Functional and structural membrane topology of rat liver microsomal glutathione transferase

摘要: Abstract The membrane topology of rat liver microsomal glutathione transferase was investigated by comparing the tryptic cleavage products from intact and permeabilized microsomes. It shown that lysine-4 is accessible at luminal surface endoplasmic reticulum, whereas lysine-41 faces cytosol. These positions are separated a hydrophobic stretch 25 amino acids (positions 11–35) which comprises likely membrane-spanning region. Reaction cysteine-49 with charged sulfhydryl reagent DTNB (2,2′-dithiobis(5-nitrobenzoic acid))) in microsomes further supports cytosolic localization this portion polypeptide chain. role two other potential membrane-spanning/associated segments C-terminal half chain examined investigating association protein to after trypsin lysine-41. Activity measurements Western blot analysis washing high concentrations salt, as well phase separation Triton X-114, indicate also binds membrane. purified Lys-41 subsequent fragments obtained yields functional expected length for product encompassing 42–154. location active site using radiolabelled together second substrate. Since isolated do not take up or release conjugate into lumen, it can be concluded side reticulum.