Protein-protein interactions between UDP-glucuronosyltransferase isozymes in rat hepatic microsomes.
The interactions between UDP-glucuronosyltransferase (UGT) isozymes, UGT1s and UGT2B1, in rat hepatic microsomes were investigated using an immunopurification technique with anti-peptide antibodies and a chemical cross-linking strategy. A 50 kDa protein coimmunopurified with UGT1s was identified as UGT2B1 by amino-terminal sequencing and immunodetection with anti-peptide antibody against UGT2B1. Evidence for direct interaction of UGT2B1 with UGT1s was obtained by the loss of UGT2B1 adsorption to immunoaffinity column in Gunn rat hepatic microsomes, which lack all UGT1 isozymes. When the microsomes were treated with the chemical cross-linking reagent 1,6-bis(maleimido)-hexane, a cross-linked product with an apparent molecular mass of 120-130 kDa was obtained that immunostained with antibodies against UGT1s and UGT2B1, indicating the formation of a heterodimer containing one of the UGT1 isozymes and UGT2B1. The effects of UGT complex formation on the stimulation of glucuronidation of testosterone and uptake of UDP-glucuronic acid (UDP-GlcUA) by UDP-N-acetylglucosamine (UDP-GlcNAc) were examined. Alkaline pH-induced dissociation of the complexes was associated with the loss of UDP-GlcNAc-dependent stimulation of glucuronidation, suggesting that two functional states of UGTs with different kinetic parameters correspond to the monomer and oligomer form of UGTs in the membranes. The UDP-GlcNAc-dependent stimulation of UDP-GlcUA uptake into the microsomal vesicles also was affected by the extent of complex formation. These results suggest that complex formation of the UGT isozymes affects the UDP-GlcNAc-dependent stimulation of glucuronidation via stimulation of UDP-GlcUA uptake.
Ikushiro
S
S
Department of Life Science, Faculty of Science, Himeji Institute of Technology, Harima Science Park City, Hyogo, Japan.
Emi
Y
Y
Iyanagi
T
T
D000818:Animals; D000906:Antibodies / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D014453:Glucuronosyltransferase / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000378:metabolism*; D008297:Male; D008301:Maleimides / Q000378:metabolism; D008862:Microsomes, Liver / Q000201:enzymology*; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D017208:Rats, Wistar; D013739:Testosterone / Q000031:analogs & derivatives / Q000378:metabolism; D000099237:UDP-Galactose Translocators; D000099148:UGT1A1 Enzyme
D000906:Antibodies; D003432:Cross-Linking Reagents; D014453:Glucuronosyltransferase; D007527:Isoenzymes; D008301:Maleimides; D009004:Monosaccharide Transport Proteins; D013739:Testosterone; D000099237:UDP-Galactose Translocators; C499946:Ugt1a1 protein, rat; D000099148:UGT1A1 Enzyme; C015529:testosterone glucuronate; C075594:1,6-bismaleimidohexane