Isolation of a mouse theta glutathione S-transferase active with methylene chloride

Abstract

A glutathione S-transferase metabolizing methylene chloride has been isolated from mouse liver using a variety of chromatographic methods. N-terminal and internal amino acid sequences show that the enzyme, designated GST T1-1*, is closely related to the rat Theta-class GST 5-5. The mouse enzyme, molecular mass 25000 Da, has been isolated to homogeneity in active form with an approximate yield of 2% of the cytosolic activity towards methylene chloride. GST T1-1* has a specific activity of about 5.5 micromol/min per mg of protein whereas the rat GST 5-5 is reported to have a specific activity of about 11 micromol/min per mg of protein [Meyer, Coles, Pemble, Gilmore, Fraser and Ketterer (1991) Biochem. J. 274, 409-414], demonstrating that both the rat and mouse enzymes have similar activity with this substrate. Limited evidence was obtained for a second enzyme, with a similar molecular mass (25400 Da), which had an N-terminal sequence identical to that of rat GST 12-12. This protein, which was sequenced from a band on a gel, was extremely labile and could not be isolated to homogeneity. The partially purified enzyme was not active with methylene chloride.

Evidence that human class Theta glutathione S-transferase T1-1 can catalyse the activation of dichloromethane, a liver and lung carcinogen in the mouse: Comparison of the tissue distribution of GST T1-1 with that of classes Alpha, Mu and Pi GST in human

“…As the formation of the GSH conjugate of DCM is catalysed by class Theta GST, these enzymes are thought to have a critical role in the carcinogenic mechanism [14]. Purification of GST isoenzymes from mouse liver has demonstrated that the murine GST T1-1 enzyme has high conjugating activity towards DCM [23]. The risk posed to human health by DCM is uncertain.…”

“…The activation of DCM is catalysed by a unique member of the class Theta transferases, namely GST T1-1 [14,23]. Unlike the class Alpha, Mu and Pi GSTs, the class Theta enzymes are not abundant, stable or readily purified by affinity chromatography [2,[24][25][26][27].…”

Glutationa e enzimas relacionadas: papel biológico e importância em processos patológicos

“…74,75 A atividade genotóxica do diclorometano está relacionada à bioativação deste por glutationa transferases citossólicas hGSTT1-1 e rGSTT5-5 (Figura 12). 70,72,76 A conjugação do diclorometano (CH 2 Cl 2 ) ou de outros dialoalcanos se dá, inicialmente, pela substituição nucleofílica de um dos áto-mos de cloro por glutationa (GSH), fornecendo o aduto Sclorometilglutationa (52). 77 O aduto S-clorometilglutationa 52 é instável e reativo, 78,79 sendo que evidências diretas de sua formação só foram possíveis utilizando-se ressonância magnética nuclear de 19 F e clorofluorometano (CH 2 ClF) como substrato para as glutationa transferases (GSTs).…”

Preliminary X-ray crystallographic studies of a newly defined human theta-class glutathione transferase

“…In some cases the resultant metabolite may become mutagenic and carcinogenic. For example, methylene chloride causes lung and liver cancer in mice via a glutathione metabolite (Mainwaring et al, 1996). Because of their inability to bind to glutathione affinity matrices and the difficulties in their purification, the theta-class enzymes are the least studied of the GST enzyme superfamily.…”

“…i! ;; 1998 International Union of Crystallography Printed in Great Britain -all rights reserved Meyer et al, 1991;Hussey & Hayes, 1992;Mainwaring et al, 1996;Whittington et al, 1996). Theta-class GSTIs can utilize sullhte esters and dichloromethane as substrates and are suggested to be important in the prevention of hepatocarcinogenesis (Hiratsuka et al, 1994).…”