Purification and kinetic mechanism of the glutathione S-transferase from C6/36, an Aedes albopictus cell line.
Glutathione S-transferase from an Aedes albopictus cell line, C6/36, was purified to apparent homogeneity by a single glutathione-Sepharose 4B affinity chromatography, with an overall yield of 66% and 226-fold purification. The enzyme is presumably a homodimer with subunit M(r) 23,000, which is similar to the enzyme isolated from other sources. Under native conditions, the enzyme exhibited multiple forms upon isoelectric focusing or polyacrylamide gel electrophoresis, and all these forms retained enzymatic activity. The pI value of the purified enzyme was distributed between approximately 4.7 and 5.4 with major form at 4.9. The purified enzyme lost 63% activity at -20 degrees C within 1 week. Stability of the purified enzyme was greatly improved by the addition 10 mg/ml of bovine serum albumin, under which only 7% activity was lost after 1 week at -20 degrees C. Activation energy for the enzyme-catalyzed conjugation of glutathione with 1-chloro-2,4-dinitrobenzene was found to be 49.1 kJ/mol. The enzyme could utilize 1-chloro-2,4-dinitrobenzene and ethacrynic acid as substrate, but not bromosulfophthalein, cumene hydroperoxide, or trans-4-phenyl-3-buten-2-one. The initial-velocity and product-inhibition studies indicated that the enzyme reaction conformed to a steady-state random Bi-Bi kinetic mechanism, which was similar to the glutathione S-transferase from other sources. The kinetic data also indicated a synergistic effect between the binding of glutathione G-site and that of organic electrophilic substrate in the H-site of the enzyme active center. The biological significance of the conjugation reaction is discussed.
Chang
G G
GG
Institute of Zoology, Academia Sinica, Taipei, Taiwan, Republic of China.
Tsai
L N
LN
Tang
S S
SS
Wang
T C
TC
D000330:Aedes / Q000166:cytology / Q000201:enzymology*; D000818:Animals; D002460:Cell Line; D004137:Dinitrochlorobenzene / Q000378:metabolism; D004795:Enzyme Stability; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008954:Models, Biological; D008956:Models, Chemical; D008970:Molecular Weight; D013379:Substrate Specificity
D004137:Dinitrochlorobenzene; D005982:Glutathione Transferase; D005978:Glutathione