A novel glutathione transferase (13-13) isolated from the matrix of rat liver mitochondria having structural similarity to class theta enzymes.
A rat liver mitochondrial-matrix fraction was prepared and shown to have 1-chloro-2,4-dinitrobenzene(CDNB)-metabolizing glutathione transferase (GST) activity. Further fractionation by sequential gel filtration, isoelectric focusing or chromatofocusing and hydroxyapatite chromatography yielded three GSTs of pI 9.3, 8.9 and 7.5, none of which bound to a GSH-agarose affinity matrix. Most of the activity was associated with the pI-9.3 form, which was selected for further study. Its activity was tested with the following potential substrates in addition to CDNB: 1,2-dichloro-4-nitrobenzene, p-nitrobenzyl chloride, trans-4-phenylbut-3-en-2-one, 1,2-epoxy-3-(p-nitrophenoxy)propane, ethacrynic acid, menaphthyl sulphate, cumene hydroperoxide, linoleic acid hydroperoxide and 4-hydroxynon-2-enal. Appreciable activity was obtained only with CDNB and ethacrynic acid (82 and 26 mumol/min per mg of protein respectively). The apparent Km for GSH, using 1 mM-CDNB, was 1.9 mM. The enzyme is a dimer of subunit Mr 26,500. It has a free N-terminus, which has enabled the first 33 amino acids to be sequenced. This portion of primary structure has a sequence in common with members of the Theta class of GSTs (eg. 36% identity with subunit 12) and also a sequence which might function as a mitochondrial import signal. It is novel and has been named 'GST 13-13'.
Harris
J M
JM
Department of Biochemistry, University College, London, U.K.
Meyer
D J
DJ
Coles
B
B
Ketterer
B
B
D000595:Amino Acid Sequence; D000818:Animals; D002845:Chromatography; D004137:Dinitrochlorobenzene / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry / Q000302:isolation & purification*; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D046911:Macromolecular Substances; D008297:Male; D008854:Microscopy, Electron; D008930:Mitochondria, Liver / Q000201:enzymology* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D051381:Rats; D011919:Rats, Inbred Strains; D012689:Sequence Homology, Nucleic Acid
D004137:Dinitrochlorobenzene; D046911:Macromolecular Substances; D005982:Glutathione Transferase