The identification, solubilization, and characterization of microsome-associated glutathione S-transferases.

Glutathione S-transferase activity has been shown to be firmly associated with microsomal fractions. This activity could not be removed by repeated washing, by sonication, or by treatment of the microsomal fractions with high salt concentrations or urea. Treatment of the microsomal fraction with puromycin and MgCls to remove the ribosomes with any associated newly synthesized proteins did not dissociate microsomal glutathione S-transferase activity from microsomes. Glutathione S-transferase activity in highly purified endomembranes and plasma membrane fractions was highest in rough endoplasmic reticulum, followed by microsomes and smooth endoplasmic reticulum. Nuclei, nuclear envelope, Golgi apparatus, plasma membrane, and unbroken mitochondria had much lower specific activities. Three microsomal glutathione S-transferases were solubilized using Emulgen 911 at a concentration sufficient to solubilize integral microsomal proteins. The isoelectric points were similar to those of the cytoplasmic transferases A, B, and C. The relative amounts of the three enzymes separated by isoelectric focusing was similar in solubilized microsomal and cytoplasmic fractions, and in both fractions about 50% of the activity was precipitated by antiserum raised against cytoplasmic glutathione S-transferase A which cross-reacted with transferase C. These findings suggest that the pattern of glutathione S-transferases in the microsomal fraction is similar to that in the cytoplasm. However, the specific activity of glutathione S-transferase in the 100,000 x g supernatant was increased 2-fold following phenobarbital treatment while the specific activity in the microsomal fraction remained unchanged. Thus, the glutathione S-transferase activity in the microsomes appears to represent a stable and controlled localization of these enzymes in the microsomal membranes. This may be important in the control of some reactive metabolites generated by microsomal monooxygenases within this membrane which preferentially remain within the membrane because of their lipophilicity.