alamethicin and 4-nitrophenol

Addition of p-nitrophenol and UDP-glucuronic acid to rat hepatic microsomes enhanced the MgATP-stimulated Ca2+ sequestration. This stimulatory effect was more explicit in the presence of the activator of glucuronidation, UDP-N-acetylglucosamine. The stimulation of Ca2+ uptake was dependent on the p-nitrophenol concentration and showed a good correlation 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 extravesicular osmolarity due to the addition of UDP-glucuronic acid to microsomes resuspended in an hyposmotic medium caused a rapid and prolonged shrinking as revealed by light-scattering measurements. This indicates a poor permeability of microsomal membrane to UDP-glucuronic acid. The subsequent addition of the pore-forming compound alamethicin resulted in an immediate swelling of vesicles indicating a rapid entry of UDP-glucuronic acid. Alamethicin also caused an about 15-fold increase in p-nitrophenol UDP-glucuronosyltransferase activity. These results support the hypothesis of the intravesicular compartmentation of the microsomal UDP-glucuronosyltransferase catalytic site.

Topics: Adenosine Triphosphate; Alamethicin; Animals; Binding Sites; Calcium; Enzyme Activation; Glucuronosyltransferase; Male; Microsomes, Liver; Nitrophenols; Rats; Rats, Sprague-Dawley; Uridine Diphosphate Glucuronic Acid

The glucuronidation of p-nitrophenol was measured in intact, saponin- and alamethicin-treated isolated mouse hepatocytes. In saponin-permeabilized cells the elevation of extrareticular UDP-glucuronic acid concentration enhanced the rate of glucuronidation threefold. When intracellular membranes were also permeabilized by alamethicin, a further tenfold increase in the glucuronidation of p-nitrophenol was present. Parallel measurements of the ER mannose 6-phosphatase activity revealed that saponin selectively permeabilized the plasma membrane, whereas alamethicin permeabilized both plasma membrane and ER membranes. The inhibition of p-nitrophenol glucuronidation by dbcAMP in intact hepatocytes was still present in saponin-treated cells and disappeared in alamethicin-permeabilized hepatocytes. It is suggested that the permeability of the endoplasmic reticulum membrane is a major determinant of glucuronidation not only in microsomes but in isolated hepatocytes as well.

Topics: Alamethicin; Animals; Cell Membrane Permeability; Endoplasmic Reticulum; Glucuronosyltransferase; Liver; Male; Mice; Nitrophenols; Phosphoric Monoester Hydrolases; Saponins; Uridine Diphosphate Glucuronic Acid