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Title: Glutathione S-transferases : catalytic and molecular properties of mu-class and theta-class isoenzymes
Author: Hussey, Amanda J.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1992
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The cytosolic Mu-class glutathione S-transferases (GST) present in human liver, skeletal muscle and testis comprise three distinct neutral-type subunits (N1 N2 and N3) which hybridize to form homodimers or heterodimers. The two N1-type subunits, which represent allelic variants encoded by the hepatic Mu-class GST locus, are designated N1a and N1b. Three isoenzymes containing N1-type subunits have been purified from one liver specimen by sequential affinity chromatography, hydroxyapatite chromatography and chromatofocusing. The skeletal muscle GST N1aN2, N1bN2, N2N2 and N2N3 were purified by a combination of affinity chromatography and anion-exchange f.p.l.c. followed by either chromatofocusing f.p.l.c. or hydroxyapatite h.p.l.c. In muscle the expression of the N1-type subunit, but not of the N2 and N3 subunits, was found to differ from specimen to specimen. The N1-type subunits were absent from about 50&37 of samples analyzed, and the purification results from four different specimens are presented to illustrate the phenotypic variation of skeletal muscle GST. The isolation of a homodimeric Mu-class isoenzyme, called GST N3N3, from human testicular tissue was achieved in two chromatographic steps; namely affinity chromatography followed by anion-exchange f.p.l.c. The neutral-type GST subunits have been defined by the decreasing isoelectric points of the homodimeric enzymes; GST N1aN1a, N1bN1b, N2N2 and N3N3 have pl values of 6.1, 5.5, 5.3 and 5.0. SDS/PAGE showed that N1, N2 and N3 have Mr values of 26700, 26000 and 263000 respectively. The N1, N2 and N3 subunits are catalytically distinct, with the N1-type subunits possessing high activity for trans-4-phenyl-3-buten-2-one and N2 having high activity towards 1,2-dichloro-4-nitrobenzene. The N-type subunits may also be distinguished immunochemically; antisera raised against the testicular GST N3N3 cross-reacted with the N3 subunit, and showed no reactivity towards either the N1-type or the N2 subunit. N-terminal amino acid sequence analysis supported the electrophoretic evidence that the N2 subunit in GST N1aN2, N1bN2, N2N2 and N2N3 represents the same polypeptide. The peptides obtained from CNBr digests of N2 were subjected separately to automated amino acid sequencing, and the results indicate that N2 is distinct but closely related to the protein encoded by the human Mu-class cDNA clone GTH4 [DeJong, Chang, Whang-Peng, Knutsen & Tu (1988) Nucleic Acids Res. 16, 8542-8554]. GST N2N2 is probably identical with GST4 [Board, Suzuki & Shaw (1988) Biochim. Biophys. Acta 953, 214-217], as over the 24 N-terminal residues of GST4 there is complete identity between the two enzymes. The N3 subunit, which is expressed in skeletal muscle and testis possesses a blocked N-terminus. Automated amino acid sequencing of a CNBr-derived peptide allowed 14 residues of the N3 subunit to be identified. This data indicated that testicular GST N3N3 is likely to be identical to brain/testis μ [Campbell, Takahashi, Abramovitz, Peretz & Listowsky (1990) J Biol. Chem. 265, 9188-9195].
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available