Purification and catalytic properties of glutathione transferase from the hepatopancreas of crayfish macrobrachium vollenhovenii (herklots).
Glutathione transferase from the hepatopancreas of fresh water crayfish Macrobrachium vollenhovenii was purified to apparent homogeneity by ion-exchange chromatography on DEAE-cellulose and by gel filtration on Sephadex G-100. The enzyme appeared to be a homodimer with molecular weight (Mr) of 46.0 +/- 1.4 kDa and a subunit Mr of 24.1 +/- 0.35 kDa. Chromatofocusing of the apparently pure enzyme revealed microheterogeneity and resolved it into two isozymic peaks, which were eluted at pH 8.36 and 8.22 respectively. Inhibition studies showed that the I50 value for cibacron blue, S-hexylglutathione, hematin, and N-ethylmaleimide (NEM) were 0.01 microM, 340 microM, 5 microM and 33 mM respectively. Out of the several substrates tested, only 1-chloro-2,4-dinitrobenzene (CDNB) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole could be conjugated with glutathione. Chemical modification studies with DTNB revealed that two sulphydryl groups per dimer were essential to the activity of the enzymes. On the basis of structural and catalytic characteristics, M. vollenhovenii GST seems close, tentatively, to the omega and zeta classes of GST. Initial-velocity studies of the enzyme are consistent with a steady-state random kinetic mechanism. Denaturation and renaturation studies with guanidine HCl (Gdn-HCl) revealed that though low Gdn-HCl concentrations (less than 0.5 M) denatured the enzyme, the enzyme was able to renature completely (100%). At higher concentration of the denaturant (0.5-4 M), refolding studies indicated that complete renaturation was not achieved. The extent of renaturation was however a function of protein concentration. Our results are consistent with a three-state unfolding process.
Adewale
Isaac Olusanjo
IO
Department of Biochemistry, Obafemi Awolowo University, Ile-Ife, Nigeria. olusanjo2002@yahoo.co.uk
Afolayan
Adeyinka
A
D000818:Animals; D003400:Astacoidea; D002374:Catalase / Q000378:metabolism; D002384:Catalysis; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005979:Glutathione Peroxidase / Q000378:metabolism; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D043143:Hepatopancreas / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D011489:Protein Denaturation; D013379:Substrate Specificity
D002374:Catalase; D005979:Glutathione Peroxidase; D005982:Glutathione Transferase