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Beta-glucuronidase latency in isolated murine hepatocytes
1Department of Medical Chemistry, Semmelweis University of Medicine, Budapest, Hungary.
Abstract:
The physiological function of microsomal beta-glucuronidase is unclear. Substrates may be either glucuronides produced in the lumen of endoplasmic reticulum (ER) or those taken up by hepatocytes. In the latter case, efficient inward transport of glucuronides at the plasma membrane and the ER membrane would be required. Therefore, the potential role of beta-glucuronidase in ER was investigated. Isolated mouse hepatocytes and mouse and rat liver microsomal vesicles were used in the experiments. Selective permeabilization of the plasma membrane of isolated hepatocytes with saponin or digitonin resulted in an almost 4-fold elevation in the rate of beta-nitrophenol glucuronide hydrolysis, while the permeabilization of plasma membrane plus ER membrane by Triton X-100 caused a further 2-fold elevation. In microsomal vesicles, the p-nitrophenol glucuronide or phenolphthalein glucuronide beta-glucuronidase activity showed about 50% latency as revealed by alamethicin or Triton X-100 treatment. A light-scattering study indicated that the microsomes are relatively impermeable to both glucuronides and to glucuronate. On the basis of our results, the role of liver microsomal beta-glucuronidase in the deconjugation of glucuronides taken up by the liver seems unlikely. Hydrolysis of the glucuronides produced in the ER lumen may play a role in substrate supply for ascorbate synthesis or in "proofreading" of glucuronidation.
Insights
Microsomal beta-glucuronidase likely does not process external glucuronides. Its role may involve internal endoplasmic reticulum (ER) substrate hydrolysis for ascorbate synthesis or glucuronidation proofreading.
Area of Science:
- Biochemistry
- Cell Biology
- Enzymology
Background:
- The physiological role of microsomal beta-glucuronidase remains largely unknown.
- Potential substrates include lumenal endoplasmic reticulum (ER) glucuronides or hepatocyte-taken-up glucuronides.
- Efficient transport across cellular membranes is necessary for external substrate access.
Purpose of the Study:
- To investigate the potential role of beta-glucuronidase within the ER.
- To determine if liver microsomal beta-glucuronidase processes external glucuronides.
Main Methods:
- Experiments utilized isolated mouse hepatocytes and liver microsomal vesicles from mice and rats.
- Selective permeabilization of hepatocyte plasma and ER membranes was performed using saponin, digitonin, and Triton X-100.
- Enzyme activity and substrate permeability were assessed using p-nitrophenol glucuronide, phenolphthalein glucuronide, and light scattering.
Main Results:
- Permeabilization of hepatocyte membranes significantly increased glucuronide hydrolysis rates.
- Microsomal vesicles exhibited approximately 50% latency in beta-glucuronidase activity.
- Microsomes demonstrated low permeability to both glucuronides and glucuronate.
Conclusions:
- Liver microsomal beta-glucuronidase is unlikely to be involved in deconjugating glucuronides taken up by hepatocytes.
- The enzyme may hydrolyze glucuronides synthesized within the ER lumen.
- This internal hydrolysis could supply substrates for ascorbate synthesis or function in glucuronidation proofreading.