The ligand-binding function of the porcine class Pi glutathione S-transferase
Date
2016-07-20
Authors
Bico, Paula C G
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Abstract
Glutathione S-transferases are multifunctional intracellular proteins. They catalyse the
conjugation of glutathione to endogenous'or foreign electrophiles, and also bind non-substrate
ligands.
Class Pi glutathione S-transferase (pGSTPl~l) was purified from porcine lung to a specific.
activity of 6.63p.ffiol/min/mg. The homodimeric protein has a molecular weight of about
4~.7kD and an isoelectric point of 8.6.
Anionic ligand-binding properties of this isoenzyme were investigated. Steady-state
fluorescence methods were used to determine ~ values for 8-anilino··l~naphtha1enesulphonic
acid (K, == 17.1p.M and 11.1J.tM using fluorescence enhancement techniques and quenching
techniques respectively), bromosulphophtbalein (Kcl=1.1p.M at pH 6.5 and 2.4/jM at pH
7.5) and glutathione {~=1201I.M). The affinity of bromosulphophthalein for the enzyme,
in the presence of 10mM glutathione was slightly enhanced (~=O.7.uM at pH 6.5). The
energy transfer betwecz the protein's tryptophan residues and 8-anUino-l-naphthalene
sulphonic acid was observed and found to be about 56% efficient. The impact of ligand
binding on both protein structure and catalytic activity were assessed. Kinetic studies show
that the active site of the enzyme is not the primary binding site for the non-substrate ligands,
but that the binding of bromosulphophthalein and to a lesser extent 8~ani1ino-l-!.~phtha1ene
sulphonic acid, does affect the active site of the enzyme, especially aner saturating
concentrations of the ligand. This may be the result of a small ligand-induced conformational
change. Fluorescence studies also indicate that the primary site for anionic ligand binding
is not in close proximity to either Trp28 or Trp38 in domain I, Competition studies indicated
that the two anionic ligands bind the Same site, < Prorein fluorescence, chemical modification
«
and size-exclusion HPLC data indicate that ligand binding does 110t induce gross
conformational changes in the protein.
Description
A dissertation submitted in fulfilment of the requirements for the degree of Master of Science
at the University of the Witwatersrand.
Johannesburg
February 1994