title: Aggregation of pyrene-labeled microsomal glutathione S-transferase. Effect of concentration.
abstract: 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 exhibited two fluorescence bands which are typical of pyrene; one at 393 nm attributable to unassociated pyrenes, the other at 480 nm attributable to pyrene 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 degrees C and at a 1% Triton X-100 concentration, the calculated equilibrium constant of this process is 65 microM. 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.
authors.affiliation: Dipartimento di Biologia, Università di Tor Vergata, Roma, Italia.
authors.forename: F
authors.initials: F
authors.lastname: Piemonte
authors.forename: A M
authors.initials: AM
authors.lastname: Caccuri
authors.forename: R
authors.initials: R
authors.lastname: Morgenstern
authors.forename: N
authors.initials: N
authors.lastname: Rosato
authors.forename: G
authors.initials: G
authors.lastname: Federici
mesh_terms: D000818:Animals; D001665:Binding Sites; D005456:Fluorescent Dyes / Q000737:chemistry*; D005982:Glutathione Transferase / Q000302:isolation & purification / Q000378:metabolism*; D008301:Maleimides / Q000737:chemistry*; D008862:Microsomes, Liver / Q000201:enzymology*; D011721:Pyrenes / Q000737:chemistry*; D051381:Rats
chemical_list: D005456:Fluorescent Dyes; D008301:Maleimides; D011721:Pyrenes; C030984:pyrene; C003944:N-(3-pyrene)maleimide; D005982:Glutathione Transferase