GLUTATHIONE-S-TRANSFERASE SUBUNITS PATTERN IN RAINBOW-TROUT ISOLATED HEPATOCYTES

Cytosolic GSTs are homo- or heterodimeric enzymes which have been assi gned to four separate families designated Alpha, Mu, Pi and Theta on t he basis of primary structure, substrate specificity and immunological properties. In fish, there are several cytosolic forms of the enzyme. The objective of the present work was to compare the GST subunit patt erns in cytosolic fractions from liver and freshly isolated hepatocyte s. Trout hepatocytes were isolated from male immature rainbow trout (O ncorhynchus mykiss) weighting 250-300 g, using a collagenase perfusion procedure. Cytosolic fractions were obtained from sonicated hepatocyt es or homogenized liver by differential centrifugation. GSTs were puri fied by a glutathione affinity chromatography and the activity of reta ined fractions was detected with chlorodinitrobenzene as substrate. Th e GST subunits were separated and characterized by a combination of HP LC and electrospray-ionization mass spectrometry analysis. HPLC analys is of affinity fractions from freshly isolated hepatocytes exhibited f ive GST subunits with highly reproducible retention times of 14, 19, 2 1, 25 and 26 min respectively. This profile was qualitatively identica l to that obtained from the cytosol prepared from the liver. Quantitat ively, the major form in isolated cells as well as in the liver corres ponded to the peak eluting at 14 min and could correspond to a Pi-clas s GST subunit. The less polar peaks, eluting at 25 and 26 min are mino r in the liver cytosol and often present as traces in hepatocytes. (C) 1998 Elsevier Science Ltd. All rights reserved.