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Intermediate Mr cytosolic components potentiate hepatic 5'-deiodinase activation by thiols
Abstract:
The role in the activation of microsomal 5'-deiodinase (5'-DI) of rat hepatic cytosolic components of Mr approx. 13,000 (Fraction B) was studied in the presence of various concentrations of thiol compounds such as dithiothreitol (DTT), dihydrolipoamide (DHLA), GSH, and 2-mercaptoethanol (2-ME). Although Fraction B (which was prepared by gel filtration to exclude GSH and GSSG) had no intrinsic 5'-DI activity, could not stimulate microsomal 5'-DI activity in the absence of added thiol and did not contain GSH as a mixed disulphide, it could produce a 3-fold increase in the maximal deiodinase activity achievable with DTT as well as other thiols, with the order being the same as the activation potency of these thiols in the absence of Fraction B (i.e. DHLA greater than DTT greater than 2-ME greater than GSH). These observations suggest that: a component of cytosolic Fraction B, designated 'deiodination factor B' (DFB), operates as an efficient intermediary to enhance activation of microsomal 5'-DI by thiols through a mechanism independent of GSH; thiols may participate in a non-specific thiol-disulphide exchange with inactive (oxidized) DFB to convert it into an active form that contains one or more thiol groups and is more effective than GSH or other thiols in facilitating the re-activation of inactive (oxidized) microsomal 5'-DI thiol (ESI) to its active state (ESH).
Insights
A cytosolic factor enhances microsomal 5'-deiodinase (5'-DI) activation by thiols. This deiodination factor B (DFB) acts as an intermediary, improving thiol-mediated activation of 5'-DI.
Area of Science:
- Biochemistry
- Molecular Endocrinology
- Enzyme kinetics
Background:
- Microsomal 5 -deiodinase (5 -DI) is crucial for thyroid hormone metabolism.
- Cytosolic factors can modulate enzyme activity, but their specific roles in 5 -DI activation are not fully understood.
Purpose of the Study:
- To investigate the role of a specific rat hepatic cytosolic fraction (Fraction B, Mr approx. 13,000) in the activation of microsomal 5 -DI.
- To elucidate the mechanism by which cytosolic components interact with thiol compounds to influence 5 -DI activity.
Main Methods:
- Gel filtration chromatography to isolate cytosolic Fraction B.
- Enzyme assays measuring microsomal 5 -DI activity in the presence of Fraction B and various thiol compounds (dithiothreitol, dihydrolipoamide, GSH, 2-mercaptoethanol).
Main Results:
- Cytosolic Fraction B, devoid of intrinsic 5 -DI activity, significantly increased maximal deiodinase activity (3-fold) when combined with thiols.
- The order of thiol potency in activating 5 -DI in the presence of Fraction B mirrored their potency in its absence.
- A component in Fraction B, termed 'deiodination factor B' (DFB), was identified as an efficient intermediary for thiol-mediated 5 -DI activation, independent of GSH.
Conclusions:
- DFB enhances thiol-mediated activation of microsomal 5 -DI through a GSH-independent mechanism.
- Thiols likely engage in thiol-disulphide exchange with inactive DFB, converting it to an active form that efficiently re-activates oxidized microsomal 5 -DI.