Cross-linking of human placenta pi class glutathione S-transferase dimer by chlorambucil.
Interaction of chlorambucil and the glutathione-depleted human placenta pi class glutathione S-transferase (pi GST) results in the formation of a complex between the drug and the protein at physiological pH. This complex is not formed in the presence of glutathione or S-hexylglutathione. Molecular mass measurement of the reaction product using matrix-assisted laser desorption mass spectrometry indicates that one molecule of chlorambucil cross-links two subunits of the homodimeric protein. A combination of enzymic proteolysis, high performance liquid chromatography, and mass spectrometry reveals that chlorambucil alkylation occurs at cysteine 47 of one subunit and cysteine 101 of the second subunit. This result supports the idea that conformational changes occur in glutathione-depleted pi GST, which allow the bifunctional tether of chlorambucil to cross-link the two subunits of the protein.
Hathout
Y
Y
Department of Chemistry and Biochemistry, University of Maryland Baltimore Country 21228, USA.
Ellis
T
T
Fabris
D
D
Fenselau
C
C
D000345:Affinity Labels; D018906:Antineoplastic Agents, Alkylating / Q000737:chemistry; D001665:Binding Sites; D002699:Chlorambucil / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000737:chemistry*; D005978:Glutathione / Q000031:analogs & derivatives / Q000737:chemistry; D005982:Glutathione Transferase / Q000737:chemistry*; D006801:Humans; D008970:Molecular Weight; D010920:Placenta / Q000201:enzymology*; D011487:Protein Conformation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000345:Affinity Labels; D018906:Antineoplastic Agents, Alkylating; D003432:Cross-Linking Reagents; D002699:Chlorambucil; D005982:Glutathione Transferase; D005978:Glutathione; C033550:hexylglutathione