Effects of flavin-binding motif amino acid mutations in the NADH-cytochrome b5 reductase catalytic domain on protein stability and catalysis.

Porcine NADH-cytochrome b5 reductase catalytic domain (Pb5R) has the RXY(T/S)+(T/S) flavin-binding motif that is highly conserved among the structurally related family of flavoprotein reductases. Mutations were introduced that alter the Arg(63), Tyr(65), and Ser(99) residues within this motif. The mutation of Tyr(65) to either alanine or phenylalanine destabilized the protein, produced an accelerated release of FAD in the presence of 1.5 M guanidine hydrochloride, and decreased the k(cat) values of the enzyme. These results indicate that Tyr(65) contributes to the stability of the protein and is important in the electron transfer from NADH to FAD. The mutation of Ser(99) to either alanine or valine, and of Arg(63) to either alanine or glutamine increased both the K(m) values for NADH (K(m)(NADH)) and the dissociation constant for NAD(+) (K(d)(NAD+)). However, the mutation of Ser(99) to threonine and of Arg(63) to lysine had very little effect on the K(m)(NADH) and K(d)(NAD+) values, and resulted in small changes in the absorption and circular dichroism spectra. These results suggest that the hydroxyl group of Ser(99) and the positive charge of Arg(63) contribute to the maintenance of the properties of FAD and to the effective binding of Pb5R to both NADH and NAD(+). In addition, the mutation of Arg(63) to either alanine or glutamine increased the apparent K(m) values for porcine cytochrome b5 (Pb5), while changing Arg(63) to lysine did not. The positive charge of Arg(63) may regulate the electron transfer through the electrostatic interaction with Pb5. These results substantiate the important role of the flavin-binding motif in Pb5R.

Molecular basis of perinatal changes in UDP-glucuronosyltransferase activity in maternal rat liver.

The molecular basis of perinatal changes occurring in major UDP-glucuronosyltransferase (UGT) family 1 isoforms and in UGT2B1, a relevant isoform belonging to family 2, was analyzed in rat liver. Nonpregnant, pregnant (19-20 days of pregnancy), and two groups of postpartum animals corresponding to early and middle stages of lactation (2-4 and 10-12 days after delivery, respectively) were studied. UGT activity determined in UDP-N-acetylglucosamine-activated microsomes revealed that bilirubin, p-nitrophenol, and ethynylestradiol (17beta-OH and 3-OH) but not androsterone and estrone glucuronidation rates, were decreased in pregnant rats. Decreased enzyme activities returned to control values after delivery. p-Nitrophenol, androsterone, and estrone conjugation rate increased in postpartum rats. Western blot analysis performed with anti-peptide-specific (anti-1A1, 1A5, 1A6, and 2B1) antibodies revealed decreased levels of all family 1 isoforms and UGT2B1 during pregnancy. In postpartum animals, protein level recovered (1A5 and 2B1) or even increased (1A1 and 1A6) with respect to control rats. Northern blot analysis suggested that expression of UGT proteins is down-regulated at a post-translational level during pregnancy and that increased levels of 1A1 and 1A6 observed in postpartum rats were associated to increased mRNA. To establish whether prolactin is involved in up-regulation of UGT1A1 and 1A6 postpartum, ovariectomized rats were treated with 300 microg of ovine prolactin per day for 7 days. The data indicated that prolactin was able to increase expression of UGT1A6 (protein and mRNA) but not 1A1. Thus, prolactin is the likely mediator of the increased expression of UGT1A6 observed in maternal liver postpartum.

One-electron reduction of quinones by the neuronal nitric-oxide synthase reductase domain.

Flavin electron transferases can catalyze one- or two-electron reduction of quinones including bioreductive antitumor quinones. The recombinant neuronal nitric oxide synthase (nNOS) reductase domain, which contains the FAD-FMN prosthetic group pair and calmodulin-binding site, catalyzed aerobic NADPH-oxidation in the presence of the model quinone compound menadione (MD), including antitumor mitomycin C (Mit C) and adriamycin (Adr). Calcium/calmodulin (Ca2+/CaM) stimulated the NADPH oxidation of these quinones. The MD-mediated NADPH oxidation was inhibited in the presence of NAD(P)H:quinone oxidoreductase (QR), but Mit C- and Adr-mediated NADPH oxidations were not. In anaerobic conditions, cytochrome b5 as a scavenger for the menasemiquinone radical (MD*-) was stoichiometrically reduced by the nNOS reductase domain in the presence of MD, but not of QR. These results indicate that the nNOS reductase domain can catalyze a only one-electron reduction of bivalent quinones. In the presence or absence of Ca2+/CaM, the semiquinone radical species were major intermediates observed during the oxidation of the reduced enzyme by MD, but the fully reduced flavin species did not significantly accumulate under these conditions. Air-stable semiquinone did not react rapidly with MD, but the fully reduced species of both flavins, FAD and FMN, could donate one electron to MD. The intramolecular electron transfer between the two flavins is the rate-limiting step in the catalytic cycle [H. Matsuda, T. Iyanagi, Biochim. Biophys. Acta 1473 (1999) 345-355). These data suggest that the enzyme functions between the 1e- <==> 3e- level during one-electron reduction of MD, and that the rates of quinone reductions are stimulated by a rapid electron exchange between the two flavins in the presence of Ca2+/CaM.

A 66-base-pair enhancer module activates the expression of a distinct isoform of UDP-glucuronosyltransferase family 1 (UGT1A2) in primary hepatocytes.

UGT1A2, an isoform of the UDP-glucuronosyltransferase family 1 (UGT1), is not expressed in the rat liver, but its expression was highly induced in primary cultures of rat hepatocytes. In primary hepatocytes that had been cultured for 70 h, the amount of UGT1A2 mRNA was 100 times higher than that in the rat liver. Deletion analysis of a 4.8-kb promoter region of the UGT1A2 gene revealed that a 66-nucleotide region between -307 and -242 upstream of the transcription start site was required for induction of UGT1A2 expression. The 66-nucleotide region acted on a heterologous promoter in a manner independent of its position and orientation in reporter constructs. Gel mobility shift assay showed that a specific binding protein to this region appeared in the nuclei of cultured hepatocytes, but was not present in the rat liver. DNase I protection analysis revealed the existence of a CTGGCAC core sequence between -274 and -268 of the UGT1A2 promoter. Methylation interference assay showed that the guanine residues at -294 and -287 on the upper strand and the guanine residue at -267 on the lower strand as well as the core sequence were required for the DNA-protein interaction. These results suggest that the 66-nucleotide region, which was designated culture-associated expression responsive enhancer module (CEREM), interacts with a specific nuclear protein and enhances the expression of UGT1A2 in cultured hepatocytes.

Calmodulin activates intramolecular electron transfer between the two flavins of neuronal nitric oxide synthase flavin domain.

The neuronal NO synthase (nNOS) flavin domain, which has similar redox properties to those of NADPH-cytochrome P450 reductase (P450R), contains binding sites for calmodulin, FAD, FMN, and NADPH. The aim of this study is to elucidate the mechanism of activation of the flavin domain by calcium/calmodulin (Ca(2+)/CaM). In this study, we used the recombinant nNOS flavin domains, which include or delete the calmodulin (CaM)-binding site. The air-stable semiquinone of the nNOS flavin domains showed similar redox properties to the corresponding FAD-FMNH(&z.ccirf;) of P450R. In the absence or presence of Ca(2+)/CaM, the rates of reduction of an FAD-FMN pair by NADPH have been investigated at different wavelengths, 457, 504 and 590 nm by using a stopped-flow technique and a rapid scan spectrophotometry. The reduction of the oxidized enzyme (FAD-FMN) by NADPH proceeds by both one-electron equivalent and two-electron equivalent mechanisms, and the formation of semiquinone (increase of absorbance at 590 nm) was significantly increased in the presence of Ca(2+)/CaM. The air-stable semiquinone form of the enzyme was also rapidly reduced by NADPH. The results suggest that an intramolecular one-electron transfer between the two flavins is activated by the binding of Ca(2+)/CaM. The F(1)H(2), which is the fully reduced form of the air-stable semiquinone, can donate one electron to the electron acceptor, cytochrome c. The proposed mechanism of activation by Ca(2+)/CaM complex is discussed on the basis of that provided by P450R.

Chemical modification of rat hepatic microsomes with N-ethylmaleimide results in inactivation of both UDP-N-acetylglucosamine-dependent stimulation of glucuronidation and UDP-glucuronic acid uptake.

Chemical modification of rat hepatic microsomes with N-ethylmaleimide (NEM) resulted in inactivation of UDP-N-acetylglucosamine (UDP-GlcNAc)-dependent stimulation of glucuronidation of p-nitrophenol. Inactivation kinetics and pH dependence were in agreement with the modification of a single sulfhydryl group. NEM also inactivated the uptake of UDP-glucuronic acid (UDP-GlcUA) but not UDP-glucose. With various sulfhydryl-modifying reagents, the inactivation of UDP-GlcUA uptake was linked to that of glucuronidation. UDP-GlcUA protected against NEM-sensitive inactivation of both UDP-GlcNAc-dependent stimulation of glucuronidation and UDP-GlcUA uptake, suggesting that the sulfhydryl group is located within or near the UDP-GlcUA binding site of the microsomal protein involved in the stimulation. Using microsomes labeled with biotin-conjugated maleimide and immunopurification with anti-peptide antibody against UDP-glucuronosyltransferase family 1 (UGT1) isozymes, immunopurified UGT1s were found to be labeled with the maleimide and UDP-GlcUA protected against the labeling as it did with the NEM-sensitive inactivation. These data suggest the involvement of a sulfhydryl residue of microsomal protein in the UDP-GlcNAc-dependent stimulation mechanism via the stimulation of UDP-GlcUA uptake into microsomal vesicles.

Systematic mutations of highly conserved His49 and carboxyl-terminal of recombinant porcine liver NADH-cytochrome b5 reductase solubilized domain.

The cDNA encoding solubilized porcine liver NADH-cytochrome b5 reductase catalytic domain (Pb5R) was cloned and overexpressed in Escherichia coli. A highly conserved His49 and a C-terminal Phe272 of Pb5R, which are located near the isoalloxazine moiety of the FAD, were systematically modulated by site-directed mutagenesis. Large structural change was not detected on the absorption and circular dichroism spectra of mutant proteins. Drastic changes in enzymatic properties were not observed, but the apparent Km value for soluble form of porcine liver cytochrome b5 (Pb5) was affected by the substitutions of His49 with glutamic acid and with lysine, deletion of C-terminal Phe272, and addition of Gly273. The values of the catalytic constant (kcat) were obviously decreased by the substitution of His49 with glutamic acid or the addition of Gly273. In these two mutants, the rate for reduction of FAD was decreased, and the rate for autoxidation of reduced FAD was increased. These results showed that His49 and C-terminal carboxyl group in Pb5R are not critical for the electron transfer to Pb5, but the electrostatic environmental changes at these positions could affect the recognition of Pb5 and modulate the catalytic function of the enzyme by changing the stability of reduced FAD.

Biochemical and molecular aspects of genetic disorders of bilirubin metabolism.

Bilirubin, the oxidative product of heme in mammals, is excreted into the bile after its esterification with glucuronic acid to polar mono- and diconjugated derivatives. The accumulation of unconjugated and conjugated bilirubin in the serum is caused by several types of hereditary disorder. The Crigler-Najjar syndrome is caused by a defect in the gene which encodes bilirubin UDP-glucuronosyltransferase (UGT), whereas the Dubin-Johnson syndrome is characterized by a defect in the gene which encodes the canalicular bilirubin conjugate export pump of hepatocytes. Animal models such as the unconjugated hyperbilirubinemic Gunn rat, the conjugated hyperbilirubinemic GY/TR-, and the Eisai hyperbilirubinemic rat, have contributed to the understanding of the molecular basis of hyperbilirubinemia in humans. Elucidation of both the structure of the UGT1 gene complex, and the Mrp2 (cMoat) gene which encodes the canalicular conjugate export pump, has led to a greater understanding of the genetic basis of hyperbilirubinemia.

The UDP glycosyltransferase gene superfamily: recommended nomenclature update based on evolutionary divergence.

This review represents an update of the nomenclature system for the UDP glucuronosyltransferase gene superfamily, which is based on divergent evolution. Since the previous review in 1991, sequences of many related UDP glycosyltransferases from lower organisms have appeared in the database, which expand our database considerably. At latest count, in animals, yeast, plants and bacteria there are 110 distinct cDNAs/genes whose protein products all contain a characteristic 'signature sequence' and, thus, are regarded as members of the same superfamily. Comparison of a relatedness tree of proteins leads to the definition of 33 families. It should be emphasized that at least six cloned UDP-GlcNAc N-acetylglucosaminyltransferases are not sufficiently homologous to be included as members of this superfamily and may represent an example of convergent evolution. For naming each gene, it is recommended that the root symbol UGT for human (Ugt for mouse and Drosophila), denoting 'UDP glycosyltransferase,' be followed by an Arabic number representing the family, a letter designating the subfamily, and an Arabic numeral denoting the individual gene within the family or subfamily, e.g. 'human UGT2B4' and 'mouse Ugt2b5'. We recommend the name 'UDP glycosyltransferase' because many of the proteins do not preferentially use UDP glucuronic acid, or their nucleotide sugar preference is unknown. Whereas the gene is italicized, the corresponding cDNA, transcript, protein and enzyme activity should be written with upper-case letters and without italics, e.g. 'human or mouse UGT1A1.' The UGT1 gene (spanning > 500 kb) contains at least 12 promoters/first exons, which can be spliced and joined with common exons 2 through 5, leading to different N-terminal halves but identical C-terminal halves of the gene products; in this scheme each first exon is regarded as a distinct gene (e.g. UGT1A1, UGT1A2, ... UGT1A12). When an orthologous gene between species cannot be identified with certainty, as occurs in the UGT2B subfamily, sequential naming of the genes is being carried out chronologically as they become characterized. We suggest that the Human Gene Nomenclature Guidelines (http://www.gene.acl.ac.uk/nomenclature/guidelines.html++ +) be used for all species other than the mouse and Drosophila. Thirty published human UGT1A1 mutant alleles responsible for clinical hyperbilirubinemias are listed herein, and given numbers following an asterisk (e.g. UGT1A1*30) consistent with the Human Gene Nomenclature Guidelines. It is anticipated that this UGT gene nomenclature system will require updating on a regular basis.

Protein-protein interactions between UDP-glucuronosyltransferase isozymes in rat hepatic microsomes.

The interactions between UDP-glucuronosyltransferase (UGT) isozymes, UGT1s and UGT2B1, in rat hepatic microsomes were investigated using an immunopurification technique with anti-peptide antibodies and a chemical cross-linking strategy. A 50 kDa protein coimmunopurified with UGT1s was identified as UGT2B1 by amino-terminal sequencing and immunodetection with anti-peptide antibody against UGT2B1. Evidence for direct interaction of UGT2B1 with UGT1s was obtained by the loss of UGT2B1 adsorption to immunoaffinity column in Gunn rat hepatic microsomes, which lack all UGT1 isozymes. When the microsomes were treated with the chemical cross-linking reagent 1,6-bis(maleimido)-hexane, a cross-linked product with an apparent molecular mass of 120-130 kDa was obtained that immunostained with antibodies against UGT1s and UGT2B1, indicating the formation of a heterodimer containing one of the UGT1 isozymes and UGT2B1. The effects of UGT complex formation on the stimulation of glucuronidation of testosterone and uptake of UDP-glucuronic acid (UDP-GlcUA) by UDP-N-acetylglucosamine (UDP-GlcNAc) were examined. Alkaline pH-induced dissociation of the complexes was associated with the loss of UDP-GlcNAc-dependent stimulation of glucuronidation, suggesting that two functional states of UGTs with different kinetic parameters correspond to the monomer and oligomer form of UGTs in the membranes. The UDP-GlcNAc-dependent stimulation of UDP-GlcUA uptake into the microsomal vesicles also was affected by the extent of complex formation. These results suggest that complex formation of the UGT isozymes affects the UDP-GlcNAc-dependent stimulation of glucuronidation via stimulation of UDP-GlcUA uptake.

Xenobiotic responsive element-mediated transcriptional activation in the UDP-glucuronosyltransferase family 1 gene complex.

We have isolated genomic DNA clones containing rat UDP-glucuronosyltransferase family 1 (UGT1) sequences and have shown drug-responsive and tissue-specific alternative expression of multiple first exons (Emi, Y., Ikushiro, S., and Iyanagi, T. (1995) J. Biochem. (Tokyo) 117, 392-399). The UGT1 locus encodes at least nine UGT1 isoforms. UGT1A1 is a major 3-methylcholanthrene (MC)-inducible form in rat liver. In this report, we have identified a cis-acting element necessary for transcriptional activation of UGT1A1 in hepatocytes. A promoter region was fused to a chloramphenicol acetyltransferase gene, and the resultant construct was transiently transfected into hepatocytes. A DNA fragment carrying 1,100 nucleotides derived from the 5'-flanking region of the UGT1A1 gene was enough for MC induction. Unidirectional deletion of this region revealed that there existed one xenobiotic responsive element (XRE), TGCGTG, between -134 and -129. When a single base substitution was introduced into the XRE, MC-induced expression of the UGT1A1 gene was completely abolished. In addition, an XRE-deleted construct failed to respond to MC. Gel mobility shift assays showed MC-inducible binding of the nuclear aromatic hydrocarbon receptor-ligand complex to this motif. Gel shift-coupled DNase I protection analyses revealed that the GCGTG-core sequence was a target site of the liganded aromatic hydrocarbon receptor. These results suggest that the XRE participates in induction of the rat UGT1A1 gene by MC.

Identification and analysis of drug-responsive expression of UDP-glucuronosyltransferase family 1 (UGT1) isozyme in rat hepatic microsomes using anti-peptide antibodies.

Expression of rat hepatic UDP-glucuronosyltransferase family 1 (UGT1) isozymes has been examined using anti-peptide antibodies raised against a conserved carboxyl-terminal portion of all isozymes and variable amino-terminal portions of each isozyme of the phenol cluster (UGT1A) and bilirubin cluster (UGT1B). Among the isozymes expressed in rat hepatic microsomes, UGT1B1 (54 kDa) of bilirubin cluster was found to be a major form and minor forms were identified as UGT1A1 (53 kDa), UGT1B2 (56 kDa), and UGT1B5 (57 kDa). Using a combination of 2D sodium dodecyl sulfate gel electrophoresis and immunoblotting, all the isozymes were found to be simultaneously lacked in Gunn rat hepatic microsomes. The effects of various drugs as inducer on the expression of each UGT1 isozyme were analyzed. The UGT1A1 and UGT1A2 of the phenol cluster isozymes were significantly induced in 3-methylcholanthrene-treated rats. The expression of UGT1B1 and the glucuronidation activity toward bilirubin in rat hepatic microsomes were induced two- to threefold by clofibrate and dexamethasone administration. On the other hand, the regulation of UGT1B2 and UGT1B5 expression was different from that of UGT1B1. These results clearly show the drug-responsive expression of each UGT1 isozyme using isozyme-specific antibodies for the first time.

cDNA cloning of a putative G protein-coupled receptor from brain.

Using degenerate oligonucleotide primers corresponding to conserved regions of the G-protein coupled receptor superfamily, we carried out a low-stringency polymerase chain reaction and obtained two novel partial-length clones from a rat brain cDNA library. We used one of these clones for conventional library screening and isolated a longer cDNA clone, designated as RBU-15, from another rat brain library. Although RBU-15 was truncated at its 5' end, Northern blot analysis revealed that the gene was expressed in the brain and spleen. Next, we isolated a full-length cDNA clone, designated as HB-954, from a human fetal brain library, using RBU-15 as a probe. The deduced amino acid sequence of HB-954 contained four putative glycosylation sites in the N-terminal part, seven transmembrane domains, and a large cytosolic domain in the C-terminal part. The protein products of RBU-15 and HB-954 likely belong to a distinctive subfamily, because no receptors in the superfamily were found to be closely related to them.

Drug-responsive and tissue-specific alternative expression of multiple first exons in rat UDP-glucuronosyltransferase family 1 (UGT1) gene complex.

Genomic clones of UDP-glucuronosyltransferase family 1 (UGT1) were isolated from wild-type Wistar rats. The UGT1 locus spans > 120 kb and forms a gene complex. In this locus nine unique first exons encoding NH2-terminal portions of each isoform were located at intervals of approximately 10 kb and followed by only one set of commonly used exons (exons II, III, IV, and V) encoding the COOH-terminal portion. From sequence analyses of the unique first exons, the amino acid sequences of the isoforms were deduced and they were divided into two groups: the Bilirubin cluster (B cluster) and the Phenol cluster (A cluster). A and B clusters consisted of four (A1-A4) and five (B1-B5) isoform-specific exons, respectively. A2, A3, B3, and B4 were identified as previously uncharacterized forms, while A4 and B4 were pseudogenes. Isoform B1 was a major component in hepatic microsomes of untreated rats and was induced in clofibrate- and dexamethasone-administered rats. A slight but a significant amount of B1 mRNA was also detected in various tissues such as intestine. mRNAs coding for isoform A1 and isoform A2 were induced in livers of methylcholanthrene (MC)-treated rats. Induction of A1 mRNA was also observed in kidneys of MC-treated rats. A genomic clone containing the commonly used exons was also isolated from Gunn rats and a single base deletion was identified in exon IV. Isoforms of the UGT1 family are made from the complex gene locus by an alternative combination of one of the unique first exons with the commonly used exons.

Glucuronidation of carcinogen metabolites by complementary DNA-expressed uridine 5'-diphosphate glucuronosyltransferases.

Five UDP glucuronosyltransferases (UGT) were synthesized from complementary DNAs expressed in COS 7 cells and were tested for their capacities to glucuronidate a range of 2-acetylaminofluorene and benzo(a)pyrene-hydroxylated metabolites. Three forms, UGT1*06, UGT2B1, and UGT2B2 [names of UGT forms follow recommended nomenclature (B. B. Burchell et al., DNA Cell Biol., 10: 487-494, 1991)], had similar capacities to glucuronidate the reactive metabolite, N-hydroxy-2-acetylaminofluorene. The less reactive 1-, 3-, 5-, and 8-hydroxy derivatives of this aromatic amine were glucuronidated by UGT1*06 and UGT2B2 to varying degrees, but these were not substrates of UGT2B1. The three isozymes also glucuronidated phenolic metabolites of benzo(a)pyrene. UGT1*06 was more active toward 2- and 5-hydroxybenzo(a)pyrene, whereas UGT2B1 preferentially glucuronidated the 4- and 11-hydroxy derivatives and UGT2B2 preferentially glucuronidated the 1-, 2-, 8-, and 9-hydroxy metabolites. Two other UDP glucuronosyltransferases, UGT2B3 and UGT2B6, that glucuronidated testosterone when expressed in COS 7 cells were both inactive toward all the carcinogen metabolites tested. These results demonstrate that the glucuronidation of metabolites of 2-acetylaminofluorene and benzo(a)pyrene is mediated by at least three UDP glucuronosyltransferases and that each form glucuronidates a unique spectrum of metabolites.

Superoxide-producing cytochrome b. Enzymatic and electron paramagnetic resonance properties of cytochrome b558 purified from neutrophils.

Molecular properties of superoxide (O2-)-producing cytochrome b558 purified from neutrophils were investigated focusing on the mechanism of the catalytic reaction. The purified cytochrome, which was depleted of FAD, exhibited high O2(-)-generating activity with consumption of NADPH in the presence of microsomal NADPH-cytochrome P-450 reductase. Exogenous additions of CO, CN-, or N3- had no effect on the enzymatic activity. Potentiometric titration of the ferric-ferrous couple of the cytochrome showed that the midpoint reduction potential was -255 mV at pH 7.4. When the reaction of the reduced cytochrome with O2 was analyzed by stopped flow and rapid scanning spectrophotometry, the ferrous form was found to be converted to the ferric form at a rate constant of 9.3 x 10(6) M-1 s-1 at 10 degrees C without showing formation of an oxygenated intermediate. EPR measurement of the ferric cytochrome at 10 K showed that the electronic spin state was in a low spin with g values of 3.2, 2.05, and 1.5. These results suggest that the heme in a six-coordinated low spin state catalyzes one electron reduction of O2 without ligation of O2 to the heme iron during the catalytic cycle.

Intracellular localization of UDP-glucuronosyltransferase expressed from the transfected cDNA in cultured cells.

Liver UDP-glucuronosyltransferase (UDPGT) is localized in the endoplasmic reticulum (ER), with its catalytic domain exposed to the lumenal side of the membrane structure. The proteins expressed from the transfected UDPGT cDNA in cultured cells were found to be localized in the ER membrane. Its enzyme activity was in a latent state and was fully expressed in an in vitro assay system when the membrane integrity was disrupted by a detergent, Triton X-100, suggesting that the orientation of the expressed enzyme in the membrane was the same as that of the liver enzyme. To investigate how the expressed UDPGT was retained in the ER, we constructed chimeric plasmids of cDNAs of UDPGT and ErbB2 which is a receptor protein localized in the cell membranes. Analysis of chimeric proteins expressed in the stable transformants of the cultured cells transfected with these plasmids to reveal that the cytoplasmic tail of UDPGT is responsible for the ER retention of the expressed proteins. Deletion and mutation analysis in the cytoplasmic tail of the enzyme demonstrated that the two lysine residues positioned at 3 and 5 from the C-terminus of the molecule are important for conferring the ER residency. Furthermore, the distance of the ER retention signal composed of the two lysine residues from the transmembrane domain may be influential for the efficiency of the ER retention activity.

Effect of chronic hypoxia on detoxication enzymes in rat liver.

Studies were performed to determine the effects of chronic hypoxia on enzymes that catalyze various detoxication reactions. Rats were exposed to room air or 10.5% O2 for 10 days, and microsomes and postmicrosomal supernatants were isolated from liver. Detoxication enzyme activities were measured by radiochemical and spectrophotometric assays, and immunoreactive protein amounts were measured by Western blot analysis. Total cytochrome P450, as measured by the CO-difference spectrum, and activities of superoxide dismutase (EC 1.15.1.1), epoxide hydrolase (EC 4.2.1.63), catalase (EC 1.11.1.6), glutathione disulfide reductase (EC 1.6.4.2), and glutathione (GSH) S-transferase (EC 2.5.1.18) were not affected by this extent of hypoxia. In contrast, 10 days of hypoxia decreased activities or immunoreactivities (% of aerobic) of GSH peroxidase (EC 1.11.1.9) (54%), cytochrome P450EtOH2 (42%), CYP3A1 (53%), sulfotransferase (EC 2.8.2.1) (77%) and UDP-glucuronosyltransferase (EC 2.4.1.17) (65%). Activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49), an important enzyme in NADPH production was also decreased to 56% of the aerobic value, but Western blot analysis showed that the amount of protein reactive with antibodies to glucose-6-phosphate dehydrogenase was not affected by hypoxia. Thus, hypoxia may decrease activity of enzymes by regulatory mechanisms even though the amount of immuno-detectable enzyme is unchanged. Liver cells isolated from rats exposed to hypoxia also gave lower GSH synthetic rates than cells from normoxic rats. This result, together with the effect of hypoxia on glucose-6-phosphate dehydrogenase, indicates that the GSH supply for GSH-dependent detoxication reactions may be limited due to chronic hypoxia. To test directly whether chronic hypoxia increased sensitivity to a compound normally detoxified by a GSH-dependent reaction, sensitivity to tert-butyl hydroperoxide (t-BuOOH) of hepatocytes from rats exposed to in vivo hypoxia was compared to that from normoxic rats. The results showed that the cells from the hypoxic rats were much more sensitive to injury. Taken together, these results suggest that decreases in amounts and/or activities of detoxication enzymes during chronic hypoxia may result in increased susceptibility of cells to chemical injury.