Functional and structural membrane topology of rat liver microsomal glutathione transferase.
The membrane topology of rat liver microsomal glutathione transferase was investigated by comparing the tryptic cleavage products from intact and permeabilized microsomes. It was shown that lysine-4 of microsomal glutathione transferase is accessible at the luminal surface of the endoplasmic reticulum, whereas lysine-41 faces the cytosol. These positions are separated by a hydrophobic stretch of 25 amino acids (positions 11-35) which comprises the likely membrane-spanning region. Reaction of cysteine-49 of the microsomal glutathione transferase with the charged sulfhydryl reagent DTNB (2,2'-dithiobis(5-nitrobenzoic acid)) in intact microsomes further supports the cytosolic localization of this portion of the polypeptide chain. The role of two other potential membrane-spanning/associated segments in the C-terminal half of the polypeptide chain was examined by investigating the association of the protein to the membrane after trypsin cleavage at lysine-41. Activity measurements and Western blot analysis after washing with high concentrations of salt, as well as after phase separation in Triton X-114, indicate that this portion of the protein also binds to the membrane. It is also shown that cleavage of the purified protein at Lys-41 and subsequent separation of the fragments obtained yields a functional C-terminal polypeptide with the expected length for the product encompassing positions 42-154. The location of the active site of microsomal glutathione transferase was investigated using radiolabelled glutathione together with a second substrate. Since isolated rat liver microsomes do not take up glutathione or release the glutathione conjugate into the lumen, it can be concluded that the active site of rat liver microsomal glutathione transferase faces the cytosolic side of the endoplasmic reticulum.
Andersson
C
C
Division of Toxicology, Karolinska Institutet, Stockholm, Sweden.
Weinander
R
R
Lundqvist
G
G
DePierre
J W
JW
Morgenstern
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003600:Cytosol / Q000201:enzymology; D004721:Endoplasmic Reticulum / Q000201:enzymology; D005982:Glutathione Transferase / Q000737:chemistry / Q000302:isolation & purification*; D007425:Intracellular Membranes / Q000201:enzymology; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D014357:Trypsin
D010446:Peptide Fragments; D005982:Glutathione Transferase; D014357:Trypsin