TY - JOUR
T1 - Membrane topology of recombinant rat liver microsomal glutathione transferase expressed in E. coli
AU - Raza, Haider
AU - Weinander, Rolf
AU - Ekström, Lena
AU - Morgenstern, Ralf
N1 - Funding Information:
This work was supported by the Swedish Cancer Society, the Swedish National Board of Laboratory Animals, and funds from the Karolinska Institute. Support from The Wennergren Foundation (to H.R.) is gratefully acknowledged.
PY - 1996/11/1
Y1 - 1996/11/1
N2 - Rat liver microsomal glutathione transferase is a mammalian membrane protein that can be successfully expressed in Escherichia coli in an enzymatically active form. The protein does not form inclusion bodies and is recovered in the membrane fraction. The membrane topology of recombinant rat liver microsomal glutathione transferase expressed in E. coli was investigated by comparing the proteolytic cleavage products from intact and permeabilized spheroplasts. It was shown that lysine-4 of microsomal glutathione transferase is directed towards the outside, whereas lysine-41 faces the inside of the E. coli inner membrane. This shows that microsomal glutathione transferase has an inside-out orientation in E, coli spheroplasts as compared to liver microsomes. This fact enables us to make topology experiments that were previously not possible. Intact spheroplasts treated with pronase yielded a cleavage pattern consistent with two additional exposed segments closer to the C-terminus. Thus a polytopic model is suggested for the membrane association of microsomal glutathione transferase.
AB - Rat liver microsomal glutathione transferase is a mammalian membrane protein that can be successfully expressed in Escherichia coli in an enzymatically active form. The protein does not form inclusion bodies and is recovered in the membrane fraction. The membrane topology of recombinant rat liver microsomal glutathione transferase expressed in E. coli was investigated by comparing the proteolytic cleavage products from intact and permeabilized spheroplasts. It was shown that lysine-4 of microsomal glutathione transferase is directed towards the outside, whereas lysine-41 faces the inside of the E. coli inner membrane. This shows that microsomal glutathione transferase has an inside-out orientation in E, coli spheroplasts as compared to liver microsomes. This fact enables us to make topology experiments that were previously not possible. Intact spheroplasts treated with pronase yielded a cleavage pattern consistent with two additional exposed segments closer to the C-terminus. Thus a polytopic model is suggested for the membrane association of microsomal glutathione transferase.
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U2 - 10.1006/bbrc.1996.1633
DO - 10.1006/bbrc.1996.1633
M3 - Article
C2 - 8912653
AN - SCOPUS:0030296348
SN - 0006-291X
VL - 228
SP - 165
EP - 170
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 1
ER -