Topology and regulation of bilirubin UDP-glucuronyltransferase in sealed native microsomes from rat liver.
Bilirubin UDP-glucuronyltransferase displays marked latency in native microsomes. To examine whether this latency correlates with structural integrity of the microsomal vesicles and reflects lumenal orientation of the enzyme's catalytic center, we analyzed the relationship between transferase activity and the degree of expression of mannose (Man)-6-phosphatase, which is a marker enzyme of the cisternal face of the ER membrane. Using detergent, sonication, or the pore-forming Staphylococcus aureus alpha-toxin to breach the microsomal membrane permeability barrier, we found that after each of these pretreatments a remarkably close direct relationship existed between latency changes for bilirubin UDP-glucuronyltransferase and Man-6-phosphatase. This finding suggested that the transferase may have the same transverse topology as the phosphohydrolase. We also compared the effects of membrane-impermeant proteinases on bilirubin UDP-glucuronyltransferase activity in native and disrupted microsomes. Whereas the unspecific proteinase nagarse markedly inactivated (to less than 30% of activities in controls) the transferase in disrupted microsomes, treatment with the proteinase had little effect on transferase activity in sealed microsomal vesicles. The results suggest that the active site of bilirubin UDP-glucuronyltransferase is on the lumenal face of the endoplasmic reticulum membrane. It was also found that activation of transferase activity by UDP N-acetylglucosamine, which is the presumed allosteric effector of UDP-glucuronyltransferase, was markedly altered by relatively small changes in structural integrity of the microsomes and totally abolished when latency of Man-6-P hydrolysis fell below approximately 80%. Collectively, these findings demonstrate that the microsomal membrane permeability barrier is a major determinant of expression of microsomal UDP-glucuronyltransferase activity and that quantitative assessment of integrity of the microsomes is essential for studying kinetic properties and regulation of microsomal UDP-glucuronyltransferase.
D000818:Animals; D001663:Bilirubin / Q000378:metabolism*; D002463:Cell Membrane Permeability; D014453:Glucuronosyltransferase / Q000378:metabolism*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D010447:Peptide Hydrolases / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013010:Sonication; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism
D014537:Uridine Diphosphate N-Acetylglucosamine; D014453:Glucuronosyltransferase; D010447:Peptide Hydrolases; D001663:Bilirubin