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Identity of microsomal glutathione S-transferases
Abstract:
Mouse liver microsomes were prepared by repeated washing, homogenization, and centrifugation until almost no more soluble enzymes were found in the supernatant of the last centrifugation. About 0.09% of the total glutathione S-transferase activity and comparable amount of soluble enzymes were detected in microsomes solubilized with Emulgen 913. By double immunodiffusion, microsomal glutathione S-transferase were shown to have a complete immunological identity with cytosolic F2 and F3 transferase from mouse liver. By Sephadex gel filtration chromatography in 1% Emulgen 913, part of the microsomal transferase activity (20 to 50%) was shown to be associated with the microsomal membrane protein fraction and appeared in the void volume. Partially purified microsomal transferases were found to have molecular weights, isoelectric points and Km's for substrate and GSH which are comparable to those of soluble liver transferases. This study seems to suggest that the presence of glutathione S-transferases in microsomes is the result of specific and nonspecific association between the microsomal membrane and soluble liver transferases.
Insights
Glutathione S-transferases found in mouse liver microsomes are immunologically identical to soluble forms. Their presence suggests association with the microsomal membrane, not unique microsomal enzymes.
Area of Science:
- Biochemistry
- Cell Biology
- Enzymology
Background:
- Microsomes are critical cellular components involved in various metabolic processes.
- Glutathione S-transferases (GSTs) are a superfamily of enzymes crucial for detoxification.
- The localization and origin of GSTs within microsomes remain incompletely understood.
Purpose of the Study:
- To investigate the presence and characteristics of glutathione S-transferase activity in mouse liver microsomes.
- To determine if microsomal GSTs are distinct entities or associated with soluble forms.
- To elucidate the relationship between microsomal membranes and GSTs.
Main Methods:
- Preparation of mouse liver microsomes through rigorous washing and centrifugation.
- Solubilization of microsomal proteins using Emulgen 913.
- Double immunodiffusion assays to compare microsomal and cytosolic GSTs.
- Sephadex gel filtration chromatography to analyze microsomal transferase activity and membrane association.
- Characterization of kinetic parameters (Km) and physical properties (molecular weight, isoelectric point) of purified enzymes.
Main Results:
- A small fraction (0.09%) of total glutathione S-transferase activity was detected in purified microsomes.
- Microsomal GSTs exhibited complete immunological identity with cytosolic F2 and F3 transferases.
- 20-50% of microsomal transferase activity was associated with the microsomal membrane fraction.
- Purified microsomal transferases shared comparable molecular weights, isoelectric points, and kinetic parameters with soluble liver transferases.
Conclusions:
- The glutathione S-transferases found in mouse liver microsomes are likely not unique microsomal enzymes.
- Their presence is attributed to specific and nonspecific association with the microsomal membrane.
- This association involves soluble liver transferases interacting with the microsomal membrane proteins.