Factors affecting the response of microsomal UDP-glucuronyltransferase to membrane perturbants

Graham, Allan B.; Wood, G. C.

doi:10.1016/0005-2736(73)90253-8

Cited by 36 publications

(11 citation statements)

References 13 publications

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“…Interestingly, incubations performed with microsomes prepared in phosphate buffer gave 2-to 3-fold greater estradiol-3-glucuronidation than those prepared in sucrose (Table 1). This agrees with the work of Graham and Wood (1973), who observed a similar trend in rat and guinea pig liver microsomes. Perhaps phosphate activates UGTs by increasing the permeability of the microsomal membrane to substrates and UDPGA.…”

Section: Compound Enzymessupporting

confidence: 93%

The Effect of Incubation Conditions on the Enzyme Kinetics of Udp-Glucuronosyltransferases

Soars

Ring²,

Wrighton³

2003

Drug Metab Dispos

136

105

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This article is available online at http://dmd.aspetjournals.org ABSTRACT:Traditionally, the Michaelis-Menten equation has been used to determine kinetic parameters for in vitro glucuronidation assays. Recently, estradiol-3-glucuronide formation was shown to exhibit non-Michaelis-Menten kinetics consistent with autoactivation. A concern with the observation of nontraditional kinetics is that they may result as an artifact of the incubation conditions. To examine this concern, the formation of estradiol-3-glucuronide was investigated using human liver microsomes prepared by two different methods, a range of assay conditions, and activation by alamethecin, sonication, or Brij 58 (polyoxyethylene monocetyl ether). Interestingly, holding the other assay components constant, estradiol-3-glucuronide formation was up to 2.5-fold greater using microsomes prepared in phosphate buffer compared with those prepared in sucrose. Incubations activated by alamethecin consistently exhibited the highest rates of estradiol glucuronidation versus the other activators. Furthermore, estradiol-3-glucuronidation exhibited autoactivation kinetics in all of the conditions investigated (n ‫؍‬ 1.2-1.7). Nontraditional kinetics were also observed when other UGT1A1 substrates such as ethinylestradiol, buprenorphine, and anthraflavic acid were studied with both human liver microsomes and recombinant UGT1A1. Naphthol, propofol, morphine, and androstanediol were used as probe UGT substrates selective for UGT1A6, UGT1A9, UGT2B7, and UGT2B15, respectively. Of these substrates, only androstanediol exhibited nontraditional kinetics using human liver microsomes. In conclusion, the Hill and/or Michaelis-Menten equations should be used to fit kinetic data to obtain an accurate assessment of in vitro glucuronidation.

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Section: Compound Enzymessupporting

confidence: 93%

The Effect of Incubation Conditions on the Enzyme Kinetics of Udp-Glucuronosyltransferases

Soars

Ring²,

Wrighton³

2003

Drug Metab Dispos

136

105

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“…Microsomal protein was determined with a biuret reagent (Graham & Wood, 1974). The UDP-glucuronyltransferase activities (assayed with 4mM-UDP-glucuronate) at 37°C fell within the ranges reported for similar preparations (Graham & Wood, 1973).…”

Section: Microsomal Fractionsmentioning

confidence: 59%

“…Male guinea pigs (Hartley strain, 250-400g) were from David Hall, Newchurch, Burton-on-Trent, Staffs., U.K. The animals were starved overnight, killed and liver microsomal fractions prepared with 0.25M-sucrose or 154mM-KCl (Graham & Wood, 1973). The washed microsomal membranes were resuspended, adjusted with the appropriate medium to 20 or 40mg of protein/ml, stored at -200C and used within 24h.…”

Section: Microsomal Fractionsmentioning

confidence: 99%

“…Transferase activities of native and perturbed microsomal membranes were determined with 0.6mM-p-nitrophenol and 4mMor 25mM-UDPglucuronate by the method of Graham & Wood (1973) with the following improvements. The incubation volume during assay was increased to 0.8 ml and, at seven appropriate times, 0.1 ml portions were removed and added to 2ml of cold 0.1 Mtrichloroacetic acid.…”

Section: Udp-glucuronyltransferase Assaysmentioning

confidence: 99%

See 1 more Smart Citation

The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Temperature-dependence of microsomal enzyme activity and thermotropic changes in membrane structure

Pechey¹,

Graham²,

Wood³

1978

Biochemical Journal

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Arrhenius plots of the non-latent UDP-glucuronyltransferase (p-nitrophenol acceptor) activity of guinea-pig microsomal membranes prepared with 154 mM-KCl were linear from 5 to 40 degrees C. Arrhenius plots for other microsomal preparations from guinea pig and rat liver that show various degrees of transferase latency, exhibited two linear regions intersecting at a sharp transition point near 20-25 degrees C. This discontinuity was abolished or greatly decreased when transferase latency was removed by treating the membranes with perturbants of phospholipid bilayer strucutre. The fluorescent probe N-phenyl-1-naphthyl-amine detected a thermotropic change in the fluidity of the phospholipid acyl chains of all the microsomal membrane preparations studied, at temperatures close to those of the Arrhenius-plot transitions. It is concluded that the thermotropic change in the structure of the membrane bilayer probably is a 'phase separation' or clustering of phospholipids, which affects a permeability barrier that restricts access of substrate to the transferase molecules.

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“…Activation of enzyme activity following treatment with surfactants has been reported previously, particularly with microsomal proteins. For example, mammalian liver UDP-glucuronyl transferase is firmly bound to microsomal membranes and its activity has been shown to be strongly dependent on the presence of compounds that perturb membranes (Chan 1967;Graham and Wood 1973).…”

Section: Controls Increasing Surfactant Concentrationmentioning

confidence: 99%

Disrupting mitochondrial function with surfactants inhibits MA-10 Leydig cell steroidogenesis

et al. 2007

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It is well established that surfactants can elicit cytotoxic effects at threshold concentrations by changing the permeability and solubilizing components of cell membranes. The purpose of this study was to characterize the relationship between perturbation of the mitochondrial membrane resulting from treatment with representative cationic, nonionic, and anionic surfactants and the extent to which this perturbation affects steroid formation and StAR protein expression and activity in MA-10 Leydig cells. The StAR protein is synthesized as an active 37 kDa extramitochondrial form, which is processed into a 30 kDa intramitochondrial form after cholesterol transfer and mitochondrial import and processing. It has been shown in several in vitro studies that the mitochondrial electrochemical gradient is required for the StAR protein to transfer cholesterol to the inner mitochondrial membrane. Each substance that was tested produced a concentration-dependent decrease in steroid formation in hCG-stimulated MA-10 cells. Decreases in progesterone production were accompanied by loss of mitochondrial membrane potential and by a decrease in the levels of the 30 kDa form of the StAR protein. However, levels of the 37 kDa form of the StAR protein did not decrease, indicating no effect on StAR protein expression. These results demonstrate how perturbation of the mitochondrial membrane by surfactants inhibits import, processing, and cholesterol transfer activity and underscore the importance of including sensitive assays that evaluate mitochondrial function when screening for potential effects on steroidogenesis with in vitro test systems.

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Factors affecting the response of microsomal UDP-glucuronyltransferase to membrane perturbants

Cited by 36 publications

References 13 publications

The Effect of Incubation Conditions on the Enzyme Kinetics of Udp-Glucuronosyltransferases

The Effect of Incubation Conditions on the Enzyme Kinetics of Udp-Glucuronosyltransferases

The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Temperature-dependence of microsomal enzyme activity and thermotropic changes in membrane structure

Disrupting mitochondrial function with surfactants inhibits MA-10 Leydig cell steroidogenesis

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