Substrate specificity and enantioselectivity of arylcarboxylic acid glucuronidation

A general and sensitive HPLC method using a precolumn switching system was developed for the separation and quantification of the individual diastereoisomeric glucuronides of the 2-phenylpropionic acid optical isomers. Kinetic properties of rat liver glucuronidation of several arylcarboxylic acids (1- and 2-naphthylacetic acids, clofibric acid, (R)-(-)- and (S)-(+)-2-phenylpropionic acids) are characterized. The results show that rat liver microsomes glucuronidate 1-naphthylacetic acid more efficiently than its regioisomer (higher Vmax/Km ratio because of a 6-fold lower Km value). Furthermore, 2-phenylpropionic acid glucuronidation occurs stereoselectively and is characterized by an enantiomeric ratio R/S = 1.60. Specific inducers of different UDP-glucuronosyltransferase isoforms and the Gunn rat strain are used to define the substrate specificity of the conjugation reaction towards arylcarboxylic aglycones. Acyl glucuronide formation is induced by phenobarbital. Gunn rats are not deficient in conjugation of these arylcarboxylic acids. These results indicate that these compounds behave similarly to classically defined group 2 substrates. In addition, the stereospecificity of 2-phenylpropionic acid conjugation is unchanged by pretreatment of animals with inducers, in vitro detergent activation, and enantiomeric inhibition. This suggests that the optical isomers of 2-phenylpropionic acid can be either conjugated by the same form or very closely regulated forms of UDPGT.     

Inhibition studies of microsomal UDP-glucuronosyltransferase activities by furosemide and salicylamide

Contrarily to cytochrome P-450, a few inhibitors of UDP-glucuronosyltransferase have been described. We verified the nature of the in vitro inhibition due to furosemide, using 4 different aglycones (morphine, p-nitrophenol, borneol and eugenol) presumably belong may to different clusters of UDP-glucuronosyltransferase activities. The variations of these corresponding kinetic parameters (Km, Vmax, specific activities) must correspond to different inhibition mechanisms of furosemide, for example different site(s) of fixation in the area of the active site of UDPGT. Because these variations were not as classically described, we checked the impact of furosemide pretreatment on in vitro levels of different UDPGT activities. We compared these result, with another inhibitor (salicylamide). The apparent induction due to the both molecules enforced the hypothesis of a complexe inhibition mechanism.     

The effect of malaria infection on 3'-azido-3'-deoxythymidine and paracetamol glucuronidation in rat liver microsomes.

The effect of malaria infection on UDP-glucuronosyltransferase (UDPGT) activity was investigated in rat liver microsomes using 3'-azido-3'-deoxythymidine and paracetamol. The Michaelis-Menten parameters, Km and Vmax were calculated and intrinsic clearance values were estimated for normal and infected livers. The results show that malaria infection alters the activity of UDPGT.     

Mercury-induced UDP-glucuronyltransferase (UDPGT) activity in mouse kidney

Administration of mercuric chloride to young adult mice produced a significant increase in the activity of renal UDP-glucuronyltransferase (UDPGT) measured with harmol as the acceptor substrate. This was observed 10 days after a daily oral dose of HgCl2 (6 micrograms Hg2+/g body wt.). The increase in UDPGT activity was correlated with an accumulation of mercury in the renal tissues and was accompanied by an increase in the apparent Vmax of the glucuronidation reaction without a change in the apparent Km values for harmol or UDPGA. Parallel studies with mercuric sulfide however showed negligible retention of mercury in both the liver or kidney nor was there any change in UDPGT activity compared to control values. The difference in solubilities of the two mercuric salts may be responsible for this observation. The possible mode of activation of UDPGT by mercury treatment is discussed.     

Heterogeneity of hepatic microsomal UDP-glucuronosyltransferase activities: Conjugations of phenolic and monoterpenoid aglycones in control and induced rats and guinea pigs

In this report we present evidence that the heterogeneity of hepatic microsomal UDP-glucuronosyltransferase(s) (UDPGT) activities depends on the chemical structures of the aglycones as well as their biophysical constants. Three animal models were used: Wistar rats, which have active UDPGTs; Gunn rats, in which some of the UDPGT activities are reduced, but which can be induced by phenobarbital; and guinea pigs. In Wistar rats, we found that some coumarins were poor substrates of UDPGT (GT₁) and that twenty monoterpenoid alcohol activities showed typical phenobarbital-inducible behavior. In Gunn rats, we showed that substitution of the phenolic aglycone by bulky (alkyl-or methoxy-) groups in the 2-position of the phenolic ring decreased UDPGT (GT₁) activity, whereas substitution in the 4-position resulted in an increase in this activity. We also showed that, in this particular strain, activities toward terpenes were less affected than activities toward flat (aromatic) aglycomes. Induction by phenobarbital in Gunn rats increased the activity and limited the deficiency for monoterpenoid alcohols. In guinea pigs, we confirmed that phenobarbital selectively increased the activities of UDPGT towards twenty monoterpenoid alcohols without affecting other typical phenobarbital-induced activities such as those for conjugation of morphine. Finally, we showed that orientation of the aglycone molecule in the active site was apparently related to its dipole moment and that the distance between “acceptor-oxygen” (hydroxyl) and the carbons out of the general plane of the molecule was an important factor. These studies clearly suggest that rat and guinea pig contain a UDPGT (monoterpenoid alcohols) with restricted specificities and also that UDPGT (GT₁) comprises at least two or three different isoenzymes, each with a slightly different restricted specificity flat aromatic aglycones.     

Glucuronidation of 3'-azido-3'-deoxythymidine catalyzed by human liver UDP-glucuronosyltransferase. Significance of nucleoside hydrophobicity and inhibition by xenobiotics

The enzymatic glucuronidation of 3'-azido-3'-deoxythymidine (AZT) catalyzed by human liver microsomal UDP-glucuronosyltransferase (EC 2.4.1.17, UDPGT) was inhibited by a number of nucleoside analogs. The inhibitory potency of these nucleoside analogs correlated with their hydrophobicity (r2 = 0.90, N = 13). Since similar results were obtained with solubilized UDPGT (r2 = 0.87, N = 7), the affinity of the nucleosides for UDPGT was probably being assessed rather than the ability of the compounds to access the membrane-bound enzyme. Three homologous inhibitors, 3'-azido-2',3'-dideoxyuridine (AzddU), 5-ethyl-AzddU, and 5-propyl-AzddU, were also studied as substrates of UDPGT. The substrate efficiency (Vmax/Km) of these three compounds and AZT also correlated with their hydrophobicity (r2 = 0.94). Sixteen drugs that are structurally unrelated to nucleosides also inhibited the glucuronidation of AZT. The mechanism of inhibition was competitive for seven compounds tested. Ki values were estimated from Dixon plots for nine other less soluble inhibitors; their mechanism of inhibition was assumed to be competitive. Since the peak physiological drug concentrations of the tested inhibitors are considerably less than their Ki values, none of these compounds are expected to strongly inhibit AZT glucuronidation in humans. However, the rank order of these drugs with respect to their inhibitory potential is probenecid greater than chrloramphenicol greater than naproxen greater than phenylbutazone much greater than other drugs tested.     

Kinetic characteristics of UDP-glucuronosyltransferases towards a dithiol metabolite of malotilate in hepatic microsomes of rats and rabbits

1. The kinetic activity of UDP-glucuronosyltransferases (UDPGT) towards a dithiol metabolite of malotilate, 2,2-di(isopropoxycarbonyl)ethylene-1,1-dithiol, was investigated using rat and rabbit hepatic microsomes. The thio-glucuronide formed was analysed by h.p.l.c. The Km values obtained using rat and rabbit UDPGT were 36.3 +/- 3.3 and 443 +/- 43 microM, respectively. The Vmax values were 7.14 +/- 0.61 and 29.2 +/- 6.4 nmol/min per mg (mean +/- SD, n = 3). 2. Phenobarbital, an inducer of the GT2 isoform of UDPGT, increased rat microsomal UDPGT activity towards the dithiol. In inhibitory studies, menthol and borneol (specific substrates for GT2a isoform) competitively inhibited glucuronidation of the dithiol. Thus it was concluded that formation of the thio-glucuronide was catalysed mainly by the GT2a isozyme of UDPGT, which is involved in glucuronidation of monoterpenoid alcohols.     

Microsomal UDP-glucuronyltransferase in rat liver: oxidative activation

Activation of microsomal UDP-glucuronyltransferase (UDPGT) activity by treatment of hepatic microsomes with either detergents or Fe(3+)/ascorbate pro-oxidant system has been reported; however, definite mechanisms underlying these effects have not been clarified. In this work, we characterize Fe(3+)/ascorbate-induced activation of UDPGT activity prior to solubilization with Triton X-100 and after the oxidation process provoked the solubilization of the enzyme. We observed a time-dependent increase in UDPGT activity up to 20 min. incubation of the microsomes with Fe(3+)/ascorbate (3-times); after 20 min. incubation, however, we observed a time-dependent decrease in this activity to basal levels after 4 hr incubation. Treatment of microsomes with 0.1% Triton X-100 (5 min.) lead to a similar increase in UDPGT activity; higher detergent concentrations produced a dose-dependent decrease in this activity to basal levels with 1% Triton X-100. Interestingly, UDPGT activity was susceptible to activation only when associated to microsomal membranes and the loss of activation correlated with the solubilization of this activity. UDPGT activation by either Fe(3+)/ascorbate or Triton X-100 was correlated with an increase in p-nitrophenol apparent K(m) and V(max) values. This activation was prevented or reversed by the reducing agents glutathione, cysteine or dithiothreitol when it was induced by the Fe(3+)/ascorbate. Furthermore, the latter provoked a significant decrease in microsomal thiol content, effect not observed after treatment with Triton X-100. Our results suggest that the main mechanism responsible for Fe(3+)/ascorbate-induced UDPGT activation is likely to be the promotion of protein sulfhydryl oxidation; this mechanism appears to be different from detergent-induced UDPGT activation.     

Suppression of aromatase activity in vitro by PCBs 28 and 105 and Aroclor 1221

The effects of polychlorinated biphenyls (PCBs) on human cytochrome P450 aromatase activity in vitro were investigated using a commercially available microsomal fraction obtained from baculovirus infected insects that had been transfected with the human CYP19 gene and cytochrome P450 reductase. The assay measured the conversion of tritiated testosterone to estradiol in Tris buffer at pH 7.4. When aroclors, commercial preparations of PCBs, were added to aromatase assays at a 10 microM concentration, Aroclor 1221 caused a reduction in the aromatase activity, whereas other aroclors (1016, 1232, 1242, 1248, 1254, 1260, 5432, 5442 and 5460) were without effect. Further investigation of the effect of Aroclor 1221 on aromatase activity showed that the inhibition was dose dependent. When a reconstituted mixture (RM) of PCBs that represented the congeneric content of human milk was investigated, no inhibition of aromatase activity at the maximum treatment of 15.0 microM was observed. None of the congeners present in the reconstituted mixture, except PCB 28 and 105, affected P450 arom activity. PCB 28 showed a statistically significant inhibition of aromatase activity (P<0.05) at 1.5 and 15 microM and a significant inhibition of aromatase activity by PCB 105 was also observed, but only at 15 microM. In three separate kinetic analyses the Km(app) for aromatase was 64, 89 and 69 nM (mean 74 nM). In addition, PCB 28 resulted in an increase in the Km(app) without a significant effect on Vmax(app), suggesting competitive inhibition by this congener. This conclusion was supported by slope (Km(app)/Vmax(app) versus [inhibitor]) and intercept (1/Vmax(app) versus [inhibitor]) replots. The slope replots gave Ki(app) values for PCB 28 of 0.9, 1.3 and 2.0 microM (mean 1.4 microM), whereas intercept replots were almost horizontal. Thus, PCB 28 is a competitive inhibitor of aromatase with a Ki(app) value approximately 20-fold the Km(app) value. Based on these studies, we conclude that most PCBs are not inhibitors of aromatase activity in vitro. However, as being inhibitors of aromatase activity, Aroclor 1221, PCB 28 and PCB 105 would remain a priority for further study as possible endocrine disrupters.     

水胺硫磷在大鼠肝微粒体的生物转化和代谢动力学

目的 探讨水胺硫磷在大鼠肝微粒体的体外代谢活化产物及代谢动力学特征。方法 应用液相色谱-四级杆-飞行时间质谱(LC/QTOF MS)筛查并鉴定水胺硫磷在大鼠肝微粒体孵育液中的氧化产物。用乙酰胆碱酯酶抑制法考察水胺硫磷及其氧化产物水胺氧磷的抑酶活性。应用液相色谱-三重四级杆串联质谱(LC/Triple-Q MS/MS)定量检测肝微粒体中的水胺硫磷及其代谢产物水胺氧磷,研究水胺硫磷及其产物的消长动力学和氧化产物生成的酶动力学。结果 筛查并鉴定了水胺硫磷在大鼠肝微粒体的氧化脱硫产物水胺氧磷。水胺氧磷对乙酰胆碱酯酶的抑制活性远高于水胺硫磷,IC₅₀值比水胺硫磷低4个数量级,表明水胺硫磷的氧化脱硫反应是一个代谢活化过程。水胺硫磷在大鼠肝微粒体中的半衰期(t_1/2)为14.6 min,外推得到体内肝清除率Cl_H为43.8 ml·min^-1·kg^-1。水胺氧磷的生成符合双相酶动力学模型,K_m,app1为1.12 μmol·L^-1,产物生产最大速率(V_max1)为0.43 μmol·min^-1·g ^-1;蛋白K_m,app2为67.92 μmol·L^-1,V_max2为1.28 μmol·min^-1·g ^-1蛋白。结论 水胺硫磷在大鼠肝微粒体中能快速代谢消除,生成抑酶活性更高的产物水胺氧磷而产生毒性。     

Relationship between sulfotransferase activity and susceptibility to acetaminophen-induced liver necrosis in the hamster

The effect of pretreatments which modulate acetaminophen sulfotransferase activity on the hepatotoxicity of acetaminophen have been examined in the hamster. Co-administration of sodium sulfate modestly enhanced the formation of acetaminophen sulfate, but provided little protection against liver injury. In isolated hepatocyte studies, sodium sulfate enhanced the Vmax of acetaminophen sulfotransferase activity, but did not alter the apparent Km toward acetaminophen. Administration of 2,6-dichloro-4-nitrophenol with acetaminophen selectively depressed acetaminophen sulfate formation in vivo and significantly exacerbated acetaminophen hepatotoxicity. In kinetic studies using isolated hamster hepatocytes, 2,6-dichloro-4-nitrophenol competitively inhibited acetaminophen sulfotransferase with a Ki of 2.5 X 10(-6) M. The data indicate that in the hamster, acetaminophen sulfotransferase activity plays a relatively minor role in the modulation of acetaminophen hepatotoxicity, and that, at hepatotoxic doses, the capacity limitation on this enzyme system is determined to a greater extent by its Km (app) value than by limitation in cofactor (3'-phosphoadenosine 5'-phosphosulfate) availability.     

Evidence that tacrolimus augments the bioavailability of mycophenolate mofetil through the inhibition of mycophenolic acid glucuronidation

We previously reported an unexpected augmentation of mycophenolic acid (MPA) levels (trough and AUC0-12) in patients receiving mycophenolate mofetil (MMF) in combination with tacrolimus versus patients receiving the same dose of MMF in combination with cyclosporin A (CsA). This finding was accompanied by a corresponding reduction of the inactive glucuronide metabolite of MPA (MPAG) in patients, suggesting that tacrolimus may effect the conversion of MPA to MPAG by the enzyme UDP-glucuronosyltransferase (UDPGT). To investigate this possibility directly, UDPGT was extracted from human liver and kidney tissue and its activity was characterized using MPA as a substrate in vitro, assessing the conversion of MPA to MPAG using analysis by high-performance liquid chromatography. With crude microsomal preparations, amounts of UDPGT at least 100 times higher in specific activity (i.e., units to milligrams of protein) could be extracted per gram of tissue from kidney as opposed to liver. This result did not appear to be related to the coextraction of a liver-specific UDPGT inhibitor because initial enzyme kinetic values (Vmax and km) were identical for kidney and liver extracts, and further purification of the liver enzyme did not enhance activity (as is seen when inhibitors are removed during purification). With further UDPGT purification (approximately 200-fold) from kidney extracts using a combination of ammonium sulfate precipitation, followed by anion exchange, hydroxyapatite, and size exclusion chromatography, the enzyme was more than 80% pure when assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Initial enzyme kinetic analysis of this purified product showed a km value for MPA of 35.4+/-5.7 microg/mL and a Vmax of 2.87+/-0.31 MPAG produced per hour (n = 7). The addition of clinically relevant concentrations of CsA (200-1,000 ng/mL) or tacrolimus (10-25 ng/mL) resulted in a dose-dependent inhibition of the UDPGT enzyme by both agents with tacrolimus, which was approximately 60-fold more efficient as an inhibitor. The calculated inhibition constants (KI) of tacrolimus and CsA for the purified UDPGT were 27.3+/-5.6 ng/ml and 2,518+/-1473 ng/ml. respectively. Both agents displayed an inhibition profile characteristic of a competitive inhibitor (substrate) that could be demonstrated in a reciprocal experiment with CsA as a substrate, but not with tacrolimus. This finding suggested that the significantly more efficient inhibition of UDPGT by tacrolimus may occur by a more complicated mechanism that is yet to be determined.     

Kinetic modeling of beta-chloroprene metabolism: I. In vitro rates in liver and lung tissue fractions from mice, rats, hamsters, and humans.

Beta-chloroprene (2-chloro-1,3-butadiene, CD) is carcinogenic by inhalation exposure to B6C3F1 mice and Fischer F344 rats but not to Wistar rats or Syrian hamsters. The initial step in metabolism is oxidation, forming a stable epoxide (1-chloroethenyl)oxirane (1-CEO), a genotoxicant that might be involved in rodent tumorigenicity. This study investigated the species-dependent in vitro kinetics of CD oxidation and subsequent 1-CEO metabolism by microsomal epoxide hydrolase and cytosolic glutathione S-transferases in liver and lung, tissues that are prone to tumor induction. Estimates for Vmax and Km for cytochrome P450-dependent oxidation of CD in liver microsomes ranged from 0.068 to 0.29 micromol/h/mg protein and 0.53 to 1.33 microM, respectively. Oxidation (Vmax/Km) of CD in liver was slightly faster in the mouse and hamster than in rats or humans. In lung microsomes, Vmax/Km was much greater for mice compared with the other species. The Vmax and Km estimates for microsomal epoxide hydrolase activity toward 1-CEO ranged from 0.11 to 3.66 micromol/h/mg protein and 20.9 to 187.6 microM, respectively, across tissues and species. Hydrolysis (Vmax/Km) of 1-CEO in liver and lung microsomes was faster for the human and hamster than for rat or mouse. The Vmax/Km in liver was 3 to 11 times greater than in lung. 1-CEO formation from CD was measured in liver microsomes and was estimated to be 2-5% of the total CD oxidation. Glutathione S-transferase-mediated metabolism of 1-CEO in cytosolic tissue fractions was described as a pseudo-second order reaction; rates were 0.0016-0.0068/h/mg cytosolic protein in liver and 0.00056-0.0022 h/mg in lung. The observed differences in metabolism are relevant to understanding species differences in sensitivity to CD-induced liver and lung tumorigenicity.     

Characterization and regulation of atrial natriuretic peptide (ANP)-R1 receptors in the human neuroblastoma cell line NB-OK-1.

We characterized in membranes from the human neuroblastoma cell line NB-OK-1, an ANP-R1 receptor (Mr 130 kDa) for the atrial natriuretic peptide (ANP). This receptor recognized biologically active forms of ANP with high affinity but showed no affinity for truncated ANP forms. It was functional in that binding correlated with guanylate cyclase activation (a 2-fold increase in Vmax) with the following rank order of potency: rat ANP-(99-126) greater than human ANP-(99-126) greater than human ANP-(102-126) greater than porcine BNP (brain natriuretic peptide). The enzyme required free Mn2+ in addition to the Mn-GTP substrate (Km of about 0.3 mM for both basal and ANP-stimulated activity). In the presence of dithiothreitol, the dose-response curve of guanylate cyclase activation was shifted rightward by a factor of 30. ANP-R1 receptors were upregulated through protein synthesis in cells exposed to 1 mM carbamylcholine or 1 mM dibutyryl cyclic AMP for 8-24 h (ANP was ineffective).     

白首乌C21甾苷元C11α-羟化的两种微生物同步转化

目的对白首乌C21甾苷元告达庭甾苷元和开德甾苷元（Ⅱ）实现C11α-羟基化,要求副产物少,收率高。方法采用黑根霉（Rhizopus nigricans）和犁头霉（Absidia sp.）两种微生物在水-正丁醇双相体系中同步转化的方法,羟化反应中控制值pH在5.0～6.0,于30℃温度下振荡培养70h。结果产物经IR、MS、NMR分析确认是C11α-羟基化了的告达庭甾苷元（Ⅲ）和开德甾苷元（Ⅲ）,副产物少,收率达70.1%。结论用黑根霉（Rhizopus nigricans）和犁头霉（Absidia sp.）两种微生物在水-正丁醇双相体系中同步转化的方法是实现C11α-羟基化的有效方法。     

Effects of coexpression of UGT1A9 on enzymatic activities of human UGT1A isoforms.

We established stable HEK293 cell lines expressing double isoforms, UGT1A1 and UGT1A9, UGT1A4 and UGT1A9, or UGT1A6 and UGT1A9, as well as stable cell lines expressing each single isoform. To analyze the protein-protein interaction between the UGT1As, we investigated the thermal stability and resistance to detergent. UGT1A9 uniquely demonstrated thermal stability, which was enhanced in the presence of UDP-glucuronic acid (>90% of control), and resistance to detergent. Interestingly, UGT1A1, UGT1A4, and UGT1A6 acquired thermal stability and resistance to detergent by the coexpression of UGT1A9. An immunoprecipitation assay revealed that UGT1A6 and UGT1A9 interact in the double expression system. Using the single expression systems, it was confirmed that estradiol 3-O-glucuronide, imipramine N-glucuronide, serotonin O-glucuronide, and propofol O-glucuronide formations are specific for UGT1A1, UGT1A4, UGT1A6, and UGT1A9, respectively. By kinetic analyses, we found that the coexpressed UGT1A9 significantly affected the kinetics of estradiol 3-O-glucuronide formation (decreased Vmax), imipramine N-glucuronide formation (increased Km and Vmax), and serotonin O-glucuronide formation (decreased Vmax) catalyzed by UGT1A1, UGT1A4, and UGT1A6, respectively. On the other hand, the coexpressed UGT1A1 increased Km and decreased the Vmax of the propofol O-glucuronide formation catalyzed by UGT1A9. The coexpressed UGT1A4 and UGT1A6 also increased the Vmax of the propofol O-glucuronide formation by UGT1A9. This is the first study showing that human UGT1A isoforms interact with other isoforms to change the enzymatic characteristics.     

Relationship between forskolin and calcium-calmodulin stimulation of rat cerebral cortex adenylate cyclase: enzyme activation modulates substrate (MgATP) affinity

In membranes prepared from the rat cerebral cortex EGTA 0.4-100 mumol/l was found to dose-dependently inhibit adenylate cyclase activity, both during basal conditions and when the cyclase activity had been stimulated with 10 mumol/l forskolin. Addition of calcium 2-30 mumol/l (in excess of EGTA) totally prevented the inhibition induced by EGTA, both in the absence and presence of forskolin. These data are consistent with the notion that in the absence of EGTA the rat cortex adenylate cyclase is considerably stimulated by endogenous calcium-calmodulin. The data also show that stimulation with endogenous calcium-calmodulin is more than additive with that of forskolin indicating an action on identical cyclase units. The kinetics of the rat cortex adenylate cyclase for its substrate (MgATP) was also investigated. Activation of the cyclase with endogenous calcium-calmodulin, induced a marked increase in the Vmax of the enzyme and a concomitant almost 2-fold increase in the Km. Stimulation with 10 microM forskolin also induced a marked increase in the Vmax as well as an almost 3-fold increase in the Km. A combination of the two stimulants caused a further increase in Vmax as well as Km; the Vmax being increased more than 17-fold and the Km being increased 3.5-fold over the basal. The data indicates that both calcium-calmodulin and forskolin induces a similar type of activation of the adenylate cyclase which is associated with a lowered affinity for its substrate.     

Formation and transport of xenobiotic glutathione-S-conjugates in red cells

In vitro studies with freshly drawn human erythrocytes showed 4-dimethylaminophenol, a cyanide antidote, to be rapidly metabolized with the formation of a transient S,S-(2-dimethylamino-5-hydroxy-1,3-phenylene)bis-glutathione conjugate and a stable S,S,S-(2-dimethylamino-5-hydroxy-1,3,4-phenylene)tris-glutathione conjugate. The stable tri-glutathionyl derivative was actively transported across the red cell membrane with an apparent Vmax = 1 nmol/min/ml red cell suspension (15 g hemoglobin/100 ml) and Km = 0.5 mM. The transport system was strictly unidirectional, inhibited completely by sodium fluoride and reduced to one-fifth by lowering the temperature from 37 to 22 degrees. Similarly S-(2,4-dinitrophenyl)-glutathione, the glutathione-S-transferase mediated glutathione-S-conjugate with 1-chloro-2,4-dinitrobenzene, was unidirectionally transported, a process which was inhibited by sodium fluoride. Kinetic analysis revealed two different transport processes: Vmax = 0.9 nmol/min/ml, Km = 1.4 microM and Vmax = 4.5 nmol/min/ml, Km = 700 microM. Mutual inhibition of the low affinity transport system was found for both glutathione-S-conjugates. The apparent energies of activation for all these transport processes and for GSSG were identical (70 kJ/mol) suggesting at least one common carrier for the excretion of the three glutathione-S-conjugates.     

The cyanide-metabolizing enzyme rhodanese in human nasal respiratory mucosa

The cyanide-metabolizing enzyme rhodanese is present in rat nasal epithelium at high activity levels. Cyanide is a common environmental pollutant. It is both toxic and an odorant. The high rhodanese activity in rat nasal epithelium may provide a mechanism for detoxicating inhaled hydrogen cyanide and may also play a role in olfaction by limiting the concentrations of cyanide in the nasal epithelium. The objective of this study was to determine whether high levels of rhodanese activity are also present in human nasal epithelium. On a per milligram mitochondrial protein basis, the rhodanese in human nasal tissue exhibited both a lower affinity (higher Km) for cyanide and a lower maximum velocity (Vmax) for cyanide metabolism than did rhodanese from rat nasal tissue. As in human liver, the human nasal enzyme appeared to exhibit substrate activation by cyanide. Rhodanese activity in the maxilloturbinates of nonsmokers was statistically higher than in smokers although only three samples per group were available. The Vmax/Km ratios for rhodanese from the nasal tissue of nonsmokers were consistently greater, thus suggesting the possibility of higher rates of cyanide metabolism in nonsmokers than in smokers.     

Evidence for a saturable, energy-dependent and carrier-mediated uptake of oral antidiabetics into rat hepatocytes.

The hepatic uptake of two sulfonylureas, glisoxepide and glibenclamide, was investigated in isolated rat hepatocytes. Two transport processes were defined: passive physical diffusion and saturable carrier transport. For diffusion at pH 7.4 the permeability coefficients were 3.3 x 10(-6) cm/s for glisoxepide and 10.6 x 10(-6) cm/s for glibenclamide. Saturable uptake differed among the sulfonylureas. Glibenclamide uptake was neither energy- nor sodium-dependent and temperature dependence was linear. The apparent activation energy for saturable glibenclamide uptake was 15.2 kJ/mol and Q10 values for uptake between 7 and 37 degrees C were 1.17 +/- 0.12. Saturable glibenclamide uptake exhibited a Km = 3.1 microM and a Vmax = 416 pmol/mg cell protein per min. Thus glibenclamide uptake was defined kinetically as a facilitated diffusion process. Glisoxepide uptake revealed two Km values: Km1 = 2-3 microM and Vmax1 = 200 pmol/mg protein per min, and Km2 = 110 microM and Vmax2 = 1600 pmol/mg protein per min. Uptake at low and high substrate concentration was energy-dependent, sodium-dependent and was inhibited by ouabain. Temperature dependence increased markedly beyond 22 degrees C and the apparent activation energy was 59.7 kJ/mol at low Km1 glisoxepide concentrations and 60.3 kJ/mol at high Km2 concentrations. Whereas glisoxepide was slowly taken up into AS-30D hepatoma cells, glibenclamide was not. The hepatic uptake of glibenclamide was not inhibited by glisoxepide but glibenclamide inhibited glisoxepide uptake. The inhibition by glibenclamide was noncompetitive. Isolated hepatocytes accumulated the sulfonylureas markedly and metabolized both. The metabolized radioligands were slowly released into the incubation buffer. The results indicate that the hepatic uptake of the two sulfonylureas is by carrier-mediated transport. The uptake processes are, however, strikingly different, indicating heterogeneity of sulfonylurea transporters.