Glutathione-S-transferase subunits pattern in rainbow trout isolated hepatocytes

Cytosolic GSTs are homo- or heterodimeric enzymes which have been assigned to four separate families designated Alpha, Mu, Pi and Theta on the 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 patterns in cytosolic fractions from liver and freshly isolated hepatocytes. Trout hepatocytes were isolated from male immature rainbow trout (Oncorhynchus mykiss) weighing 250–300 g, using a collagenase perfusion procedure. Cytosolic fractions were obtained from sonicated hepatocytes or homogenized liver by differential centrifugation. GSTs were purified by a glutathione affinity chromatography and the activity of retained fractions was detected with chlorodinitrobenzene as substrate. The GST subunits were separated and characterized by a combination of HPLC and electrospray-ionization mass spectrometry analysis. HPLC analysis of affinity fractions from freshly isolated hepatocytes exhibited five GST subunits with highly reproducible retention times of 14, 19, 21, 25 and 26 min respectively. This profile was qualitatively identical to that obtained from the cytosol prepared from the liver. Quantitatively, the major form in isolated cells as well as in the liver corresponded to the peak eluting at 14 min and could correspond to a Pi-class GST subunit. The less polar peaks, eluting at 25 and 26 min are minor in the liver cytosol and often present as traces in hepatocytes.