Evidence for the Intraluminal Positioning of p-Nitrophenol UDP-glucuronosyltransferase Activity in Rat Liver Microsomal Vesicles

Fulceri, Rosella; Bánhegyi, Gábor; Gamberucci, Alessandra; Giunti, Roberta; Mandl, József; Benedetti, Angelo

doi:10.1006/abbi.1994.1081

Cited by 79 publications

(57 citation statements)

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“…The alamethicin-permeabilized microsomes were recovered on filters and washed as above. More than 95% of the microsomal protein was retained by filters, indicating that the alamethicin treatment did not affect the vesicular structure of microsomes as al-ready reported (41). The alamethicin-permeabilized microsomes retained amounts of radioactivity Յ 20% of that associated to untreated microsomes.…”

Section: Methodsmentioning

confidence: 66%

“…The radioactivity associated with microsomes was measured in vesicles incubated both in the presence and absence of the pore-forming antibiotic alamethicin (41,44) to determine net intravesicular accumulation. Because, in addition to [ 14 C]glucose-6-phosphate, [ 14 C]glucose (produced by glucose-6-phosphatase activity) can contribute to the intravesicular measured radioactivity (as it was indeed the case, see below), we expressed as "glucose-6-phosphate ϩ glucose" the 14 C-radioactivity accumulated (see Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Osmotically induced changes in microsomal vesicle size and shape were initiated by adding 0.1 ml (black arrowhead) of concentrate solutions of sucrose or glucose-6-phosphate (G-6-P) to 1.5 ml of the microsomal suspensions (in a hypoosmotic buffer; 70 or 35 g of protein/ml, for rat and human microsomes, respectively), giving the final concentration 75 mM (sucrose) or 30 mM (glucose-6-phosphate). Alamethicin (10 g per ml; white arrowheads) was then added to fully permeabilize microsomal vesicles (41). The addition of the poorly permeable sucrose resulted in a sustained shrinkage of vesicles indicating intactness of microsomal membrane (30, 45).…”

Section: Discussionmentioning

confidence: 99%

“…In each experiment, alamethicin (0.05 mg/ml) was added to the parallel incubates to distinguish the intravesicular and the bound radioactivity (41,44). The alamethicin-permeabilized microsomes were recovered on filters and washed as above.…”

Section: Methodsmentioning

confidence: 99%

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Demonstration of a Metabolically Active Glucose-6-phosphate Pool in the Lumen of Liver Microsomal Vesicles

Bánhegyi

Marcolongo

Fulceri

et al. 1997

Journal of Biological Chemistry

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Glucose-6-phosphate transport was investigated in rat or human liver microsomal vesicles using rapid filtration and light-scattering methods. Upon addition of glucose-6-phosphate, rat liver microsomes accumulated the radioactive tracer, reaching a steady-state level of uptake. In this phase, the majority of the accumulated tracer was glucose, but a significant intraluminal glucose-6-phosphate pool could also be observed. The extent of the intravesicular glucose pool was proportional with glucose-6-phosphatase activity. The relative size of the intravesicular glucose-6-phosphate pool (irrespective of the concentration of the extravesicular concentration of added glucose-6-phosphate) expressed as the apparent intravesicular space of the hexose phosphate was inversely dependent on glucose-6-phosphatase activity. The increase of hydrolysis by elevating the extravesicular glucose-6-phosphate concentration or temperature resulted in lower apparent intravesicular glucose-6-phosphate spaces and, thus, in a higher transmembrane gradient of glucose-6-phosphate concentrations. In contrast, inhibition of glucose-6-phosphate hydrolysis by vanadate, inactivation of glucose-6-phosphatase by acidic pH, or genetically determined low or absent glucose-6-phosphatase activity in human hepatic microsomes of patients suffering from glycogen storage disease type 1a led to relatively high intravesicular glucose-6-phosphate levels. Glucose-6-phosphate transport investigated by light-scattering technique resulted in similar traces in control and vanadate-treated rat microsomes as well as in microsomes from human patients with glycogen storage disease type 1a. It is concluded that liver microsomes take up glucose-6-phosphate, constituting a pool directly accessible to intraluminal glucose-6-phosphatase activity. In addition, normal glucose-6-phosphate uptake can take place in the absence of the glucose-6-phosphatase enzyme protein, confirming the existence of separate transport proteins.

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Section: Methodsmentioning

confidence: 66%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Demonstration of a Metabolically Active Glucose-6-phosphate Pool in the Lumen of Liver Microsomal Vesicles

Bánhegyi

Marcolongo

Fulceri

et al. 1997

Journal of Biological Chemistry

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“…Since the UGT active site is on the interior of the microsomal vesicles, most glucuronidation screening preparations include a detergent or pore-forming antibiotic, such as alamethicin, to disrupt the membrane barrier and enhance access of both substrate and cofactor to the UGT active site (30)(31)(32)(33). Detergents have been shown to alter intrinsic UGT activity and, while alamethicin appears to have less of an impact on intrinsic enzyme activity than detergent-based enhancers, there is still some concern that in vitro glucuronidation rates determined in the presence of activity enhancers may not reflect in vivo activity (34,35).…”

Section: Expressed Enzyme Study Caveatsmentioning

confidence: 99%

Reaction Phenotyping: Current Industry Efforts to Identify Enzymes Responsible for Metabolizing Drug Candidates

Harper

Brassil

2008

AAPS J

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Abstract. Reaction phenotyping studies to identify specific enzymes involved in the metabolism of drug candidates are increasingly important in drug discovery efforts. Experimental approaches used for CYP reaction phenotyping include incubations with cDNA expressed CYP enzyme systems and incubations containing specific CYP enzyme inhibitors. Since both types of experiments present specific advantages as well as known drawbacks, these studies are generally viewed as complementary approaches. Although glucuronidation pathways are also known to present potential drug-drug interaction issues as well as challenges related to their polymorphic expression, reaction phenotyping approaches for glucuronidation are generally limited to cDNA expressed systems due to lack of availability of specific UGT inhibitors. This article presents a limited review of current approaches to reaction phenotyping studies used within the pharmaceutical industry.

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Endoplasmic Reticulum

B��nhegyi¹,

Benedetti

Csala

2008

Wiley Encyclopedia of Chemical Biology

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Several pathways are compartmentalized in the endoplasmic reticulum (ER). These intraluminal activities require the passage of substrates, cofactors, and products through the ER membrane. The arguments for a general permeability of the ER membrane are contradicted by strong biochemical, pharmacological, clinical, and genetic evidence, which indicates that the lipid bilayer has a barrier function and that specific transport activities are needed in the membrane. Consequently, the ER lumen can be regarded as a separate metabolic compartment. This article overviews the best characterized intraluminal processes in which the compartmentation is important either by defining an intraluminal milieu, by limiting the rate of the reaction, by determining the specificity, or by creating a common substrate pool because of the colocalization.

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Evidence for the Intraluminal Positioning of p-Nitrophenol UDP-glucuronosyltransferase Activity in Rat Liver Microsomal Vesicles

Cited by 79 publications

References 0 publications

Demonstration of a Metabolically Active Glucose-6-phosphate Pool in the Lumen of Liver Microsomal Vesicles

Demonstration of a Metabolically Active Glucose-6-phosphate Pool in the Lumen of Liver Microsomal Vesicles

Reaction Phenotyping: Current Industry Efforts to Identify Enzymes Responsible for Metabolizing Drug Candidates

Endoplasmic Reticulum

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