Purification and characterisation of glutathione transferase from the giant African snail, Archachatina marginata.
1. Glutathione-S-transferase has been purified from the hepatopancreas of Archachatina marginata to homogeneity. 2. The enzyme was found to be a dimer with a molecular weight of 44,000. The subunits sizes were 22,500 and 23,500 respectively. The isoelectric points of the enzyme were 8.35, 7.95 and 4. The enzyme was most stable at temperature below 40 degrees C. Upon denaturation by 4 M urea, only 56% of the activity could be recovered. 3. The Kms for glutathione and 1-chloro-2,4-dinitrobenze (CDNB) were 0.23 mM and 0.4 mM respectively. The specific activity of the enzyme with CDNB and p-nitrophylacetate as substrates were 47 mumol/mg and 38 mumol/mg respectively. 4. Inhibition studies showed that S-hexylglutathione, Rose Bengal, iodoacetamide, sodium azide and Procion Blue H-B were good inhibitors with I50 values ranging from 18.5 microM to 299 mM. 5. The amino acid composition showed that the enzyme had a relatively high content of hydrophobic and acidic amino acid residues. The peptide maps of the tryptic digests of the native and performic acid-oxidised enzyme indicated that there might be about two disulphide bridges per molecule of the enzyme.
Ajele
J O
JO
Department of Biochemistry, Federal University of Technology, Akure, Nigeria.
Afolayan
A
A
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D004137:Dinitrochlorobenzene / Q000378:metabolism; D004795:Enzyme Stability; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D007526:Isoelectric Point; D046911:Macromolecular Substances; D008970:Molecular Weight; D010449:Peptide Mapping; D011489:Protein Denaturation; D012908:Snails / Q000201:enzymology*; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism
D000596:Amino Acids; D004137:Dinitrochlorobenzene; D046911:Macromolecular Substances; D005982:Glutathione Transferase; D014357:Trypsin; D005978:Glutathione