GLUTATHIONE S-TRANSFERASES OF THE YEAST YARROWIA-LIPOLYTICA HAVE UNUSUALLY LARGE MOLECULAR-MASS

Two similar glutathione S-transferases (GSTs), which do not bind to gl utathione- or S-hexylglutathione-agarose affinity resins, have been pu rified from the yeast Yarrowia lipolytica. An approx. 400-fold purific ation was obtained by a combination of DEAE-Sephadex, phenyl-Sepharose , hydroxyapatite and Mono-Q anion-exchange chromatography. The native molecular mass of both proteins was estimated as approx. 110 kDa by bo th Superose-12 gel-filtration chromatography and non-denaturing electr ophoresis. SDS/PAGE indicated a subunit mass of 50 kDa. Reverse-phase HPLC of purified proteins gave a single, well-resolved, peak, suggesti ng that the proteins are homodimers. Identical behaviour on HPLC, nati ve electrophoresis and SDS/PAGE, N-terminal sequencing, sensitivity to a panel of inhibitors and identical specific activities with 1-chloro -2,4-dinitrobenzene as substrate suggest that the two isoenzymes are v ery similar. The enzymes do not immunoblot with antisera to any of the main GST classes, and N-terminal sequencing suggests no clear relatio nship with previously characterized enzymes, such as that of the fungu s, Phanerochaete chrysosporium [Dowd, Buckley and Sheehan (1997) Bioch em. J. 324, 243-248]. It is possible that the two isoenzymes arise as a result of post-translational modification of a single GST isoenzyme.