Observation of an intact noncovalent homotrimer of detergent-solubilized rat microsomal glutathione transferase-1 by electrospray mass spectrometry.
Microsomal glutathione transferase-1 (MGST1) is a membrane-bound enzyme involved in the detoxification of xenobiotics and the protection of cells against oxidative stress. The proposed active form of the enzyme is a noncovalently associated homotrimer that binds one substrate glutathione molecule/trimer. In this study, this complex has been directly observed by electrospray mass spectrometry analysis of active rat liver MGST1 reconstituted in a minimum amount of detergent. The measured mass of the homotrimer is 53 kDa, allowing for the mass of three MGST molecules in complex with one glutathione molecule. Collision-induced dissociation of the trimer complex resulted in the formation of monomer and homodimer ion species. Two distinct species of homodimer were observed, one unliganded and one identified as a homodimer.glutathione complex. Activation of the enzyme by N-ethylmaleimide through modification of Cys(49) (Svensson, R., Rinaldi, R., Swedmark, S., and Morgenstern, R. (2000) Biochemistry 39, 15144-15149) was monitored by the observation of an appropriate increase in mass in both the denatured monomeric and native trimeric forms of MGST1. Together, the data correspond well with the proposed functional organization of MGST1. These results also represent the first example of direct electrospray mass spectrometry analysis of a detergent-solubilized multimeric membrane protein complex in its native state.
Lengqvist
Johan
J
Department of Medical Biochemistry and Biophysics, Lüdwig Institute for Cancer Research, Stockholm, Sweden.
Svensson
Richard
R
Evergren
Emma
E
Morgenstern
Ralf
R
Griffiths
William J
WJ
D000818:Animals; D003902:Detergents; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008861:Microsomes / Q000201:enzymology*; D017830:Octoxynol; D051381:Rats; D012995:Solubility; D021241:Spectrometry, Mass, Electrospray Ionization
D003902:Detergents; D008565:Membrane Proteins; D017830:Octoxynol; C122107:microsomal glutathione S-transferase-I; D005982:Glutathione Transferase; D005978:Glutathione