Turnover of murine beta-glucuronidase. Comparison among liver, kidney, and spleen and between lysosomes and microsomes

Smith, Kathy Beckingham; Ganschow, Roger E.

doi:10.1016/s0021-9258(17)30391-5

Cited by 34 publications

(1 citation statement)

References 32 publications

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“…Although the egasyn-13-glucuronidase complex segregated with lumenal secretory proteins in these in vitro studies, a fascinating in vivo difference is that while secretory proteins are rapidly released from the liver, the microsomal glucuronidase complex is stabile within the lumen of the endoplasmic reticulum with a half-life of several days (43). One possible explanation of these findings is that secretory proteins and the 13-glucuronidase complex are in separate vesicles and that the microsomal vesicle containing the [5-glucuronidase complex is unusual in that it is not on the secretory pathway.…”

Section: Discussionmentioning

confidence: 99%

Lumenal location of the microsomal beta-glucuronidase-egasyn complex.

Brown¹,

Novak²,

Takeuchi³

et al. 1987

The Journal of Cell Biology

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Abstract. Mouse liver l~-glucuronidase is stabilized within microsomal vesicles by complexation with the accessory protein egasyn. The location of the 13-glucuronidase-egasyn complex and free egasyn within microsomal vesicles was investigated. Surprisingly, it was found that neither the complex nor free egasyn are intrinsic membrane components. Rather, both are either free within the vesicle lumen or only weakly bound to the inside of the vesicle membrane. This conclusion was derived from release studies using low concentrations of Triton X-100 or controlled sonication. Both the intact complex and free egasyn were released in parallel with lumenal proteins, not with intrinsic membrane components. Also, 13-glucuronidase was protected from digestion by proteinase K by the membrane of microsomal vesicles. The hydrophilic nature of both the complex and free egasyn was confirmed by phase separation experiments with the detergent Triton X-114. Egasyn is one of an unusual group of esterases that, despite being located within the lumen or only weakly bound to the lumenal surface of the endoplasmic reticulum, do not enter the secretory pathway. ~-Glucuronidase is an unusual acid hydrolase in that large amounts (20-40%) of stable enzyme exist in the microsomal comparmaent of liver in addition to the usual lysosomal location. A wide variety of biochemical and genetic information indicates that the same polypeptide is found at both subcellular sites and that complex formation with the accessory protein, egasyn, is required for stabilization of ~glucuronidase in microsomes (reviewed in 24). The system thus serves as a model for the specific subceUular localization of enzymes. Mutant mice that lack egasyn have no stable microsomal I~-glucuronidase (14). More recent studies have shown that egasyn is associated exclusively with the precursor form of 13-glucuronidase in microsomes (9) and that egasyn is identical with mouse esterase-22 (26,27).It has been postulated that egasyn stabilizes the l~-glucuronidase complex in microsomes by serving as an integral membrane anchor peptide (24). Others (12) have suggested that microsomal I~-glucuronidase serves in a membrane structural role. In this paper we present evidence that the microsomal 13-glucuronidase-egasyn complex, rather than being an integral membrane component, surprisingly is either free within the lumen of microsomal vesicles or very weakly attached to the inside of these vesicles. These results suggest, in turn, that the binding protein, egasyn, stabilizes 13-glucuronidase within the microsomal lumen. Materials and Methods AnimalsC57BL/6J, Mus musculus molossinus and Mus spretus mice were raised in the animal facilities of Roswell Park Memorial Institute. Mice 2-4 mo old and of both sexes were used. Unless otherwise indicated, C57BL/6J mice were used. Radiolabeling ExperimentsRadiolabeling of membrane and secretory components of microsomes was done according to the method of Kreibich and Sabatini (20). Membrane phospholipids were labeled by injecting mice intraperitoneal...

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Section: Discussionmentioning

confidence: 99%

Lumenal location of the microsomal beta-glucuronidase-egasyn complex.

Brown¹,

Novak²,

Takeuchi³

et al. 1987

The Journal of Cell Biology

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Turnover and Degradation of Mitochondria and Their Proteins

Grisolı́a

Knecht

Hernández-Yago

et al. 1980

Ciba Foundation Symposium 75 ‐ Protein Degradation in Health and Disease

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Quantitative importance of biliary excretion to the turnover of hepatic lysosomal enzymes

et al. 1995

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The turnover rate of an individual protein is a function of the rates of synthesis and loss of that protein. For most intracellular proteins, loss occurs through digestion by lysosomal or cytosolic proteases. Although a significant proportion of hepatic lysosomal enzymes is released from the hepatocyte by excretion into bile, the contribution of biliary excretion to the turnover of hepatic lysosomal enzymes has never been measured. Thus, we used in vivo pulse-labeling to determine the half-lives of two hepatic hydrolases, beta-galactosidase (beta-gal) and beta-glucuronidase (beta-glu). Each enzyme was purified by immunoisolation from hepatic lysosomes that were isolated at various times after injection of rats with 3H-labeled leucine. The decay curves for the specific radioactivities of beta-gal and beta-glu were used to calculate the half-lives of the proteins, which were 3.8 and 5.1 days, respectively. To determine the percent of total hepatic contents of each enzyme that was lost per day by biliary excretion, we collected bile from bile fistula rats for 24 hours and then used radioimmunoassays to quantitate the amounts of beta-gal and beta-glu in bile and liver samples of the same rats. We found that approximately 4% of the total hepatic contents of both beta-gal and beta-glu was excreted into bile per day. Finally, we used these data to calculate that 31% and 41% of hepatic losses of beta-gal and beta-glu, respectively, were due to biliary excretion. These results suggest that extracellular release through biliary excretion is a major mechanism contributing to the turnover of lysosomal hydrolases.

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Turnover of murine beta-glucuronidase. Comparison among liver, kidney, and spleen and between lysosomes and microsomes

Cited by 34 publications

References 32 publications

Lumenal location of the microsomal beta-glucuronidase-egasyn complex.

Lumenal location of the microsomal beta-glucuronidase-egasyn complex.

Turnover and Degradation of Mitochondria and Their Proteins

Quantitative importance of biliary excretion to the turnover of hepatic lysosomal enzymes

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