistry , physics , quantum mechanics
Microsomal glutathione S ‐transferase was labeled by the fluorescence probe N ‐(1‐pyrenyl)maleimide which modified 1 mol thiol residue/mol protein. The enzyme activity increased about tenfold after the binding. The pyrene‐labeled microsomal glutathione S ‐transferase exibited two fluorescence bands which are typical of pyrene; one at 393 nm attributable to unassociated pyrenes, the other at 480 nm attributable to pyrenes excimers (excited dimers). The excimeric fluorescence increased at high protein concentrations indicating a shift of the equilibrium of labeled polypeptide chains from trimeric complexes, the functional unit of microsomal glutathione S ‐transferase, to larger aggregates. At 25°C and at a 1% Triton X‐100 concentration, the calculated equilibrium constant of this process is 65 μM. Along with the formation of large aggregates, a progressive increase of the enzymic activity was observed. Thus, N ‐(1‐pyrenyl)maleimide appears to be a very useful probe to study the supramolecular structure of this enzyme.