EVIDENCE FOR THE INTRALUMINAL POSITIONING OF P-NITROPHENOL UDP-GLUCURONOSYLTRANSFERASE ACTIVITY IN RAT-LIVER MICROSOMAL VESICLES

Addition of p-nitrophenol and UDP-glucuronic acid to rat hepatic micro somes enhanced the MgATP-stimulated Ca2+ sequestration. This stimulato ry effect was more explicit in the presence of the activator of glucur onidation, UDP-N-acetylglucosamine. The stimulation of Ca2+ uptake was dependent on the p-nitrophenol concentration and showed a good correl ation with the rate of p-nitrophenol glucuronidation. The stimulation of Ca2+ sequestration was probably due to its coaccumulation with the intraluminar Pi originated during glucuronidation. The increase in ext ravesicular osmolarity due to the addition of UDP-glucuronic acid to m icrosomes resuspended in an hyposmotic medium caused a rapid and prolo nged shrinking as revealed by light-scattering measurements. This indi cates a poor permeability of microsomal membrane to UDP-glucuronic aci d. The subsequent addition of the pore-forming compound alamethicin re sulted in an immediate swelling of vesicles indicating a rapid entry o f UDP-glucuronic acid. Alamethicin also caused an about 15-fold increa se in p-nitrophenol UDP-glucuronosyltransferase activity. These result s support the hypothesis of the intravesicular compartmentation of the microsomal UDP-glucuronosyltransferase catalytic site. (C) 1994 Acade mic Press, Inc.