Open AccessPurification of a new acidic glutathione S ‐transferase, GST‐Yn 1 Yn 1 , with a high leukotriene‐C 4 synthase activity from rat brain
Author(s) -
TSUCHIDA Shigeki,
IZUMI Takashi,
SHIMIZU Takao,
ISHIKAWA Takashi,
HATAYAMA Ichiro,
SATOH Kimihiko,
SATO Kiyomi
Publication year - 1987
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1987.tb13681.x
Subject(s) - chromatofocusing , leukotriene c4 , isoelectric point , glutathione , molecular mass , gel electrophoresis , microbiology and biotechnology , chemistry , biochemistry , affinity chromatography , isoelectric focusing , isozyme , glutathione s transferase , polyacrylamide gel electrophoresis , enzyme , atp synthase , leukotriene , biology , immunology , asthma
A new acidic form of glutathione S ‐transferase (GST, pI 6.2) was purified from rat brain by S ‐hexylglutathione affinity chromatography followed by chromatofocusing. This form occupied 20–25% of the total activity bound to the affinity column. It had a molecular mass (subunit 26 kDa) similar to that of a major GST form of rat testis (M T or 6‐6) on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. However, it differed from the M T in isoelectric point, activity towards 1,2‐dichloro‐4‐nitrobenzene and immunological properties. On two‐dimensional gel electrophoresis the brain form gave a spot which was identical in molecular mass, isoelectric point and immunological properties to a less acidic one (Yn 1 ) of two spots (Yn 1 and Yn 2 ) of the testis GST‐M T . Therefore, the brain acidic form is a homodimer, and named GST‐Yn 1 Yn 1 . The activity was inhibited by sulfasalazine, an inhibitor of leukotriene‐C 4 synthase. This form (GST‐Yn 1 Yn 1 ) showed the highest leukotriene‐C 4 synthase activity, 496 nmol/mg protein in 5 min, among nine cytosolic GST isoenzymes from the rat. The K m values for leukotriene A 4 and glutathione were 26 μM and 3.5 mM respectively. A major GST form of rat brain, occupying about 40% of the total activity, was identical with GST‐P (7–7) purified from rat liver bearing preneoplastic hyperplastic nodules and localized at astroglias. GST‐P also showed the significant leukotriene‐C 4 synthase activity, 67.2 nmol/mg protein in 5 min, but the K m for leukotriene A 4 was 100 μM, fourfold higher than that of GST‐Yn 1 Yn 1 . These results suggest that mainly GST‐Yn 1 Yn 1 may be involved in leukotriene‐C 4 synthesis in rat brain.