Purification and characterization of lysosomal β-glucuronidase from human placenta

Abstract

1. Subcellular fractions of human placenta were prepared by nitrogen-bomb homogenization and differential centrifugation. 2. beta-Glucuronidase from placental lysosomes was purified 2100-fold on a protein basis. 3. The lysosomal enzyme, at different stages of purification, was characterized by using 4-methylumbelliferyl beta-d-glucuronide and phenolphthalein beta-d-glucuronide as substrates. 4. Only one isoenzyme of beta-glucuronidase was found in placenta; the enzyme in the endoplasmic reticulum appeared to be the same as the lysosomal enzyme. 5. The isoenzyme contained in normal plasma was different from that of the placenta. 6. The elevated beta-glucuronidase activity found in plasma obtained during pregnancy was due to increased activity of the normal plasma isoenzyme; no contribution was made by placental isoenzyme. 7. Plasma contained a heat-stable, non-diffusible activator of placental beta-glucuronidase. 8. A heat-stable competitive inhibitor of placental and plasma beta-glucuronidase was also present in plasma.

Major glucuronide metabolites of testosterone are primarily transported by MRP2 and MRP3 in human liver, intestine and kidney

“…Recent study also supports the role of microbiota in regulating the circulating T in mice, in which adult male mice with a normal microbiota had higher circulating levels of T compared to mice without gut microbiota, and transfer of cecal contents from male mice to female mice resulted in elevated circulating T in female mice [22]. In addition, endogenous β-glucuronidases have been found in several mammalian tissues such as placenta [23,24], which can synthesize T from TG. Therefore, the intracellular androgen glucuronide metabolites can be considered as precursors of T and DHT (active) and A and Etio (inactive), and the altered expression of efflux transporters by factors such as genetic polymorphism [25], drug-drug interactions [26], diseases [27], age [28,29], gender [30,31], and ethnicity [32], may affect their disposition, leading to dysregulation of steroidal homeostasis.…”

Deconjugation Kinetics of Glucuronidated Phase II Flavonoid Metabolites by β-glucuronidase from Neutrophils

“…10,[21][22][23][24] For 4MUglA, Km values reported were 330 mM 21) for human neutrophils, and ¿50 mM for placental b-glucuronidase. 22) O'Leary et al 24) presented kinetic constants at pH 7.2 for the hydrolysis of a mixture of several flavonoid glucuronides and pNPglA by human recombinant bglucuronidase isolated from COS-7 cells. Km values reported by them are of the same order of magnitude as in our study: 1400, 48, 167, and 237 mM for pNPglA, Q4?glA, Q3glA, and Q7glA, respectively, but no data were reported for Q3?glA or IRglA.…”

“…25) Human recombinant b-glucuronidase had higher activity at pH 5.5 compared to pH 7 and 7.4 when N-hydroxy-diacetylbenzidine glucuronide was used as a substrate. 26) Human b-glucuronidase derived from placenta, 22) lung tumor tissue, 27) or synovial fluid 28) all exhibited optima between pH 3.5 and 5. Contractor and Shane 22) were the only ones to determine the optimal pH of 4MUglA hydrolysis by b-glucuronidase (from placenta), which they found to be 3.…”

“…26) Human b-glucuronidase derived from placenta, 22) lung tumor tissue, 27) or synovial fluid 28) all exhibited optima between pH 3.5 and 5. Contractor and Shane 22) were the only ones to determine the optimal pH of 4MUglA hydrolysis by b-glucuronidase (from placenta), which they found to be 3. This agrees with the relatively low pH we found to be optimal for 4MUglA compared to pNPglA.…”

“…9,24) Early studies reported on a separate placental isoenzyme, with properties different from the enzyme found in plasma. 22) The placental isoenzyme was heat labile and had lower specific activity. However, these findings have not been confirmed in later studies.…”