Rabbit Liver Cytosol Research Articles

Selenium (Se) as well as mercury (Hg) may influence the methylation and toxicity of inorganic arsenic, but further research is needed with combination of Inorg-arsenic, Se, and Hg

Selenium (Se) as well as mercury (Hg) may influence the methylation and toxicity of inorganic arsenic, but further research is needed with combination of Inorg-arsenic, Se, and Hg

Tissue and interspecies comparison of catechol-O-methyltransferase mediated catalysis of 6-O-methylation of esculetin to scopoletin and its inhibition by entacapone and tolcapone

Catechol-O-methyltransferase (COMT) methylates both endogenous and exogenous catechol compounds to inactive and safe metabolites. We first optimised conditions for a convenient and sensitive continuous fluorescence-based 6-O-methylation assay of esculetin, which we used for investigating the COMT activity in human, mouse, rat, dog, rabbit, and sheep liver cytosols and microsomes and in ten different rat tissues. Furthermore, we compared the inhibition potencies and mechanisms of two clinically used COMT inhibitors, entacapone and tolcapone, in these species. In most tissues, the COMT activity was at least three times higher in cytosol than in microsomes. In the rat, the highest COMT activity was found in the liver, followed by kidney, ileum, thymus, spleen, lung, pancreas, heart, brain, and finally, skeletal muscle. Entacapone and tolcapone were characterised as highly potent mixed type tight-binding inhibitors. The competitive inhibition type dominated over the uncompetitive inhibition with entacapone, whereas uncompetitive inhibition dominated with tolcapone. Rats, dogs, pigs, and sheep are high COMT activity species, in contrast to humans, mice, and rabbits; COMT activity is highest in the liver. Both entacapone and tolcapone are potent COMT inhibitors, but their inhibition mechanisms differ.

Role of N-acetyltransferase 2 acetylation polymorphism in 4, 4′-methylene bis (2-chloroaniline) biotransformation

Arylamine N-acetyltransferase 1 (NAT1) and 2 (NAT2) catalyze the acetylation of arylamine carcinogens. Single nucleotide polymorphisms in the NAT2 coding exon present in NAT2 haplotypes encode allozymes with reduced N-acetyltransferase activity towards the N-acetylation of arylamine carcinogens and the O-acetylation of their N-hydroxylated metabolites. NAT2 acetylator phenotype modifies urinary bladder cancer risk following exposures to arylamine carcinogens such as 4-aminobiphenyl. 4, 4′-methylene bis (2-chloroaniline) (MOCA) is a Group 1 carcinogen for which a role of the NAT2 acetylation polymorphism on cancer risk is unknown. We investigated the role of NAT2 and the genetic acetylation polymorphism on both MOCA N-acetylation and N-hydroxy-MOCA O-acetylation. MOCA N-acetylation exhibited a robust gene dose response in rabbit liver cytosol and in cryopreserved human hepatocytes derived from individuals of rapid, intermediate and slow acetylator NAT2 genotype. MOCA exhibited about 4-fold higher affinity for recombinant human NAT2 than NAT1. Recombinant human NAT2*4 (reference) and 15 variant recombinant human NAT2 allozymes catalyzed both the N-acetylation of MOCA and the O-acetylation of N-hydroxy-MOCA. Human NAT2 5, NAT2 6, NAT2 7 and NAT2 14 allozymes catalyzed MOCA N-acetylation and N-hydroxy-O-acetylation at rates much lower than the reference NAT2 4 allozyme. In conclusion, our results show that NAT2 acetylator genotype has an important role in MOCA metabolism and suggest that risk assessments related to MOCA exposures consider accounting for NAT2 acetylator phenotype in the analysis.

Rabbit N-acetyltransferase 2 genotyping method to investigate role of acetylation polymorphism on N- and O-acetylation of aromatic and heterocyclic amine carcinogens.

The rabbit was the initial animal model to investigate the acetylation polymorphism expressed in humans. Use of the rabbit model is compromised by lack of a rapid non-invasive method for determining acetylator phenotype. Slow acetylator phenotype in the rabbit results from deletion of the N-acetyltransferase 2 (NAT2) gene. A relatively quick and non-invasive method for identifying the gene deletion was developed and acetylator phenotypes confirmed by measurement of N- and O-acetyltransferase activities in hepatic cytosols. Rabbit liver cytosols catalyzed the N-acetylation of sulfamethazine (p=0.0014), benzidine (p=0.0257), 4-aminobiphenyl (p=0.0012), and the O-acetylation of N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP; p=0.002) at rates significantly higher in rabbits possessing NAT2 gene than rabbits with NAT2 gene deleted. In contrast, hepatic cytosols catalyzed the N-acetylation of p-aminobenzoic acid (an N-acetyltransferase 1 selective substrate) at rates that did not differ significantly (p>0.05) between rabbits positive and negative for NAT2. The new NAT2 genotyping method facilitates use of the rabbit model to investigate the role of acetylator polymorphism in the metabolism of aromatic and heterocyclic amine drugs and carcinogens.

Interspecies differences in the metabolism of methotrexate: An insight into the active site differences between human and rabbit aldehyde oxidase

Several drug compounds have failed in clinical trials due to extensive biotransformation by aldehyde oxidase (AOX) (EC 1.2.3.1). One of the main reasons is the difficulty in scaling clearance for drugs metabolised by AOX, from preclinical species to human. Using methotrexate as a probe substrate, we evaluated AOX metabolism in liver cytosol from human and commonly used laboratory species namely guinea pig, monkey, rat and rabbit. We found that the metabolism of methotrexate in rabbit liver cytosol was several orders of magnitude higher than any of the other species tested. The results of protein quantitation revealed that the amount of AOX1 in human liver was similar to rabbit liver. To understand if the observed differences in activity were due to structural differences, we modelled rabbit AOX1 using the previously generated human AOX1 homology model. Molecular docking of methotrexate into the active site of the enzyme led to the identification of important residues that could potentially be involved in substrate binding and account for the observed differences. In order to study the impact of these residue changes on enzyme activity, we used site directed mutagenesis to construct mutant AOX1 cDNAs by substituting nucleotides of human AOX1 with relevant ones of rabbit AOX1. AOX1 mutant proteins were expressed in Escherichia coli. Differences in the kinetic properties of these mutants have been presented in this study.

A 27.368 kDa retinal reductase in New Zealand white rabbit liver cytosol encoded by the peroxisomal retinol dehydrogenase–reductase cDNA: purification and characterization of the enzymeThis paper is one of a selection of papers in this Special Issue, entitled International Symposium on Recent Advances in Molecular, Clinical, and Social Medicine, and has undergone the Journal's usual peer-review process.

We obtained a full-length cDNA based on a sequence deposited in GenBank (accession No. AB045133), annotated as rabbit peroxisomal NADP(H)-dependent retinol dehydrogenase-reductase (NDRD). The rabbit NDRD gene, like its mouse and human homologs, harbors 2 initiation sites, one of which theoretically encodes a 29.6 kDa protein with 279 amino acids, and the other encodes a 27.4 kDa protein with 260 amino acids. The purification of a rabbit cytosolic retinol oxidoreductase with a subunit molecular mass of 34 kDa and an N terminus that is not completely identical to that of NDRD, has been reported. An enzyme responsible for the all-trans retinal reductase activity in the liver cytosol of New Zealand white rabbit was purified to homogeneity using differential centrifugation and successive chromatographic analyses. The subunit molecular mass of the purified enzyme, revealed by SDS-PAGE, was approximately 27 kDa. The intact molecular mass, measured by MALDI-TOF mass spectrometry, was 27.368 kDa. The 60 kDa relative mobility observed in size-exclusion chromatography indicates that the native protein probably exists as a dimer. The purified enzyme was positively confirmed to be the product of NDRD by peptide mass fingerprinting, tandem mass spectrometry, and N-terminal sequencing. Taken together, the results suggested that the native protein is truncated at the N terminus.

Identification of Aldehyde Oxidase as the Neonicotinoid Nitroreductase

Imidacloprid (IMI), the prototypical neonicotinoid insecticide, is used worldwide for crop protection and flea control on pets. It is both oxidatively metabolized by cytochrome P450 enzymes and reduced at the nitroguanidine moiety by a previously unidentified cytosolic "neonicotinoid nitroreductase", the subject of this investigation. Two major metabolites are detected on incubation of IMI with rabbit liver cytosol: the nitrosoguanidine (IMI-NO) and the aminoguanidine (IMI-NH2). Three lines of evidence identify the molybdo-flavoenzyme aldehyde oxidase (AOX, EC 1.2.3.1) as the neonicotinoid nitroreductase. First, classical AOX electron donor substrates (benzaldehyde, 2-hydroxypyrimidine, and N-methylnicotinamide) dramatically increase the rate of formation of IMI metabolites. Allopurinol and diquat are also effective electron donors, while NADPH and xanthine are not. Second, AOX inhibitors (potassium cyanide, menadione, and promethazine) inhibit metabolite formation when N-methylnicotinamide is utilized as an electron donor. Without the addition of an electron donor, rabbit liver cytosol reduces IMI only to IMI-NO at a slow rate. This reduction is also inhibited by potassium cyanide, menadione, and promethazine, as well as by additional AOX inhibitors, cimetidine and chlorpromazine. Finally, IMI nitroreduction by AOX is sensitive to an aerobic atmosphere, but to a much lesser extent than cytochrome P450 2D6. Large species differences are observed in the IMI nitroreductive activity of liver cytosol. While rabbit and monkey (Cynomolgus) give the highest levels of total metabolite formation, human, mouse, cow, and rat also metabolize IMI rapidly. In contrast, dog, cat, and chicken liver cytosols do not reduce IMI at appreciable rates. AOX, as a neonicotinoid nitroreductase, may limit the persistence of IMI, and possibly other neonicotinoids, in mammals.

Effects of antirheumatic gold compounds on the conversion of xanthine dehydrogenase to oxidase in rabbit liver cytosol in vitro

Effects of auranofin (AUR), aurothioglucose (AuTG) and aurothiomalate (AuTM) on the conversion of xanthine dehydrogenase (XD) to oxidase (XO) in the cytosolic fraction from rabbit liver were examined. AUR had no effect on the conversion of XD to XO at concentrations up to 50 μM, whereas at concentrations ranging from 10 to 25 μM, AuTG and AuTM induced the conversion of XD to XO. The constituents of AuTG and AuTM, aurous ion (Au +), but not mercaptosuccinic acid and 1-thio-β-D-glucose, converted XD to XO in a similar degree to AuTG and AuTM. This means that Au (I) moiety has an important role in the AuTG- and AuTM-induced conversion of XD to XO. Furthermore, N-acetyl-L-cysteine (NAC) and British anti-Lewisite (BAL) reconverted AuTG and AuTM-induced XO to XD, implying that clinical activity of NAC and BAL against toxic reactions of AuTG and AuTM is partially due to the XO reconversion. These results suggest that AuTG and AuTM have the potential to convert XD to its reactive oxygen species-generating form, XO, and that this effect may be correlated with cytotoxic actions of these drugs.

Aryl hydrocarbon receptor activation and cytochrome P450 1A induction by the mitogen-activated protein kinase inhibitor U0126 in hepatocytes.

The aryl hydrocarbon receptor (AhR) is involved in various processes such as cytochrome P450 (P450) 1A induction after xenobiotic exposure. It is also considered to play a major role in cell proliferation and differentiation. Recent evidences have suggested a cross-talk between AhR functions and the mitogen-activated protein kinase (MAPK) cascade. We now report that 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126), a specific inhibitor of MAPK kinase (MEK) MEK1/2, elicits a marked increase in CYP1A1 expression at both mRNA and protein levels associated with a significant increase of enzyme activity in primary rat hepatocytes and a human hepatoma cell line. This induction occurred independently of MEK/extracellular signal-regulated kinase (ERK) activation and in the absence of ERK1 and ERK2 expression. The effect of U0126 was mediated by its ability to transactivate xenobiotic responsive element (XRE)-driven genes, as demonstrated by transfection assays with an XRE-driven luciferase construct in the human B16A2 hepatoma cell line. CYP1A1 modulation was abolished by a cotreatment with resveratrol, an established AhR antagonist, arguing for AhR activation by U0126. Such an effect was demonstrated by direct in vitro ligand binding competition assays using rabbit liver cytosol, showing that this compound binds AhR with an EC(50) = 25 x 10(-6) M. Moreover, we demonstrated that U0126 is a substrate for several P450s including human CYP1A2, -1A1, and -1B1. We conclude that the widely used specific inhibitor of MEK/ERK, U0126, also acts as a potent AhR activator and an inducer of related genes. Such effects on the AhR may have an impact on biological functions attributed previously to MAPK inhibition.

Neonicotinoid insecticides: reduction and cleavage of imidacloprid nitroimine substituent by liver microsomal and cytosolic enzymes.

The major insecticide imidacloprid (IMI) is known to be metabolized by human cytochrome P450 3A4 with NADPH by imidazolidine hydroxylation and dehydrogenation to give 5-hydroxy-imidacloprid and the olefin, respectively, and by nitroimine reduction and cleavage to yield the nitrosoimine, guanidine, and urea derivatives. More extensive metabolism by human or rabbit liver microsomes with NADPH or rabbit liver cytosol without added cofactor reduces the IMI N-nitro group to an N-amino substituent, i.e., the corresponding hydrazone. A major metabolite on incubation of IMI in the human microsome-NADPH system is tentatively assigned by LC/MS as a 1,2,4-triazol-3-one derived from the hydrazone; the same product is obtained on reaction of the hydrazone with ethyl chloroformate. The hydrazone and proposed triazolone are considered here together (referred to as the hydrazone) for quantitation. Only a portion of the microsomal reduction and cleavage of the nitroimine substituent is attributable to a CYP450 enzyme. The cytosolic enzyme conversion to the hydrazone is inhibited by added cofactors (NAD > NADH > NADP > NADPH) and enhanced by an argon instead of an air atmosphere. The responsible cytosolic enzyme(s) does not appear to be DT-diaphorase (which is inhibited by several neonicotinoids), aldose reductase, aldehyde reductase, or xanthine oxidase. However, the cytosolic metabolism of IMI is inhibited by several aldo-keto-reductase inhibitors (i.e., alrestatin, EBPC, Ponalrestat, phenobarbital, and quercetin). Other neonicotinoids with nitroimine, nitrosoimine, and nitromethylene substituents are probably also metabolized by "neonicotinoid nitro reductase(s)" since they serve as competitive substrates for [(3)H]IMI metabolism.

Enzymatic and non-enzymatic reduction of brucine N -oxide by aldehyde oxidase and catalase

1. Brucine N -oxide was reduced by aldehyde oxidase in rabbit liver cytosol in the presence of an electron donor, 2-hydroxypyrimidine, under anaerobic conditions. The flavoprotein purified from rabbit liver exhibited significant reductase activity in the presence of electron donors. 2. Brucine N -oxide was also reduced by rabbit liver cytosol and blood in the presence of both a reduced pyridine nucleotide and FAD under anaerobic conditions. The N -oxide reductase activities were inhibited by carbon monoxide and air. However, these activities were not abolished when liver cytosol and blood were boiled. Rabbit erythrocytes exhibited the reductase activity, but not plasma. 3. When liver cytosol or blood was separated by DEAE-cellulose column chromatography, the fractions with the reducing activity in the presence of both NADH and FAD also showed catalase activity. 4. Catalase catalysed the brucine N -oxide reduction in the presence of both NAD(P)H and FAD. Hematin also exhibited the reductase activity in the presence of both NAD(P)H and FAD. Photochemically reduced FAD was effective in the reduction instead of NAD(P)H and FAD. 5. Brucine N -oxide reduction proceeds via two routes in liver cytosol and blood. One is enzymatic reduction by aldehyde oxidase; the other is non-enzymatic reduction catalysed by the haem group of catalase in the presence of reduced flavin.

Monitoring of microcystin-protein phosphatase adduct formation with immunochemical methods

Using anti-microcystin-LR monoclonal antibodies, an immunoblotting procedure was developed to monitor the formation of microcystin-protein phosphatase adducts in vitro and in vivo. The detection limits for the covalent binding of MCYST-LR with the recombinant protein phosphatase 1 (PP1) and rabbit liver cytosol proteins were found to be 0.1 ng and 0.3 ng per assay, respectively. MCYST-PP1 adducts were detected 30 s after the addition of MCYST-LR into the reaction mixture. Reduction of the methyldehydroalanine (Mdha) residue of MCYST-LR with ethanethiol totally abolished the covalent binding of the toxin to PP1, but retained its inhibitory toxicity on PP1. Immunoblotting analyses and enzyme-linked immunosorbant assay showed that between 5 min to 16 h after i.p. injection of single dose (35 μg/kg) of MCYST-LR into mice, approximately 0–27% of the injected toxin was found covalently bound while 0.2–9.2% existed as free form in liver cytosol.

Deamidation of Asparagine Residues in a Recombinant Serine Hydroxymethyltransferase

Serine hydroxymethyltransferase purified from rabbit liver cytosol has at least two Asn residues (Asn5 and Asn220) that are 67 and 30% deamidated, respectively. Asn5 is deamidated equally to Asp and isoAsp, while Asn220 is deamidated only to isoAsp. To determine the effect of these Asn deamidations on enzyme activity and stability a recombinant rabbit liver cytosolic serine hydroxymethyltransferase was expressed in Escherichia coli over a 5-h period. About 90% of the recombinant enzyme could be isolated with the two Asn residues in a nondeamidated form. Compared with the enzyme isolated from liver the recombinant enzyme had a 35% increase in catalytic activity but exhibited no significant changes in either affinity for substrates or stability. Introduction of Asp residues for either Asn5 or Asn220 did not significantly alter activity or stability of the mutant forms. In vitro incubation of the recombinant enzyme at 37°C and pH 7.3 resulted in the rapid deamidation of Asn5 to both Asp and isoAsp with a t1/2 of 50–70 h, which is comparable to the rate found with small flexible peptides containing the same sequence. The t1/2 for deamidation of Asn220 was at least 200 h. This residue may become deamidated only after some unfolding of the enzyme. The rates for deamidation of Asn5 and Asn220 are consistent with the structural environment of the two Asn residues in the native enzyme. There are also at least two additional deamidation events that occur during prolonged incubation of the recombinant enzyme.

Molecular Cloning of Retinal Oxidase/Aldehyde Oxidase cDNAs from Rabbit and Mouse Livers and Functional Expression of Recombinant Mouse Retinal Oxidase cDNA inEscherichia coli

Retinal oxidase (EC 1.2.3.11) is a molybdenum-containing flavoenzyme with high enzymatic activity as to retinoic acid synthesis. In this study, we provide direct evidence that retinal oxidase is identical to aldehyde oxidase (EC 1.2.3.1) by cDNA cloning. Retinal oxidase and aldehyde oxidase, purified from rabbit liver cytosol using the original methods, showed completely identical HPLC patterns and amino acid sequences for three corresponding polypeptides (103 amino residues). The primary structural information obtained from the cleaved polypeptides permitted molecular cloning of the full-length cDNA of rabbit liver retinal oxidase (aldehyde oxidase). We also cloned and sequenced the full-length cDNA of mouse retinal oxidase. The cDNAs of rabbit and mouse retinal oxidase have a common sequence approximately 4.6 kb long, comprising 4-kb coding regions. The open reading frames of the cDNAs predict single polypeptides of 1334 and 1333 amino acids; the calculated minimum molecular mass of each is ∼147,000. Northern blot analysis showed that the rabbit retinal oxidase mRNA was widely expressed in tissues. Finally, we successfully constructed a prokaryotic expression system for mouse retinal oxidase. The purified recombinant retinal oxidase fromEscherichia colishowed a typical spectrum of aldehyde oxidases and a lowerKm(3.8 μM) for retinal and a higherVmax(807 nmol/min/mg protein) for retinoic acid synthesis than those of rabbit retinal oxidase (8 μM and 496 nmol/min/mg protein). This represents the first eukaryotic molybdenum-containing flavoprotein to be expressed in an active form in a prokaryotic system.

Purification and Characterization of Two AmineN-Sulfotransferases, AST-RB1 (ST3A1) and AST-RB2 (ST2A8), from Liver Cytosols of Male Rabbits

Two sulfotransferases (STs), designated as AST-RB1 (ST3A1) and AST-RB2 (ST2A8), with high a amine N-sulfonating activity, were purified from male rabbit liver cytosols. AST-RB1 and AST-RB2 were purified to homogeneity by the anion-exchange, affinity, and hydroxyapatite chromatography. The N-terminus of both enzymes were blocked. The subunit molecular mass of both enzymes was estimated to be 34 kDa on SDS–PAGE. AST-RB1 efficiently catalyzedN-sulfonation of alicyclic, alkyl, and arylamines such as 4-phenyl-1,2,3,6-tetrahydropyridine, 1-[(5-chloro-2-oxo-3(2H)-benzothiazolyl)acetyl]-piperazine, desipramine, and aniline, whereas its catalytic activities toward 2-naphthol and dehydroepiandrosterone (DHEA) were very low. On the other hand, AST-RB2 efficiently catalyzed sulfonation of desipramine and DHEA, but had no activity toward 2-naphthol. Amino acid sequences of peptide fragments derived from the purified AST-RB1 showed no significant homology with previously reported STs, but those from the purified AST-RB2 shared a high similarity with those of the ST2 family. Both enzymes were expressed specifically in the liver. The present results strongly suggest that the purified AST-RB1 is a novel enzyme in terms of structure and catalytic properties showing high selectivity for amine substrates, and AST-RB2 is a quite unique from among ST2A enzymes of other species in its substrate specificity.

In Vitro Methylation of Arsenite by Rabbit Liver Cytosol: Effect of Metal Ions, Metal Chelating Agents, Methyltransferase Inhibitors and Uremic Toxins

ABSTRACTThe methylation of carrier-free 74As-arsenite by liver cytosol of Flemish Giant rabbits is highly susceptible to additions of trace elements. in vitro supplementation of essential trace elements like zinc (Zn2+), vanadium (V5+), iron (Fe2+), copper (Cu2+) and selenate was shown to increase the methylation efficiency. Trivalent metal ions (e.g. Al3+, Cr3+ and Fe3+), Hg2+, Tl+ and SeO32- had a deleterious effect. The inhibitory effect of EDTA, oxime and many divalent cations (Ca2+, Mg2+, Sr2+,.) suggest a co-factor role for a specific divalent metal ion, possibly Zn2+. Chelating agents used in clinical treatment of acute and chronic inorganic arsenic poisoning lower the methylation capacity of cytosol by rendering the trivalent arsenic unavailable for the methyltransferase enzymes. S-adenosylhomocysteine and periodate-oxidized adenosine, inhibitors of s-adenosylmethionine dependent methylation pathways, inhibit the methylation of arsenite. Pyrogallol, a catechol-O-methyltransferase inhibitor, blocks the action of arsenite- and monomethylarsonic methyltransferase enzymes, suggesting a close structural relationship between the active sites of the different enzymes. Some uraemic toxins, namely oxalate, p-cresol, hypoxanthine, homocysteine and myo-inositol, inhibit arsenic methylation.

Purification and characterization of two major forms of naloxone reductase from rabbit liver cytosol, new members of aldo-keto reductase superfamily.

Rabbit liver cytosol produced approximately equal amounts of 6alpha-naloxol and 6beta-naloxol from naloxone in the presence of NADPH at pH 7.4, and contained at least four forms of naloxone reductase. The two major forms, NR1 and NR2, which catalyze the stereospecific reduction of naloxone to 6alpha-naloxol and 6beta-naloxol, respectively, were purified to apparent homogeneity by various chromatographic techniques. Both enzymes are monomeric proteins with similar molecular weights of 35000-36000, but NR1 is a basic protein with an isoelectric point (pI) of 9.3, while NR2 is an acidic protein (pI of 5.9). NR1 and NR2 gave the maximal activities at pH 8.0 and 6.1, respectively. NR1 exhibited considerable activity with NADH as well as with NADPH, whereas NR2 showed highly restricted specificity for NADPH. The Km and Vmax values of NR1 and NR2 for naloxone were 1.0 and 0.06 mM, and 76 and 162 munits/mg, respectively. In addition to naloxone, naltrexone and dihydromorphinone served as good substrates for NR2 but were poor substrates for NR1. Both enzymes reduced aliphatic and aromatic aldehydes, cyclic and aromatic ketones, and quinones at higher rates. The two enzymes catalyzed the dehydrogenation of 17beta-hydroxysteroids with low Km values, and NR2 showed an additional 3alpha-hydroxysteroid dehydrogenase activity. Amino acid sequence data of NRI (99% of whole protein) and NR2 (66%) showed that both enzymes belong to the aldo-keto reductase (AKR) superfamily and can be classified into the AKR1C subfamily. These findings therefore indicate that they are new members of the AKR superfamily and may be involved physiologically in the steroid metabolism as well as in the detoxification of xenobiotic carbonyl compounds.

Purification, Characterization and Partial Primary Structure of Morphine 6-Dehydrogenase from Rabbit Liver Cytosol

Morphinone, a toxic metabolite, was formed from morphine by NAD(P)-dependent morphine 6-dehydrogenase(s) in both the cytosol and microsomal fractions of the rabbit liver at pH 7.4. The enzyme activity in the cytosol fraction was about twice that in the microsomal fraction and NAD served as the preferred cofactor in both fractions. The enzyme in the cytosol fraction was purified to a homogeneous protein by the use of various chromatographic techniques. The enzyme is a monomeric protein with a molecular weight of 36,000 and an isoelectric point of 6.4. The enzyme had a dual cofactor specificity but NAD was more efficiently utilized than NADP. With NAD, the enzyme showed an optimal pH of 9.4, and theKmandVmaxvalues toward morphine were 0.72 mmand 0.59 unit/mg protein, respectively. The enzyme also exhibited a significant activity for morphine analogs having an unsaturated bond at C-7,8 (codeine, ethylmorphine, and normorphine), alicyclic alcohols (3-hydroxyhexobarbital, 1-indanol, and cyclohexene-2-ol) and benzenedihydrodiol. In the reverse reaction, the enzyme exhibited highly restricted specificity foro-quinones. Sulfhydryl reagents and quercetin inhibited the enzyme but pyrazole, barbital, and indomethacin had little effect on the enzyme activity. Androstanes, lithocholic acid, and estradiol potently inhibited the enzyme in a competitive manner toward morphine binding. The partial amino acid sequence of the random peptides obtained by the proteolytic digestion of the enzyme, which comprised about 40% of the whole protein, revealed a significant homology to the corresponding regions in the members of the aldo-keto reductase family. These results therefore indicate that the present enzyme is a new and unique member of the aldo-keto reductase family.

Purification and characterization of a novel cytosolic NADP(H)-dependent retinol oxidoreductase from rabbit liver

Rabbit liver cytosol exhibits very high retinol dehydrogenase activity. At least two retinol dehydrogenases were demonstrated to exist in rabbit liver cytosol, and the major one, a cytosolic NADP(H)-dependent retinol dehydrogenase (systematic name: retinol oxidoreductase) was purified about 1795-fold to electrophoretic and column chromatographic homogeneity by a procedure involving column chromatography on AF-Red Toyopearl twice and then hydroxyapatite. Its molecular mass was estimated to be 34 kDa by SDS-PAGE, and 144 kDa by HPLC gel filtration, suggesting that it is a homo-tetramer. The enzyme uses free retinol and retinal, and their complexes with CRBP as substrates in vitro. The optimum pH values for retinol oxidation of free retinol and CRBP-retinol were 8.8–9.2 and 8.0–9.0, respectively, and those for retinal reduction of free retinal and retinal-CRBP were the same, 7.0–7.6. K m for free retinol and V max for retinal formation were 2.8 μM and 2893 nmol/min per mg protein at 37°C (pH 9.0) and the corresponding values with retinol-CRBP as a substrate were 2.5 μM and 2428 nmol/min per mg protein at 37°C (pH 8.6); K m for free retinal and V max for retinol formation were 6.5 μM and 4108 nmol/min per mg protein, and the corresponding values with retinal-CRBP as a substrate were 5.1 μM and 3067 nmol/min per mg protein at 37°C, pH 7.4. NAD(H) was not effective as a cofactor. 4-Methylpyrazole was a weak inhibitor (IC 50=28 mM) of the enzyme, and ethanol was neither a substrate nor an inhibitor of the enzyme. This enzyme exhibits relatively broad aldehyde reductase activity and some ketone reductase activity, the activity for aromatic substitutive aldehydes being especially high and effective. Whereas, except in the case of retinol, oxidative activity toward the corresponding alcohols was not detected. This novel cytosolic enzyme may play an important role in vivo in maintaining the homeostasis of retinal, the substrate of retinoic acid synthesis, at least in rabbit liver, since a high concentration of retinol in liver and the lower K m of the enzyme for retinol force the oxidative reaction, while higher activity of retinal reductase at physiological pH forces the reductive reaction.

Identification of a Putative Effector for Cdc42Hs with High Sequence Similarity to the RasGAP-related Protein IQGAP1 and a Cdc42Hs Binding Partner with Similarity to IQGAP2

Cdc42 is a Ras-related GTP-binding protein that has been implicated in the regulation of the actin cytoskeleton and cell morphology. In this study, we have identified a protein with a molecular mass approximately 180 kDa from rabbit liver cytosol (designated p180), which binds preferentially to the GTP- and guanosine 5'-3-O-(thio)triphosphate-bound forms of Cdc42. Binding of p180 to GTP-bound Cdc42 maintains it in the GTP-bound state. Another cytosolic protein, with an apparent molecular mass of 175 kDa (p175), was also found to interact with Cdc42, but this association showed less dependence on guanine nucleotides. Both p180 and p175 were capable of binding to Rac1 but not to RhoA or Ha-Ras. The limit functional domain of the Cdc42-GAP protein did not compete with p180 or p175 for binding to Cdc42. However, the Cdc42-binding domain from mPAK-3, a member of the PAK (p21 activated kinase) family of serine/threonine kinases, competed with both proteins. The binding of p180 or p175 was inhibited by mutations of the putative effector loop of Cdc42. p180 and p175 also bound less effectively to a Cdc42/Ras chimera in which loop 8 from Ras was substituted for the predicted loop 8 in Cdc42 that includes a 13-amino acid insert present in all Rho family members but absent in Ras. Microsequencing of a p180 peptide revealed 92% identity with the human IQGAP1 protein, while two peptides derived from p175 were 89 and 100% identical to human IQGAP2. These findings identify IQGAP1 and IQGAP2 as a new class of target/effectors that utilize both regions of the switch I domain and an insert region distinct to Rho proteins for binding to Cdc42.