The GSTs of M. edulis provide an easily assayed activity which would be expected to respond to changes in pollution status. The main GST and a related GSH-binding protein have been purified and biochemically characterized. The former protein is most similar to the Pi class while the latter is a catalytically inactive monomer which appears to be related to the Mu class. This enzyme activity has been assessed as a potential indicator of exposure to chemical pollutants in both Cork Harbour and Venice Lagoon (using the closely related species, M. galloprovincialis). Controlled exposure studies with mussels in holding tanks have indicated that the herbicide aldicarb gives a slight but significant increase in GST activity consistent with the inducibility of these enzymes by xenobiotics in this bivalve. At present, we are studying samples which have been deliberately exposed to PAH and PCB compounds. Studies of this type are important in helping to understand the effects and fate of chemical pollutants released into estuarine environments.

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Glutathione reductase (ECl.6.4.2) is an FAD-wntaining flavoprotein, the principal function of which is to maintain a high ratio of GSHlGSSG via the reaction GSSG + NADPH + H+ -> 2GSH + NADP+. It is also involved in the biosynthesis of deoxyribonucleotides, in the cell cycle and in stress adaptation on a cellular level. The objective of this study was to show the effects of five isocyanates on the fluorescence spectrum of the enzyme, individually and in the presence of either NADPH or GSSG. lsocyanates are known to inhibit this enzyme [ I ,2] Isocyanate effects were studied at 1, 5, 10, 20 and 3Oppm. The different concentrations gave similar results for each isocyanate.Solutions of the aliphatic isocyanates, n-Octyl-. n-Butyl-and 2-Chloroethylisocyanate. made up in acetone, were inherently nonfluorescent when compared wth an acetone blank An acetone control showed no appreciable effect on the intrinsic fluorescence of glutathione reductase. n-Octylisocyanate caused a decrease in the fluorescence of the enzyme in the 300-360nm region of the spectrum. It is possible that the isocyanate is involved in a carbamylation reaction within the active site [3]. Such reactions should greatly affect the fluorescence of any tyrosine, tryptophan or phenylalanine residues that are in, or proximal to, the active site.A comparison of the fluorescence spectra of the enzyme in the presence and absence of GSSG confirms the absence of any quenching by GSSG. Since Tyrll4 is part of the GSSG binding site it is unlikely that this residue is quenched by the presence of isocyanate. However Tyrl97 is located in another area of the active site and so is more likely to be affected. There is a slight reduction in the fluorescence of the enzyme at 350nm (specific for tryptophan) when isocyanate is added. It is tempting to suggest the presence of a tryptophan residue proximal to the active site. However, models of the enzyme do not show any tryptophan residues in such a location and so the observed fluorescence decrease probably reflects the quenching of the tryptophan residue known to be at the surface. However another possibility is the fluorescence decrease may be due to Trp287 which is in close proximity to the NADPH-binding site. When NADPH is bound, the latter forms part of the active site. This decreased fluorescence was observed for all three aliphatic isocyanates with the apparent quench magnitude at 350nm decreasing with decreasing size of the n-alkyl side chain. This is Fluoresccnce Spectrum of Glutathione Reductase Effects of Alipbabic Isocynuak NADPH: OSSO: Wwdeqsth (mn) (1) (2) 4.4units (20ng) Glutathione reductase enzyme in 2mls of 125mM Phosphate-EDTA buffer, pH 7.5, with I without 2WpM GSSG Enzyme (as in (1) above) plus 1. 5, 10, 20 or 30ppm (final concentration in liquid) n-Octylisocyanale, n-Butylisocyanate or 2-Chloroelhylisocyanate Enzyme (as in (1) above) plus 2Wfl NADPH Acelone I n-Octylisocyanste / n-Butylisocyanate 12-Chloroethyl isocyanate fluorescence spectrum (3) (4) Fluorescence Spectrum of Glutahone Reductase Effe...

Biochemical Society Transactions (1 995) 23 361 S Perturbations in the cytosolic free calcium ion concentration in isolated cells exposed t o certain organic atmospheric pollutants 1.