Search

Goal: The long-term goal of our research is to develop safe and effective soluble epoxide hydrolase (sEH) inhibitors. The objective of this study is to evaluate the potency and selectivity of six natural isothiocyanates (ITCs) as sEH inhibitors., Methods: Molecular docking was used to model likely interactions between the ligands and receptors. The sEH inhibitory activity was tested using a validated fluorescence-based assay and PHOME as a substrate. To evaluate their selectivity as sEH inhibitors, the inhibitory potential of the ITCs was determined on microsomal epoxide hydrolase (mEH) and cytochrome P450 (CYP) enzymes in human liver microsomes. Probe substrates such as styrene oxide (mEH substrate) and established substrates for CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 were used in this study. The metabolites of these substrates were analyzed using validated LC-MS/MS and HPLC-UV assays., Results: Molecular Docking revealed significant differences in binding site preference among the ITCs in silico and pointed to important interactions between the ligands and the catalytic residues of the sEH enzyme. In vitro, the ITCs showed varying degrees of sEH inhibition, but sulforaphane (SFN) and phenyl isothiocyanate (PITC) were the most potent inhibitors with IC 50 values of 3.65 and 7.5 μM, respectively. mEH was not significantly inhibited by any of the ITCs. Erucin and iberin were the only ITCs that did not inhibit the activity of any of the tested CYP enzymes., Conclusion: Our results demonstrate that natural ITCs have the potential to offer safe, selective, and potent sEH inhibition., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

We examined the effects of thyrotoxic rubber antioxidants, 2-mercaptobenzimidazole (MBI, 0.3 mmol/kg/day) and its methyl derivatives, methyl-MBIs [4-methyl-MBI (4-MeMBI, 0.6 mmol/kg/day), 5-methyl-MBI (5-MeMBI, 0.6 mmol/kg/day), and 4(or 5)-methyl-MBI (4(5)-MeMBI, 0.6 or 1.2 mmol/kg/day)], on the drug-metabolizing activity in male rat liver microsomes by 8-day repeated oral administration. The weight of liver and thyroid were increased by all the test chemicals; MBI was most potent, and there was no additive or synergistic effect between 4-MeMBI and 5-MeMBI. MBI decreased the cytochrome P450 (CYP) content, NADPH-cytochrome P450 reductase (POR) activity, 7-ethoxycoumarin O-deethylation (ECOD) activity, and flavin-containing monooxygenase (FMO) activity, but increased the 7-pentoxyresorufin O-depentylation (PROD) activity, suggesting inhibition of the drug-metabolizing activity on the whole but induce some activities such as the CYP2B activity. On the contrary, all the methyl-MBIs increased the CYP content, CYB5 content, ECOD activity, 7-ethoxyresorufin O-deethylation (EROD) activity, and PROD activity, indicating that they are mostly inducible of the CYP activity. However, the methyl-MBIs decreased the FMO activity, and 5-MeMBI and 4(5)-MeMBI appeared inhibitory for CYPs 2C11 and 2C13. Between 4-MeMBI and 5-MeMBI, there was no additive or synergistic effect on the drug-metabolizing activity, but was counteraction. It was concluded that MBI and methyl-MBIs had both inhibitory and inducible effects on the drug-metabolizing activity in rat liver microsomes at thyrotoxic doses. The effects of 4(5)-MeMBI indicated that the increased liver weight alone can be a hepatotoxic sign but not an adaptive no-adverse response in toxicity studies. The present results were related to the toxicokinetic profiles of MBI and 4(5)-MeMBI in the repeated toxicity studies., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Huperzine A (HupA), one of the reversible and selective acetylcholinesterase inhibitors derived from Chinese herb Huperzia Serrata, possesses affirmative action of ameliorating cognitive dysfunction of Alzheimer's disease. Up to now, the effects of HupA on human cytochrome P450s (CYPs) have not been fully elucidated. The purpose of the present study was to clarify the metabolic pathway of HupA in vitro and in vivo, and to evaluate the CYPs inhibition/induction profile of HupA in vitro. The catalytic activity of CYP enzymes (CYP1A2, 2A6, 2C9, 2C19, 2D6, 2E1 and 3A4) was measured by the quantification of specific enzyme substrates using validated liquid chromatography-tandem mass spectrometry (LC/MS/MS) methods. The in vivo metabolic pathway evaluation was performed in an open, single-dose pharmacokinetic study of HupA in fourteen elderly subjects, with urine collecting at certain intervals. In human liver microsomes, HupA (10 ng/mL) was not metabolized within 90 min, and it showed negligible inhibition against these CYP isoforms within 0.2-100 ng/mL. In human liver hepatocytes, the activities of CYP1A2 and CYP3A4 were not significantly altered when incubated at 2 or 20 ng/mL of HupA. After oral administration of 0.1 mg HupA, the total proportion of HupA excreted through urine was relatively high, accounting to 35± 9% at the limited time period of 48 h. These results suggest that HupA is substantially excreted by kidney unchanged rather than metabolized by human liver, and is unlikely to cause clinically relevant drug-drug interaction (DDI) when co-administrated with drugs that are metabolized by CYP isoenzyme system., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Prolonged therapy with phenobarbital may cause vitamin D deficiency or osteomalacia. In the current study, we propose a novel mechanism for drug-induced osteomalacia involving impaired bioactivation of vitamin D(3) due to decreased 25-hydroxylation of vitamin D(3) in liver. The present data, using the pig as model, demonstrate direct effects by phenobarbital on the expression of CYP27A1 and CYP2D25, two important 25-hydroxylases. Treatment by phenobarbital markedly reduced the rate of 25-hydroxylation by primary hepatocytes and suppressed the cellular CYP27A1 mRNA levels. The rate of 25-hydroxylation by two different purified 25-hydroxylases, microsomal CYP2D25, and mitochondrial CYP27A1, respectively, was dose-dependently inhibited by phenobarbital. Reporter assay experiments in liver-derived HepG2 cells revealed a marked PXR-mediated transcriptional downregulation of the CYP2D25 promoter. In addition, the data indicate that phenobarbital might affect the mRNA stability of CYP2D25. Taken together, the data suggest that vitamin D(3) 25-hydroxylation may be suppressed by phenobarbital. A downregulation of 25-hydroxylation by phenobarbital may explain, at least in part, the increased risk of osteomalacia, bone loss, and fractures in long-term phenobarbital therapy.

Cytochrome CYP1A (CYP1A) enzymes catalyze bioactivation of 3-methylcholanthrene (MC) to genotoxic metabolites. Here, we tested the hypothesis that CYP1A2 catalyzes formation of MC-DNA adducts that are preferentially formed in the promoter region of CYP1A1, resulting in modulation of CYP1A1 gene expression. MC bound covalently to plasmid DNA (50 micro g) containing human CYP1A1 promoter (pGL3-1A1), when incubated with wild-type (WT) liver microsomes (2 mg) and NAPPH 37 degrees C for 2h, giving rise to 9 adducts, as determined by (32)P-postlabeling. Eighty percent of adducts was located in the promoter region. Transient transfection of the adducted plasmids into rat hepatoma (H4IIE) cells for 16h, followed by MC (1 micro M) treatment for 24h inhibited reporter (luciferase) gene expression by 75%, compared to unadducted controls. Our results suggest that CYP1A2 plays a key role in sequence-specific MC-DNA adduct formation in the CYP1A1 promoter region, leading to attenuation of CYP1A1 gene expression.

The acyl-CoA-dependent modulation of hepatic microsomal UDP-glucuronosyltransferase (UGT) function in rats was studied. Oleoyl- and palmitoyl-CoAs inhibited UGT activity toward 4-methylumbelliferone in the presence of Brij 58. However, acyl-CoAs enhanced UGT activity in untreated microsomes. A maximum activation of about 8-fold over the control was observed at 15 microM oleoyl-CoA, whereas 50 microM or more oleoyl-CoA had an inhibitory effect on UGT function. Medium- and long-chain acyl-CoAs also exhibited similar effects. On the basis of resistance to tryptic digestion of UGTs, oleoyl-CoA at 15 microM has no ability to change the permeability of the endoplasmic reticulum (ER) membrane, although perturbation of the membrane occurred with 50 microM oleoyl-CoA. N-Ethylmaleimide and 5,5'-dithiobis(2-nitrobenzoic acid) abolished the oleoyl-CoA (15 microM)-dependent activation of microsomal UGT. These results suggest that: (1) acyl-CoAs play a role as an endogenous activator of UGTs, and (2) a sulfhydryl group is required for the activation of UGT by physiological concentrations of acyl-CoAs.

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) induces cytochromes P450 (CYPs) such as CYP1A1 and CYP1A2 via activation of the aromatic hydrocarbon receptor (AHR). Herein we describe the TCDD-dependent enrichment of CYPs in liver microsomes and mitoplasts from C57BL/6J mice. TCDD-induced accumulation of CYP1A1 and CYP1A2 was observed in microsomes and mitoplasts after treatment with 15 microg TCDD/kg/d for 3d. While microsomal CYP1 proteins peaked at 1 week and diminished thereafter, mitoplast CYP1 proteins persisted 8 weeks at high levels. TCDD also induced microsomal CYP2A5, but not microsomal proteins immunoreactive to CYP2C11, CYP3A2 or CYP4A1 antibodies. Nevertheless, each of these proteins increased in mitoplasts following TCDD exposure. These results suggest that TCDD increases mitochondrial CYP immunoreactive proteins under the transcriptional control of the AHR, as well as CYPs that are not under AHR control. We speculate that such mitochondrial CYPs may be involved in the generation, or mitigation, of the well-known TCDD-inducible oxidative stress response.

Indirubin and indigo, which are thought to be natural ligands for aryl hydrocarbon receptor (AhR), showed marked AhR ligand activities in a reporter gene assay using recombinant yeast. Their activities were comparable with or more potent than that of 2,3,7,8-tetrachlorodibenzo-p-dioxin. When indirubin and indigo were administered to mice, ethoxyresorufin-O-dealkylase and methoxyresorufin-O-dealkylase activities in the liver were increased, but subsequently decreased within 2 days. Indirubin was more potent than indigo. Levels of cytochrome P450 1A1/2 proteins and mRNAs in the liver of mice dosed with indirubin were also enhanced. These enhancing effects of indirubin and indigo were not observed in AhR knock-out mice. Ethoxyresorufin-O-dealkylase and methoxyresorufin-O-dealkylase activities in rat hepatocytes and HepG2 cells were enhanced by the addition of indirubin or indigo, but less potently than by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Indigocarmine, a sulfate derivative of indigo, which is used as food additive, did not show these inducing effects on drug-metabolizing enzymes. Our results suggest that indirubin and indigo act as inducers for cytochrome P450 1A1/2 mediated by AhR in mammals in vivo.

Nicotinic acid has been used as a cholesterol-lowering agent for a few decades already, whereas the cytoprotective and antiviral properties of nicotinamide are slowly gaining attention. In both cases however, very high doses are needed to achieve a therapeutic effect, resulting in blood concentrations sometimes as high as 15 mM. Based on their common pyridine functionality, we hypothesized that these two molecules could inhibit human P450 enzymes. In vitro inhibition studies demonstrate that, at their therapeutic concentrations, both nicotinic acid and nicotinamide inhibit CYP2D6 (Ki = 3.8 +/- 0.3 and 19 +/- 4 mM, respectively). Nicotinamide also inhibits CYP3A4 (Ki = 13 +/- 3 mM) and CYP2E1 (Ki = 13 +/- 8 mM). As expected for nitrogen-containing heteroaromatic molecules, spectrophotometric analysis indicates that the inhibition occurs via coordination of the pyridine nitrogen atom to the heme iron.

The mechanism of impairment of cytochrome P450 (P450)-dependent metabolism in hamster liver during leishmaniasis is reported. A significant decrease in the level of P450 was observed on the 20th day of infection when the parasite load in the liver was maximum. The decrease in P450 level was accompanied by a significant increase in the level of marker enzymes of liver and degeneration of liver tissue. The impairment was isozyme-specific and concomitant with the induction of nitric oxide synthase. The results of in vitro experiments with generated nitric oxide and with scavengers demonstrated that the impairment is mediated by NO. Treatment of the infected animals with a combination therapy showed reduction in parasite load, reversal of P450 impairment, and recovery of liver enzymes and tissue close to normal.

In this study, the metabolic activation of 2-nitrofluorene (NF) to estrogenic compounds was examined. NF was negative in estrogen reporter assays using estrogen-responsive yeast and human breast cancer cell line MCF-7. However, the compound exhibited estrogenic activity after incubation with liver microsomes of 3-methylcholanthrene-treated rats in the presence of NADPH. Minor estrogenic activity was observed when liver microsomes of untreated or phenobarbital-treated rats were used instead of those from 3-methylcholanthrene-treated rats. When the compound was incubated with the liver microsomes of 3-methylcholanthrene-treated rats in the presence of NADPH, 7-hydroxy-2-nitrofluorene (7-OH-NF) was formed as a major metabolite. However, little of the metabolite was formed by liver microsomes of untreated or phenobarbital-treated rats. Rat recombinant cytochrome P450 1A1 exhibited a significant oxidase activity toward NF, affording 7-OH-NF. Liver microsomes of phenobarbital-treated rats also enhanced oxidase activity toward NF. In this case, 9-hydroxy-2-nitrofluorene was formed. 7-OH-NF exhibited a significant estrogenic activity, while the activity of 9-hydroxy-2-nitrofluorene was much lower. These results suggest that the estrogenic activity of NF was due to formation of the 7-hydroxylated metabolite by liver microsomes.

R-(+)-Pulegone, a monoterpene ketone, is a potent hepatotoxin. One of the major metabolites of pulegone has been shown to be p-cresol, a glutathione depletor and a known toxin. Allylic hydroxylation of 4-methyl-2-cyclohexenone results in the formation of p-cresol. The present study documents for the first time the involvement of cytochrome P-450 system and the stereochemical preference in this hydroxylation reaction. Incubation of PB-induced rat liver microsomes as well as reconstituted PB-induced cytochrome P-450 system with +/-4-methyl-2-cyclohexenone in the presence of NADPH and O(2) resulted in the formation of 4-hydroxy-4-methyl-2-cyclohexenone and p-cresol. From the assay mixture, the unreacted substrate, viz., 4-methyl-2-cyclohexenone was isolated and purified and its optical rotation was found to be 2.2 (in CHCl(3)). The observed enantiomeric excess in the recovered substrate was further confirmed by circular dichroism (CD) studies. The CD spectrum has a peak at 292nm and a trough at 270nm. The enantiomeric excess in the recovered substrate indicates that the hydroxylation at C-4 position is stereoselective. The significance of these results with respect to pulegone-mediated hepatotoxicity is discussed.

Acetaminophen is a widely used over-the-counter drug that causes severe hepatic damage upon overdose. Cytochrome P450-dependent oxidation of acetaminophen results in the formation of the toxic N-acetyl-p-benzoquinone-imine (NAPQI). Inhibition of cytochrome P450 enzymes responsible for NAPQI formation might be useful--besides N-acetylcysteine treatment--in managing acetaminophen overdose. Investigations were carried out using human liver microsomes to test whether selective inhibition of cytochrome P450s reduces NAPQI formation. Selective inhibition of CYP3A4 and CYP1A2 did not reduce, whereas the inhibition of CYP2A6 and CYP2E1 significantly decreased NAPQI formation. Furthermore, selective CYP2E1 inhibitors that are used in human therapy were tested for their inhibitory effect on NAPQI formation. 4-Methylpyrazole, disulfiram, and diethyl-dithiocarbamate were the most potent inhibitors with IC(50) values of 50 microM, 8 microM, and 33 microM, respectively. Although cimetidin is used in the therapy of acetaminophen overdose as an inhibitor of cytochrome P450, it is not able to reduce NAPQI formation.

Furanoterpenes are widely distributed in the plant kingdom. In this study we have carried out enzymatic synthesis of simple furan compounds from the molecules containing an alpha-isopropylidene ketone unit and the role of cytochrome P450 in this biotransformation has been conclusively established. Eight model compounds (acyclic, monocyclic, and bicyclic, 1-8), having an alpha-isopropylidene ketone unit, were synthesized and incubated with PB-induced rat liver microsomes in the presence of NADPH and O(2). GC-MS and NMR analyses of the product(s) indicated the formation of corresponding furano derivatives (11-18). Cytochrome P450 inhibitors, metyrapone, SKF-525, and carbon monoxide, inhibited the formation of furan (8) to 76, 62, and 97%, respectively. Incubation of dehydrofukinone (7), a naturally occurring terpene, with purified cytochrome P450, NADPH-cytochrome P450 reductase, and dilaurylphosphatidylcholine in the presence of NADPH and O(2) resulted in the formation of 10 and furanodehydrofukinone (19). Based on these observations, we propose one of the probable biosynthetic routes for lower furanoterpenoids in higher plants., ((c)2002 Elsevier Science.)

Cytochrome P4502E1 (CYP2E1) induction by ethanol contributes to alcoholic liver disease and we found that a mixture of polyunsaturated phosphatidylcholines (PPC), which protects against alcohol-induced liver injury, also decreases CYP2E1. Since dilinoleoylphosphatidylcholine (DLPC) is the major component of PPC, we assessed here whether it is responsible for the protection of PPC by feeding rats for 8 weeks our liquid diet containing ethanol (36% of energy) or isocaloric carbohydrates, with either DLPC (1.5 g/1000 cal), PPC (3 g/1000 cal), or linoleate. CYP2E1 was assessed by Western blots and by two of its enzyme activities: the microsomal ethanol-oxidizing system (MEOS) and p-nitrophenolhydroxylase (PNP). With ethanol, CYP2E1 increased 10-fold, with corresponding rises in PNP and MEOS activities. Compared to linoleate, DLPC significantly decreased cytochrome b(5), total cytochromes P450, CYP2E1 content and its corresponding activities. DLPC decreases ethanol-induced CYP2E1 and should be considered for the prevention of alcoholic liver disease., (Copyright 2001 Academic Press.)

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), an environmental contaminant, induced xanthine oxidase and xanthine dehydrogenase (XO/XDH) activities, in addition to ethoxyresorufin-O-dealkylase and methoxyresorufin-O-dealkylase activities in liver of mice. When TCDD was given to mice as a single oral dose of 40 microg/kg, the activities of XO and XDH increased about threefold within 3 days and the increased levels were maintained for 4 weeks. The treatment of mice with 3-methylcholanthrene also induced XO/XDH activities, but phenobarbital and dexamethasone had no effect. The level of aldehyde oxidase, a molybdenum flavoenzyme related to XO/XDH, in mouse liver was also enhanced about 1.5-fold by TCDD treatment. The inducing effect of TCDD and 3-methylcholanthrene was not observed in null mice (AhR(-/-)), which lack the AhR gene. XO and XDH activities were induced by TCDD in heterozygous mice (AhR(+/-)). The lipid peroxidation in liver was stimulated by TCDD. The induction of XO and XDH, which produces reactive oxygen species, may contribute to the various toxicities of TCDD., (Copyright 2001 Academic Press.)

Despite numerous studies demonstrating that microsomal triglyceride transfer protein (MTP) activity is critical to apoB secretion, there is still controversy as to whether MTP directly facilitates the translocation of apoB across the membrane of the endoplasmic reticulum (ER) through either the recruitment of lipids and/or chaperone activity. In the present study, a specific inhibitor of MTP (BMS 197636) was utilized in HepG2 cells to investigate whether a direct relationship exists between the translocation of apoB across the ER membrane and the lipid-transferring activity of MTP. Inhibition of MTP (with 10 and 50 nmol/L of the inhibitor) did not significantly affect the translocation of newly synthesized apoB (P = 0.77) or the translocational efficiency of the steady-state apoB mass (P = 0.45), despite a 49% decrease in apoB secretion and increased proteosomal degradation. These results compared well with subcellular fractionation experiments which showed no significant change in the fraction of apoB accumulated in the lumen of isolated microsomes in MTP-treated cells (P = 0.35). In summary, MTP lipid transfer activity does not appear to influence translocational status of apoB, but its inhibition is associated with an increased susceptibility to proteasome-mediated degradation and reduced assembly and secretion of apoB lipoprotein particles., (Copyright 2000 Academic Press.)

C-Phycocyanin (from Spirulina platensis) effectively inhibited CCl(4)-induced lipid peroxidation in rat liver in vivo. Both native and reduced phycocyanin significantly inhibited peroxyl radical-induced lipid peroxidation in rat liver microsomes and the inhibition was concentration dependent with an IC(50) of 11.35 and 12.7 microM, respectively. The radical scavenging property of phycocyanin was established by studying its reactivity with peroxyl and hydroxyl radicals and also by competition kinetics of crocin bleaching. These studies have demonstrated that phycocyanin is a potent peroxyl radical scavenger with an IC(50) of 5.0 microM and the rate constant ratios obtained for phycocyanin and uric acid (a known peroxyl radical scavenger) were 1.54 and 3.5, respectively. These studies clearly suggest that the covalently linked chromophore, phycocyanobilin, is involved in the antioxidant and radical scavenging activity of phycocyanin., (Copyright 2000 Academic Press.)

Resveratrol (trans-3,4',5-trihydroxystilbene) is a phytoalexin compound found in juice and wine produced from dark-skinned grape cultivars and reported to have anti-inflammatory and anticarcinogenic activities. To investigate the mechanism of anticarcinogenic activities of resveratrol, the effects on cytochrome P450 (P450) were determined in human liver microsomes and Escherichia coli membranes coexpressing human P450 1A1 or P450 1A2 with human NADPH-P450 reductase (bicistronic expression system). Resveratrol slightly inhibited ethoxyresorufin O-deethylation (EROD) activity in human liver microsomes with an IC(50) of 1.1 mM. Interestingly, resveratrol exhibited potent inhibition of human P450 1A1 in a dose-dependent manner with IC(50) of 23 microM for EROD and IC(50) of 11 microM for methoxyresorufin O-demethylation (MROD). However, the inhibition of human P450 1A2 by resveratrol was not so strong (IC(50) 1.2 mM for EROD and 580 microM for MROD). Resveratrol showed over 50-fold selectivity for P450 1A1 over P450 1A2. The activities of human NADPH-P450 reductase were not significantly changed by resveratrol. In a human P450 1A1/reductase bicistronic expression system, resveratrol inhibited human P450 1A1 activity in a mixed-type inhibition (competitive-noncompetitive) with a K(i) values of 9 and 89 microM. These results suggest that resveratrol is a selective human P450 1A1 inhibitor, and may be considered for use as a strong cancer chemopreventive agent in humans., (Copyright 1999 Academic Press.)

Microsomal glutathione transferase has hitherto only been purified from mammalian species. N-ethylmaleimide and trypsin activation (discriminating features of this enzyme) has only been observed in microsomes from mammals. In this paper we describe the first isolation and characterization of a non-mammalian microsomal glutathione transferase from frog (Xenopus laevis) liver. This protein has a molecular weight similar to that of the mammalian enzyme (approximately 17 kDa), but cannot be activated by N-ethylmaleimide or trypsin. In fact the enzyme is rapidly inactivated by this sulfhydryl reagent and protease. It thus appears that N-ethylmaleimide activation is not an obligatory property of microsomal glutathione transferase. The frog liver microsomal glutathione transferase has one of the highest specific activities towards the second substrate 1-chloro-2,4-dinitrobenzene (CDNB) (200 mumol/min mg) obtained with any glutathione transferase and accounts for the high activity found in frog liver microsomes. The kcat/K(m) for glutathione and CDNB are 0.017 and 1.1 x 10(6) M-1 s-1, respectively. The enzyme also functions as a glutathione peroxidase (dilinoleoyl phosphatidylcholine hydroperoxide is reduced (5.2 mumol/min mg)). It is now evident that a highly active microsomal glutathione transferase, with a molecular weight similar to that of the mammalian enzymes also exists in a non-mammal species.

Extracellular ATP is found to produce a variety of important biological responses. Ecto-ATPases are located on numerous cell types in many different species, regulate extracellular ATP levels and can be a key step in generating adenosine. Studies conducted on chicken ecto-ATPases from liver and cardiac and smooth muscle show a variety of differing properties including (1) different apparent Km's, (2) lectin sensitivity, (3) responses to detergents, (4) responses to lipid mediators, and (5) responsiveness to nucleotide-mimetic affinity labels. These results suggest that although each enzyme hydrolyzes extracellular ATP, they should each be viewed as a distinct subtype of the whole ecto-ATPase family due to their differential responses, largely linked to proposed regulatory phenomenon.

We show that the production of glucose from glucose-6-phosphate hydrolysis outside microsomes is a function of glucose-6-phosphatase independent of its property to form glucose inside microsomes. Indeed, during development (before 1 day of age), mouse liver microsomes had glucose-6-phosphatase producing glucose solely outside microsomes. Furthermore, in vivo treatment of rats with the glucocorticoid analogue triamcinolone resulted in increased glucose-6-phosphatase activity outside but not inside microsomes and without change in the catalytic subunit 40 kDa glucose-6-phosphatase mRNA abundance or protein level, indicating that other factors induced by triamcinolone (e.g., altered membrane lipid environment and/or a regulatory protein) were responsible for the activity change. Triamcinolone treatment also lessened the inhibition of glucose-6-phosphatase by pyridoxal 5'-phosphate (PLP), but this effect was not due to an interaction of PLP with the active site. Accordingly, reversal of the inhibition was observed after permeabilization of the microsomes. The two distinct orientations of liver microsomal glucose-6-phosphate phosphohydrolase suggest different physiological roles played by this enzyme in the endoplasmic reticulum membrane.

Upon N-demethylation of caffeine (CA) by rat and human liver microsomes, theobromine (TB), paraxanthine (PX), and theophylline (TP) are produced. The optimal pHs for the formation of TB, PX, and TP from CA by rat liver microsomes are 7.4 (most), 8.2 (minor) and 8.6 (moderate). At pH 7.4, PX is the primary metabolite formed and makes up 48% of the CA metabolites generated. In the presence of SKF525A, an inhibitor of P450 (CYP), the rates of TB, PX and TP production are inhibited by 32%, 68% and 42%, respectively. Alternatively, in the presence of methimazole, an inhibitor of flavin-containing monooxygenase (FMO), the rates of TB, PX and TP production are inhibited by 66%, 48% and 73%, respectively. In the presence of both SKF525A and methimazole, they are inhibited by 95%, 84% and 94%, respectively. With human liver microsomes, the CA is metabolized faster but is inhibited more extensively either by SKF525A (PX production) or by methimazole (TB production). Alternatively, when CA is metabolized at pH 8.6, the optimal pH of FMO catalyzed reaction, the rates of TB and TP formation are increased but the rate of PX production is decreased. Furthermore, at pH 8.6 and in the presence of methimazole, the rates of TB and TP formation are decreased by 82% and 95%, respectively. These results indicate that the FMO is responsible primarily for productions of TB and TP and the CYP for PX.

This study examined the relationship between dose- and time-dependent hepatic localization of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and expression of CYP1B1, CYP1A1 and CYP1A2 proteins. A dose-dependent increase in hepatic TCDD in female Sprague-Dawley rats treated with 0.01-30.0 microg TCDD/kg was observed. TCDD induced CYP1A1 protein in rats treated with 0.3 microg TCDD/kg or higher. TCDD induced CYP1A2 and CYP1B1 proteins in rats treated with 1.0 microg TCDD/kg or higher. The in vivo ED50 (microg TCDD/kg) for TCDD-induced CYP1A1, CYP1A2 and CYP1B1 proteins were 0.22, 0.40 and 5.19, respectively. Hepatic accumulation of TCDD reached a maximum at 8 hours post dosing with a t1/2 of approximately 10 days. TCDD-induced CYP1A1/CYP1A2 protein expression was increased time-dependently, reaching a maximum at 3 days after dosing and remaining elevated for 35 days. In contrast, TCDD-induced CYP1B1 protein showed significant expression at 3 days after dosing and decreased to basal concentrations by 35 days. This study demonstrates that TCDD exhibits differential dose-response and time-course relationships on hepatic localization and cytochrome P-450 protein expression.

Previously we have demonstrated inhibition of lipid peroxidation by reduced glutathione (GSH) in rat liver microsomes that is dependent upon the presence of alpha-tocopheral (alpha-TH) in the membranes. Glutathione disulfide (GSSG) potentiated the inhibitory effect of GSH in an enzymatic (NADPH-dependent) lipid peroxidation system in rat liver microsomes; however, inhibition by GSH + GSSG is independent of alpha-TH. When we repeated these experiments with a non-enzymatic system (ascorbate/ADP) to stimulate lipid peroxidation, GSSG did not potentiate the inhibitory effect of GSH. To delineate the mechanism of inhibition of microsomal lipid peroxidation by GSH + GSSG, we examined the effects of these compounds on cytochrome P-450 reductase (EC 1.6.2.4), an important component of the NADPH-dependent enzymatic lipid peroxidation system. It was observed that GSH alone caused about 25% and 21% inhibition of reductase activity in crude microsomes and partially purified enzyme preparations, respectively. There was no inhibition of reductase activity by GSSG alone in either crude microsomes or partially purified enzyme preparations. However, when added together, GSH and GSSG enhanced the inhibition of reductase activity in crude microsomes and partially purified enzyme by 39% and 56%, respectively. We speculate that one possible mechanism for the inhibition of NADPH-dependent lipid peroxidation by GSH + GSSG is, in part, due to inhibition of NADPH cytochrome P-450 reductase, thus affecting the initiation phase of lipid peroxidation.

A cDNA encoding vitamin K-dependent gamma-glutamyl carboxylase was cloned from a human Hep G2 cDNA library. The RNA transcript of the enzyme was found to be widely distributed in various human and rat tissues with liver showing the highest level. The carboxylase transcription in liver was not affected in rats treated with a single dose of warfarin (10 mg/kg) when measured up to 48 hours after the dose, though, at 12 hours, carboxylase activity measured in liver microsomes was elevated 5.4 fold over controls (p < 0.001). In rats fasted for 72 hours there was no affect on transcription in the liver while hepatic carboxylase activity increased 4.1 fold (p < 0.001). These data suggest that the increase in activity of the liver carboxylase in warfarin treated or fasted rats was not regulated by transcription but more likely was due to a posttranscriptional mechanism.

We studied the effect of oral supplementation with ursodeoxycholate (UDCA) or tauroursodeoxycholate (TUDCA) on the lipid content and fatty acid composition of rat hepatic microsomes. UDCA and TUDCA significantly increased the total amount of lipids with the exception of cholesteryl-esters. UDCA significantly increased the triglycerides and phosphatidylethanolamine (PE) microsomal content, and decreased the cholesterol/phospholipids and the phosphatidylcholine (PC)/PE ratio. Both treatments increased the percentage oleic acid and of polyunsaturated fatty acids (PUFA) in each class of lipids. UDCA and TUDCA had a different action on PUFA microsomal molar percentage of phospholipids: UDCA increased the relative percentage of PUFA in the PE fraction, while TUDCA increased the relative percentage of PUFA in the PC fraction. These changes in the hepatic lipid content and composition might in part explain both cytoprotective action of these hydrophillic bile acids and their effect on membrane fluidity.

The permeability of rat liver microsomes to glucose has been studied by using (14)C-labelled D-glucose and a light-scattering technique. 1) The microsomal intravesicular apparent isotope space for D-glucose (1mM; after 5 min incubation at 22 degrees C) was 2.34 microl/mg protein, i.e., approximately 72% of the apparent water space. 2) Efflux of [(14)C]D-glucose from microsomal vesicles pre-loaded as in 1) and measured by rapid Millipore filtration after dilution (100 fold) in a glucose-free medium revealed that 15 sec after dilution only 15% of intravesicular glucose was still retained by microsomes. 3) Osmotic behaviour of microsomes upon addition of D-glucose measured by a light-scattering technique revealed a glucose influx, saturable at [D-glucose] > 100 mM, and (partially) inhibited by pentamidine and cytochalasin B. Ascorbic acid, L-glucose and other monosaccharides and related compounds also permeated liver microsomes in a fashion similar to D-glucose. These data indicate the existence of a facilitative transport system(s) for glucose in the membrane of liver endoplasmic reticulum vesicles.

Induction of liver cytochrome P450 2B1 by beta-ionone was investigated in male and female Sprague Dawley rats. Administration of beta-ionone subcutaneously 72 and 48 hr before sacrificing the animals not only significantly induced the liver microsomal activity of pentoxyresorufin O-dealkylase, but also clearly increased in the level of cytochrome P450 2B1 protein. The induction of cytochrome P450 2B1 by beta-ionone was much greater in male rats than in female rats. A slot blot analysis showed that the mRNA level was increased from 6 hr after treatment with beta-ionone in male rats and from 12 hr after treatment in female rats. Taken together, the present results indicate for the first time that the induction of cytochrome P450 2B1 by beta-ionone might be regulated by the accumulation of mRNAs.

Cytochrome P4502E1 is localized in the pericentral (PC) zone of the liver acinus to a greater extent than in the periportal (PP) zone. After pyrazole treatment, PC microsomes were more active in oxidizing typical substrates of CYP2E1 than PP microsomes and had an increased content of CYP2E1. The ability of PC and PP microsomes from pyrazole-treated rats to interact with iron and generate reactive oxygen species such as the hydroxyl radical (.OH) was evaluated. A sensitive DNA strand cleavage assay was used to detect .OH; supercoiled plasmid DNA is compact but is converted by .OH-induced single strand breaks to the relaxed open circular state. Microsomes from PC hepatocytes of pyrazole-treated rats were several fold more reactive than PP microsomes in promoting NADPH-dependent DNA strand cleavage with a variety of iron catalysts, including ferric-ATP, ferric-histidine, ferric-citrate, ferric ammonium sulfate, and ferric-EDTA. DNA strand cleavage was inhibited by superoxide dismutase, catalase, and .OH scavengers such as DMSO and ethanol. Rates of H2O2 production were higher with the PC microsomes. These results indicate that rates of .OH production are higher with PC microsomes than PP microsomes after pyrazole treatment to induce cytochrome P4502E1 and suggest the possibility that elevated production of reactive oxygen species may play a role in ethanol toxicity to the PC zone of the liver acinus.

NADPH-cytochrome-P450 reductase both purified from rat hepatic microsomes and involved in microsomal fraction was inactivated by treatment with alpha-lipoic acid. Since alpha-lipoic acid contains disulfide bond, it reacts with SH-groups of the reductase via the reaction of thiol-disulfide exchange resulting in the loss of the enzyme reducing activity. NADP+ completely protected reductase from the inactivation. The modification of reductase was reversible: the modified enzyme was partially reactivated with dithiothreitol and dihydrolipoic acid in the case when cytochrome c was used as a substrate of reductase. In the case when inorganic substrate, K3Fe(CN)6, was used for assay the activity of modified reductase no reactivation was observed. It was found that the order of the reaction of inactivation of membrane-bound microsomal reductase is equal to 1.2 +/- 0.2, which is in an agreement with pseudo-first order kinetics, and the second-order-rate constant of 26 M-1min-1. The results have shown that well known therapeutic agent alpha-lipoic acid is an efficient inhibitor of both purified and microsomal reductase.

Phenobarbital, the potent inducer of CYP2B and CYP3A, and dexamethasone, that induces CYP3A, are not able to elevate p-nitrophenol hydroxylase activity of CYP2E1. However, rats treated with phenobarbital and dexamethasone in combination showed threefold increase in p-nitrophenol hydroxylation and the activity correlates with an elevated amount of a 53.000 dalton protein. Biosynthesis of mRNA and P450 protein is required for the induction. 3-amino-1,2,4-triazole and anti CYP2E1 IgG inhibition studies show that CYP2E1 is not responsible for enhanced p-nitrophenol hydroxylation, but the residual activity indicates the participation of other isozyme(s). As a result of double induction, changes in the amount of CYP2E1 of microsomes were not detected by Western blot analysis compared to untreated rat liver microsomes.

The specific binding of the archetypal cytochrome P450 3A subfamily (CYP3A) inducer dexamethasone is examined in microsomal fractions since rat and human liver CYP3A is induced by glucocorticoids through a mechanism which is apparently independent of the cytosolic glucocorticoid receptor. Dexamethasone binds in a specific and saturable manner to microsomes with an affinity constant (Kd) of 59 +/- 12.9 nM which compares to a Kd of 2.3 +/- 0.17 nM in cytosol. The total receptor concentrations ([LR]emax) in microsomes and cytosol are 9.5 +/- 1.67 pmols/mg protein and 410 +/- 167 fmol/mg protein respectively. The microsomal binding of dexamethasone is antagonised by several transcriptional and/or post-transcriptional CYP3A inducers with decreasing potency pregnenolone 16 alpha carbonitrile > metyrapone > phenobarbitone. Troleandomycin, which indirectly induces CYP3A1 in vivo and by protein stabilisation, does not antagonise the binding of dexamethasone in microsomes. The transcriptional and/or post-transcriptional induction of CYP3A may therefore be associated with the interaction of inducers with a microsomal protein.

Male F344 rats were treated with lead nitrate, nickel chloride, cobalt chloride or cadmium chloride, and changes in the expression of cytochrome P450IA2 (CYP1A2) in the livers were assessed by enzymatical, immunochemical, and molecular biological methods. All ionic metals used significantly decreased total CYP amount in the liver microsomes. However, among ionic metals used only lead showed inhibitory effects on the microsomal activity for CYP1A2-dependent mutagenesis of aromatic amines and on the expression of mRNA and protein of the enzyme, indicating that ionic lead is a unique inhibitor for the expression of CYP1A2 enzyme in the rat liver.

Anandamide (arachidonylethanolamide) has been identified as a brain constituent that selectively binds to the cannabinoid receptor and possesses cannabimimetic activity. Cytochromes P450 catalyze the oxidation of arachidonic acid to several metabolites possessing very potent pharmacological activity. We examined whether P450 would also metabolize anandamide, and whether cannabidiol (a cannabinoid which inactivates several P450s) would affect this metabolism. Mouse hepatic P450s were found to metabolize anandamide to at least 10 different metabolites, four of which were characterized by mass spectrometry. Cannabidiol selectively inhibited the formation of two of these four anandamide metabolites. The significance of anandamide metabolism remains to be explored.

The kinetic deuterium isotope effect on the microsomal oxidative demethylation of methoxyl groups was measured using methoxychlor, an insecticide, as a model compound. When the metabolic reactions were conducted in the presence of bovine serum albumin, the observed isotope effect was smaller than that in its absence. The values were unaltered when the albumin concentration was changed from 1% to 2%. The results were interpreted by the alteration of the ES-complex formation/dissociation rate with the interaction between substrate and albumin.

The effects of chronic nicotine administration on the enzymatic activities and immunoreactivities of P4501A1/2 and P4502E1 were determined in microsomal fractions from brain regions and liver. Chronic nicotine increased 7-ethoxyresorufin O-deethylase activity and P4501A1/2 immunoreactivity in hippocampus and brain stem, decreased values in cerebral cortex, thalamus, and striatum, and did not affect parameters in other brain regions. In contrast, N-nitrosodimethylamine N-demethylase activity from nicotine-injected rats increased in all brain regions examined, and this increase was associated with increased P4502E1 immunoreactivity. Chronic nicotine did not alter either enzyme activities or immunoreactivities of these isoforms in hepatic microsomes.

It has been shown that the incorporation of [14C]serine into phosphatidylserine in rat liver microsomal fractions is distinctly stimulated by ATP and Mg2+ at low (10 microM) Ca2+ concentration. This stimulation occurred in untreated microsomes, but did not occur in fully disrupted microsomal vesicles. The stimulatory effect of ATP and Mg2+ on phosphatidylserine synthesis was reduced by calcium ionophore A23187 and by thapsigargin, which is a specific blocker of Ca(2+)-ATPase. It was also diminished by caffeine and enhanced by heparin, agents known to modulate Ca2+ release by receptor channels. It is therefore postulated that the synthesis of phosphatidylserine occurs on the luminal side of the endoplasmic reticulum and can be regulated by events responsible for Ca2+ release and entry into these structures.

Oxidative metabolism of cinnarizine (CZ) and its fluorine derivative flunarizine (FZ), both of which are selective calcium entry blockers, was examined in human liver microsomes. The ring-hydroxylations and the N-desalkylations constituted primary metabolic pathways in microsomal metabolism of CZ and FZ. Among these pathways, the ring-hydroxylase (p-hydroxylation) activities at the cinnamyl moiety of both drugs were highly correlated with debrisoquine 4-hydroxylase activity and CYP2D6 content. Quinidine, a selective inhibitor of CYP2D6, suppressed the ring-hydroxylase activities of CZ and FZ. These results suggest that CYP2D6 is involved in the ring-hydroxylation of the cinnamyl moiety of both CZ and FZ in human liver microsomes.

Liver microsomes from rats pretreated with various inducers of P450 isoforms exhibit very different abilities to catalyze the oxidation of N omega-hydroxy-L-arginine (NOHA) by NADPH and O2 with formation of citrulline and nitrogen oxides. Treatment of rats with dexamethasone, a classical inducer of P450 3A, leads to a spectacular 7-fold increase of the activity found for untreated rats, while induction by phenobarbital causes a much lower increase of this activity and induction by 3-methylcholanthrene or clofibrate decreases it. Specific inhibitors of P450s3A as troleandomycin and dihydroergotamine strongly inhibit NOHA oxidation whereas metyrapone, an inhibitor of other P450 subfamilies, was without effect. These data show the particular ability of P450s of the 3A subfamily to catalyze the second step of the oxidation of L-arginine by NO synthases (NOS). This analogy between NOSs and P450s3A is further substantiated by a protein sequence comparison which shows that a 9-amino acid segment present in all NOSs exhibits a strong similarity with the sequence mainly responsible for heme binding in P450s3A which is well conserved in all P450s. This segment contains all the structural factors which are thought to be crucial for heme binding in P450s.

Cytochrome P450 (CYP450) 2E1 (CYP2E1) is induced by pure ethanol following its chronic administration, and commercial alcoholic beverages, whose major constituent is ethanol, are generally assumed to have a similar effect on this isoform of CYP450. Recently, we serendipitously discovered that beer administered to rats for six weeks had only a minimal inductive effect on hepatic microsomal CYP2E1 activity, while rats on 10% ethanol had CYP2E1 levels five-fold greater than controls. The daily ethanol intake levels for the beer fed and 10% ethanol fed rats were equivalent. In addition, CYP450 spectral features of microsomes from beer fed and ethanol fed rats were markedly different. Spectral examination of microsomes from beer fed rats revealed that about 40% of the total CYP450 content existed in the form of a metabolic intermediate (MI) complex, while no evidence was found for MI complex formation in microsomes of ethanol fed rats. We conclude that beer contains an unidentified component(s) that apparently blocks the typical ethanol induction of CYP2E1 and form an MI complex with CYP450.

The lipid peroxidation of and the O2- generation by rat liver microsomes in the presence of NADPH or both NADPH and Fe3+ were determined by thiobarbituric acid-reacting substance formation and by chemiluminescence intensities with a cypridina luciferin analog, 2-methyl-6-(p-methoxyphenyl)-3, 7-dihydroimidazo[1,2-a]pyrazin-3-one(MCLA), as a chemiluminescence probe. Judging from the experiments with various inhibitors on the O2- generation and the lipid peroxidation, O2- generated, at intramembranous site, by cytochrome P-450 system is considered to be highly involved in the iron-induced lipid peroxidation.

The effects of pyridine on renal cytochrome P4502E1 (CYP2E1) expression in rat have been examined by immunoblot and Northern blot analyses. Immunoblot analyses revealed that 2E1 protein levels were elevated from 1.4- to 4.6-fold following pyridine administration in a dose- and time-dependent manner. Northern blot analyses revealed that renal 2E1 poly(A)+ RNA levels increased from 1.4- to 3.8-fold following pyridine treatment and that these increases in 2E1 mRNA paralleled the dose- and time-dependent increases in 2E1 protein content. In contrast, hepatic 2E1 poly(A)+ RNA levels failed to increase following these same dosing regimens, suggesting that metabolic alterations, such as those associated with starvation, were not etiologic factors in renal 2E1 induction. These results show that pyridine induced CYP2E1 in kidney and that elevation of renal 2E1 protein levels accompanying pyridine administration occurred at least partly as a consequence of increased 2E1 poly(A)+ RNA levels. The results of this research reveal that regulatory mechanisms governing CYP2E1 expression may differ in hepatic and renal tissues.

Microsomal NADPH-driven electron transport is known to initiate lipid peroxidation by activating oxygen in the presence of iron. This pro-oxidant effect can mask an antioxidant function of NADPH-driven electron transport in microsomes via vitamin E recycling from its phenoxyl radicals formed in the course of peroxidation. To test this hypothesis we studied the effects of NADPH on the endogenous vitamin E content and lipid peroxidation induced in liver microsomes by an oxidation system independent of iron: an azo-initiator of peroxyl radicals, 2,2'-azobis (2,4-dimethylvaleronitrile), (AMVN), in the presence of an iron chelator deferoxamine. We found that under conditions NADPH: (i) inhibited lipid peroxidation; (ii) this inhibitory effect was less pronounced in microsomes from vitamin E-deficient rats than in microsomes from normal rats; (iii) protected vitamin E from oxidative destruction; (iv) reduced chromanoxyl radicals of vitamin E homologue with a 6-carbon side-chain, chromanol-alpha-C-6. Thus NADPH-driven electron transport may function both to initiate and/or inhibit lipid peroxidation in microsomes depending on the availability of transition metal catalysts.

Rat liver microsomes catalyze the oxidation of para-hexyloxy-benzamidoxime 1 to the corresponding arylamide 2 and NO2-, by NADPH and O2. Involvement of cytochromes P450 as catalysts of this reaction was shown by the strong inhibitory effects of CO and miconazole and the spectacular increase of the activity upon treatment of rats with dexamethasone, a specific inducer of cytochromes P450 of the 3A subfamily. Formation of NO during oxidation of 1 was shown by detection of the formation of cytochrome P450- and cytochrome P420-Fe(II)-NO complexes by visible and EPR spectroscopy. The formation of these complexes should be responsible, at least in part, for the fast decrease of the rate of microsomal oxidation of 1 with time. These results suggest that exogenous compounds containing amidine or amidoxime functions could act as precursors of NO in vivo after in situ oxidation by cytochromes P450.

Accompanying the decrease in serum cholesterol and increase in concentration of ubiquinone in liver and its microsomes, the activity, but not the protein, of HMG-CoA reductase decreased in ubiquinone-supplemented rats. A soluble 58-kDa preparation of HMG-CoA reductase was partially inhibited on addition of ubiquinone indicating a possible feedback type of action.

HRP catalyzes the oxidation of N omega-hydroxy-L-arginine (NOHA) by H2O2 with formation of citrulline and NO2- with initial rates of about 0.7 and 0.2 nmol per nmol HRP per min. In the same manner, cytochromes P450 from rat liver microsomes catalyze the oxidation of NOHA to citrulline and NO2- by cumylhydroperoxide. Inhibitors of these hemeproteins (N3- and CN- for HRP and miconazole for P450) strongly inhibit both citrulline and NO2- formation. Rates of NOHA oxidation by these hemeproteins markedly decrease with time presumably because of their denaturation by nitrogen oxides and of the formation of hemeprotein-iron-NO complexes. These results suggest that NO (and other nitrogen oxides) could be formed from oxidation of NOHA by other enzymes than NO-synthases.

The effect of musk xylene on contents of both cytochrome P-450IA1 and cytochrome P-450IA2 in rat liver was investigated using Western blotting analysis. Rats were treated i.p. for five consecutive days with either 50, 100 or 200 mg musk xylene/kg body weight. Musk xylene increased both total cytochrome P-450 and cytochrome b5 contents in rat liver microsomes. Musk xylene induced cytochrome P-450IA2 (384 pmol/mg protein) strongly and preferentially and the ratio of cytochrome P450IA2/P-450IA1 was about 12 at the lowest dose tested. Musk xylene also induced the cytochrome P-450IA1 dose-dependently, but these extents were very small (32-174 pmol/mg protein). These results suggest that musk xylene may be a more specific inducer for cytochrome P-450IA2 than any other inducers reported.

Hepatic microsomes from adult male rats representing six inbred strains catalyzed quantitatively significant, NADPH dependent reductions of progesterone to the 20 beta (20R) alcohol and S-warfarin to its 11S-OH product. Microsomes from mature females and immature rats of both sexes were essentially devoid of these activities. Two strains of rat evidenced about 21% of these activities compared with the other strains and both activities were 25-81% repressed by treatment of rats with phenobarbital (PB). An excellent linear correlation was demonstrated for the two activities considering sex, age, NADPH much greater than NADH preference, PB-repression and strain differences. However, detergent latency (71%) and resistance to trypsinolysis were only observed for the keto-reductase activity with S-warfarin. Microsomes also catalyzed the reduction of progesterone to its 20 alpha-OH derivative but this activity preferred NADH greater than NADPH, was induced 2.7-fold by PB and was essentially independent of age, sex and animal strain. Furthermore, unlike the 20 beta-OH activity, this reduction was resistant to proteolytic inactivation.

Controlled proteolytic digestion by trypsin or bacterial proteases limited to the cytosolic side of the native microsomal membrane is not efficient to inhibit glucose-6-phosphate hydrolysis. Modification of the microsomes with deoxycholate prior to protease treatment is prerequisite to allow accessibility of the integral protein and inhibition of enzyme activity. Glucose-6-phosphatase of native microsomes, however, is rapidly inactivated by micromolar concentrations of TPCK as well as TLCK. In deoxycholate-modified microsomes both reagents do not affect glucose-6-phosphate hydrolysis. These results indicate that in the native, intact microsomal membrane glucose-6-phosphatase is not accessible to proteolytic attack from the cytoplasmic surface. The putative inhibitory effect of some trypsin or bacterial protease preparations on glucose-6-phosphatase of native microsomes observed most possibly is a result of contaminating agents as TPCK or TLCK.