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Microsomal glutathione transferase: Species characteristics, catalysis and topology
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
1992 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

The membrane-bound detoxication enzyme microsomal glutathione transferase is localized predominantly in the liver of mammalian species, where it is present in high concentrations in the endoplasmic reticulum and the outer mitochondrial membrane. The enzyme conjugates a broad range of hydrophobic xenobiotics bearing reactive electrophilic centers to glutathione, which facilitates deactivation and excretion.

The microsomal glutathione transferase activity is increased several-fold upon the addition of sulfhydryl reagents, as well as by partial proteolysis. This unique property could reflect a mechanism of regulation of the enzyme in vivo. The microsomal glutathione transferase was purified from mouse and human liver and found to be similar to the rat enzyme in its structural properties, its activity profile and its ability to be activated by sulfhydryl reagents. The human enzyme displayed glutathione peroxidase/transferase activity towards products of lipid peroxidation, indicative of a functional role in protection against lipid peroxidation.

The histidine-specific reagent diethylpyrocarbonate (DEPC) rapidly inactivated the microsomal glutathione transferase and it was shown that 90 % of the activity was lost within the time period required for modification of the most reactive histidine. Inclusion of the substrate analogue S-hexylglutathione decreased the inactivation rate, indicating a functional importance for this histidine residue. Data is also presented which supports the involvement of arginine and lysine residue(s) in catalysis.

A random sequential catalytic mechanism could be defined for the microsomal glutathione transferase by the use of alternate substrate diagnosis. Chemical catalysis appears to rely on the lowering of the pKa of the thiol in glutathione to approximately 6.4. Activation of the enzyme was found to increase the catalytic efficiency towards a much broader range of substrates than was previously realized. This can be considered as a well-suited defence mechanism against toxic insult by electrophiles, when glutathione levels are usually decreased.

The thiol substrate specificity of microsomal glutathione transferase was investigated by the use of one glycyl- and eight y-glutamyl-modified glutathione analogues. The a-amino group of the glutamyl residue was demonstrated to be of preferential functional importance for the activity of the activated enzyme. The a-carboxyl of the glutamyl residue is obligatory for activity as demonstrated by the lack of activity with the descarboxy analogue 4-Abu-L-Cys-Gly. It was also noted that the activated microsomal glutathione transferase is more selective towards the thiol substrates than is the unactivated enzyme.

By comparing the tryptic cleavage products from intact and permeabilized microsomes, it was shown that the lysine-4 of the enzyme is located at the luminal side of the endoplasmic reticulum, whereas lysine-41 faces the cytosol. A hydrophobic stretch (positions 11-35) is the likely membranespanning region and additional membrane association is indicated. The active site, as well as the site of activation (cysteine-49), were demonstrated to face the cytosol.

Ort, förlag, år, upplaga, sidor
Stockholm: Stockholm University, 1992. , s. 54
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
URN: urn:nbn:se:su:diva-162686Libris ID: 7610748ISBN: 91-7153-000-2 (tryckt)OAI: oai:DiVA.org:su-162686DiVA, id: diva2:1268724
Disputation
1992-05-22, Arrheniuslaboratoriet, Svante Arrhenius väg 12, Stockholm, 10:00
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Tillgänglig från: 2018-12-06 Skapad: 2018-12-06 Senast uppdaterad: 2018-12-07Bibliografiskt granskad

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