Oxidation of 20-hydroxyleukotriene B4 to 20-carboxyleukotriene B4 by human neutrophil microsomes. Role of aldehyde dehydrogenase and leukotriene B4 omega-hydroxylase (cytochrome P-450LTB omega) in leukotriene B4 omega-oxidation

Leukotriene B4 (LTB4), a potent chemoattractant for leukocytes, is catabolized by human neutrophils via omega-oxidation. Neutrophil microsomes are known to oxidize 20-hydroxy-LTB4 (20-OH-LTB4) to its 20-oxo and 20-carboxy derivatives in the presence of NADPH. This activity has been ascribed to LTB4 omega-hydroxylase (cytochrome P-450LTB omega), a conclusion supported by our finding of the reversal of carbon monoxide inhibition by 450 nm light and by competitive inhibition studies. The oxidation of 20-oxo-LTB4 to 20-carboxy-LTB4 is also catalyzed by microsomes fortified with 1 mM NAD+, and this activity is not affected by cytochrome P-450LTB omega inhibitors. The evidence is compatible with involvement of a disulfiram-insensitive aldehyde dehydrogenase in this second oxidation pathway. Interaction of the two pathways is evidenced by facilitation of NADPH-dependent oxidation of 20-OH-LTB4 by the addition of NAD+. This synergism may be explained by removal of the aldehyde intermediate by the NAD(+)-dependent aldehyde dehydrogenase. Taken together with the finding that the NAD(+)-dependent activity is severalfold higher than the NADPH-dependent one, the dehydrogenase may be important in the oxidation of 20-OH-LTB4 to 20-carboxy-LTB4.     

Acetaldehyde-dependent oxidation of glutathione catalyzed by rat liver cytosol

We have identified a novel reaction in which acetaldehyde promotes rat hepatic cytosolic catalysis of O2 consumption coupled with glutathione oxidation without apparent release of activated forms of O2. Acetaldehyde is not consumed in the reaction. The reaction (O2 consumption or oxidized glutathione production) is saturable with respect to varying glutathione (K'm congruent to 20-45 microM) but not at high acetaldehyde concentrations. However, activity in the range of acetaldehyde found in liver from alcohol metabolism (10-100 microM) appeared to be saturable (K'm congruent to 25-50 microM). Since neither acetaldehyde-dependent glutathione loss nor O2 consumption is detectable in guinea pig hepatic cytosol or hepatic cytosol from selenium-deficient rats, we propose that acetaldehyde interacts with glutathione peroxidase, converting the enzyme into a glutathione oxidase.     

NAD+-dependent retinol dehydrogenase in liver microsomes

A microsomal NAD+-dependent retinol dehydrogenase is being described with optimal activity at physiological pH. The enzyme was present in liver microsomes of rats and also in a strain of deermice which lacks the cytosolic retinol dehydrogenase. Unlike the latter enzyme, the microsomal retinol dehydrogenase was not inhibited by either ethanol or 4-methylpyrazole; its activity was insensitive to CO and not oxygen dependent, in contradistinction with that of the microsomal cytochrome P-450 and NADPH-dependent retinol oxidase. Chronic ethanol consumption resulted in an increased activity of the microsomal retinol dehydrogenase which may contribute to hepatic retinol depletion, especially in view of the insensitivity of the enzyme to ethanol inhibition.     

Identification and characterization of Mg2+-dependent phosphotyrosyl protein phosphatase from rat liver cytosol

Although highly purified preparations of Mg2+-dependent phosphoseryl protein phosphatase (also designated phosphatase IA or phosphatase 2C) dephosphorylated phosphotyrosyl histone, the activity has been resolved from phosphatase IA by polyacrylamide gel electrophoresis at pH 9.5. This novel phosphotyrosyl-specific protein phosphatase absolutely requires Mg2+ or Mn2+ for activity, is inhibited by Zn2+, vanadate and fluoride, and has an optimal pH of 9.0 and Mr = 50,000. Certain properties of this phosphatase so closely resemble those of phosphatase IA that the two enzymes tend to be copurified through various separation procedures.     

Compartmentation of NAD+-dependent signalling

NAD(+) plays central roles in energy metabolism as redox carrier. Recent research has identified important signalling functions of NAD(+) that involve its consumption. Although NAD(+) is synthesized mainly in the cytosol, nucleus and mitochondria, it has been detected also in vesicular and extracellular compartments. Three protein families that consume NAD(+) in signalling reactions have been characterized on a molecular level: ADP-ribosyltransferases (ARTs), Sirtuins (SIRTs), and NAD(+) glycohydrolases (NADases). Members of these families serve important regulatory functions in various cellular compartments, e.g., by linking the cellular energy state to gene expression in the nucleus, by regulating nitrogen metabolism in mitochondria, and by sensing tissue damage in the extracellular compartment. Distinct NAD(+) pools may be crucial for these processes. Here, we review the current knowledge about the compartmentation and biochemistry of NAD(+)-converting enzymes that control NAD(+) signalling.     

Sirtuin:依赖NAD+的去乙酰化酶

组蛋白的乙酰化一去乙酰化修饰在基因表达调控中起重要作用。参与去乙酰化的酶除了经典的Ⅰ类和Ⅱ类组蛋白去乙酰化酶(histone deacetylase,HDAC),还有比较特殊的Ⅲ类HDAC——Sirnlin,其活性依赖于NAD^ 。酵母的Sirtuin——Sir2在交配型基因沉默、端粒区基因沉默、rDNA沉默中起重要作用．还可能参与长寿与衰老的调节。在人类,Sirtuin的底物是组蛋白、各种转录因子如p53、FOXO、NF—KB、乙酰化酶如D300和其他的各种功能蛋白质。根据底物特点推测,人类Sirtuin蛋白的生理功能可能一方面是参与调节细胞在应激条件下的存活与死亡的平衡,另一方面是参与代谢的调节。     

Purification and characterization of NADP+-dependent 3 alpha-hydroxysteroid dehydrogenase from mouse liver cytosol

A monomeric 3 alpha-hydroxysteroid dehydrogenase with a molecular weight of 34,000 was purified to apparent homogeneity from mouse liver cytosol. The enzyme catalyzed the reversible oxidation of the 3 alpha-hydroxy group of C19-, C21-, and C24-steroids, reduced a variety of carbonyl compounds, and was inhibited by SH-reagents, synthetic estrogens, anti-inflammatory drugs, prostaglandins, and delta 4-3-ketosteroids. Although these properties are similar to those of the enzyme from rat liver cytosol, the mouse enzyme exhibited low dehydrogenase activity toward benzene dihydrodiol and some alicyclic alcohols, it showed a strict cofactor specificity for NADP(H), and high substrate inhibition was observed in the reverse reaction. In addition, dexamethasone, deoxycorticosterone, and medroxyprogesterone acetate inhibited the mouse enzyme competitively at low concentrations and noncompetitively at high concentrations, whereas hexestrol, indomethacin, and prostaglandin A1 were competitive inhibitors. Steady-state kinetic measurements in both directions indicated that the reaction proceeds through an ordered bi bi mechanism with the cofactors binding to the free enzyme. The 3-ketosteroid substrates inhibited the enzyme uncompetitively at elevated concentrations, suggesting that the substrates bind to the enzyme.NADPH complex and to the enzyme NADP+ complex.     

Involvement of a single thiol group in the conversion of the NAD+-dependent activity of rat liver xanthine oxidoreductase to the O2-dependent activity.

The effects of 2-iodosobenzoic acid, 4-chloromercuribenzoate, 5,5'-dithiobis-(2-nitrobenzoic acid) and tetraethylthioperoxydicarbonic diamide (disulphiram) on the NAD+-dependent activity of xanthine oxidoreductase from rat liver were investigated. Only disulphiram converted the NAD+-dependent activity into the O2-dependent activity quantitatively, without changing the xanthine hydroxylation rate. The modification process was a first-order reaction with respect to time (min) and disulphiram concentration (microM). The kinetic data showed that modification of single thiol group is sufficient for loss of the enzymic activity towards NAD+ as electron acceptor. The complete protection afforded by NAD+ against the action of disulphiram suggests that the essential thiol group may be involved in binding of NAD+ to the xanthine oxidoreductase molecule.     

NAD+-dependent DNA ligase encoded by a eukaryotic virus.

We report the production, purification, and characterization of an NAD(+)-dependent DNA ligase encoded by the Amsacta moorei entomopoxvirus (AmEPV), the first example of an NAD(+) ligase from a source other than eubacteria. AmEPV ligase lacks the zinc-binding tetracysteine domain and the BRCT domain that are present in all eubacterial NAD(+) ligases. Nonetheless, the monomeric 532-amino acid AmEPV ligase catalyzed strand joining on a singly nicked DNA in the presence of a divalent cation and NAD(+). Neither ATP, dATP, nor any other nucleoside triphosphate could substitute for NAD(+). Structure probing by limited proteolysis showed that AmEPV ligase is punctuated by a surface-accessible loop between the nucleotidyltransferase domain, which is common to all ligases, and the N-terminal domain Ia, which is unique to the NAD(+) ligases. Deletion of domain Ia of AmEPV ligase abolished the sealing of 3'-OH/5'-PO(4) nicks and the reaction with NAD(+) to form ligase-adenylate, but had no effect on phosphodiester formation at a pre-adenylated nick. Alanine substitutions at residues within domain Ia either reduced (Tyr(39), Tyr(40), Asp(48), and Asp(52)) or abolished (Tyr(51)) sealing of a 5'-PO(4) nick and adenylyl transfer from NAD(+) without affecting ligation of DNA-adenylate. We conclude that: (i) NAD(+)-dependent ligases exist in the eukaryotic domain of the phylogenetic tree; and (ii) ligase structural domain Ia is a determinant of cofactor specificity and is likely to interact directly with the nicotinamide mononucleotide moiety of NAD(+).     

Multiple aflatoxin B1 binding proteins exist in rat liver cytosol

The in vitro binding of aflatoxin B1 to rat liver cytosolic proteins was investigated. Aflatoxin B1 binding activity was assayed with protein purified by gel permeation chromatography, ammonium sulfate fractionation, and DEAE-cellulose chromatography. Twenty-five percent of the total binding activity was associated with proteins eluted by 0 and 0.1 M NaCl. Over 50% of the total binding activity was associated with protein present in the 0.2 M NaCl fraction. Glutathione S-transferase activity was also monitored and found only in the low salt (less than 0.2 M NaCl) fractions. The proteins eluted by 0.2 M NaCl were further purified by hydroxylapatite column chromatography and binding was found predominantly in a single fraction. The protein purification steps resulted in a 20-fold increase in the specific binding activity over that initially observed in the cytosol. These results indicate that multiple proteins are capable of binding aflatoxin B1 in rat liver cytosol.     

cAMP-independent casein kinase mediates phosphorylation of many T3-dependent phosphoproteins in rat liver cytosol

Administration of T3 (20 micrograms/100 g BW) for 3 days increases phosphorylation of several proteins in rat liver cytosol in vitro. To help elucidate the mechanism of T3-induced phosphorylation, we studied which protein kinase(s) mediate phosphorylation of endogenous cytosolic proteins. Five different protein kinases were obtained by DEAE+ cellulose column chromatographic fractionation of liver cytosol. When their ability to phosphorylate heat-inactivated cytosol was investigated, casein kinase, a cAMP independent protein kinase, showed the strongest effect. Casein kinase, purified by phosphocellulose chromatography, phosphorylated more than 10 cytosolic proteins. Several T3-dependent (and cAMP independent) phosphoproteins were included among these. One protein with Mr 39 X 10(3), of which phosphorylation is stimulated by T3 within five hours after injection, was the most active substrate for casein kinase. The results suggest that casein kinase is the enzyme responsible for phosphorylation of many rat liver cytosolic proteins and that several phosphoproteins, apparently under T3-regulation, might be phosphorylated by this enzyme.     

Calcium uptake and release by rat liver mitochondria in the presence of rat liver cytosol or the components of cytosol

Summary A study has been made of factors present in rat liver cytosol that might regulate the calcium content of mitochondria. A cytosol preparation containing all the components of molecular weight greater than 10,000 prevented uptake and caused early release of accumulated calcium. These effects were due to free long-chain fatty acids and their coenzyme A derivatives present in the cytosol, and these inhibitory effects were controlled by inclusion of Mg²⁺, carnitine, and adenosine triphosphate at physiological levels in the incubation medium. Palmitoyl carnitine was a good substrate for calcium uptake and did not cause release of calcium from mitochondria. A specific fatty acid-binding protein was found in cytosol which may be the intracellular transport protein for fatty acids.     

The mechanism of conversion of rat liver xanthine dehydrogenase from an NAD+-dependent form (type D) to an O2-dependent form (type O).

Rat liver xanthine dehydrogenase, type D, has been isolated directly from crude extracts as an antibody complex and its properties compared with those of two oxidase forms of the enzyme, heat-treated type O and trypsin-treated type O, also isolated as antibody complexes. The type D antibody complex displays electron acceptor specificities and electron paramagnetic resonance properties characteristic of an NAD⁺-dependent dehydrogenase whereas the trypsin-treated type O complex behaves as an O₂-utilizing oxidase. The heat-treated type O complex displays intermediate behavior. After electrophoresis in dodecyl sulfate-urea-acrylamide gels, type D and heated type O enzymes show single polypeptide bands, each of approximately 150,000 molecular weight. The trypsinized type O also shows one major band but with an approximate molecular weight of 130,000. Purified type D enzyme, when proteolytically treated, is converted to an oxidase with increased mobility on polyacrylamide gels. The 150,000 molecular weight subunit is cleaved into smaller subunits during proteolysis. Treatment with 5,5′-dithiobis-(2-nitrobenzoic acid) converts the type D enzyme, whether isolated as the purified enzyme or as the immune precipitate, to type O enzyme in a time-dependent manner. Titration of type D and the two type O antibody complexes with 5,5′-dithiobis-(2-nitrobenzoic acid) reveals that type D and heated type O each has approximately 28 reactive sulfhydryls, whereas the trypsinized type O has only 8 such groups. Many of the free sulfhydryls are vicinal and form disulfide bonds during the conversion to an oxidase by this reagent. Unproteolyzed preparations of type O rat liver enzyme and milk xanthine oxidase are converted to type D enzymes by treatment with dithiothreitol. The converted enzymes display electron acceptor specificities and epr properties characteristic of an NAD⁺-dependent dehydrogenase molecule.     

Specific and potent inhibition of NAD+-dependent DNA ligase by pyridochromanones

Pyridochromanones were identified by high throughput screening as potent inhibitors of NAD+-dependent DNA ligase from Escherichia coli. Further characterization revealed that eubacterial DNA ligases from Gram-negative and Gram-positive sources were inhibited at nanomolar concentrations. In contrast, purified human DNA ligase I was not affected (IC50 > 75 microm), demonstrating remarkable specificity for the prokaryotic target. The binding mode is competitive with the eubacteria-specific cofactor NAD+, and no intercalation into DNA was detected. Accordingly, the compounds were bactericidal for the prominent human pathogen Staphylococcus aureus in the low microg/ml range, whereas eukaryotic cells were not affected up to 60 microg/ml. The hypothesis that inhibition of DNA ligase is the antibacterial principle was proven in studies with a temperature-sensitive ligase-deficient E. coli strain. This mutant was highly susceptible for pyridochromanones at elevated temperatures but was rescued by heterologous expression of human DNA ligase I. A physiological consequence of ligase inhibition in bacteria was massive DNA degradation, as visualized by fluorescence microscopy of labeled DNA. In summary, the pyridochromanones demonstrate that diverse eubacterial DNA ligases can be addressed by a single inhibitor without affecting eukaryotic ligases or other DNA-binding enzymes, which proves the value of DNA ligase as a novel target in antibacterial therapy.     

Characterization of rat and mouse NAD+-dependent 3alpha/17beta/20alpha-hydroxysteroid dehydrogenases and identification of substrate specificity determinants by site-directed mutagenesis

In this study, we characterized rat and mouse aldo-keto reductases (AKR1C16 and AKR1C13, respectively) with 92% sequence identity. The recombinant enzymes oxidized non-steroidal alcohols using NAD⁺ as the preferred coenzyme, and showed low 3α/17β/20α-hydroxysteroid dehydrogenase (HSD) activities. The substrate specificity differs from that of rat NAD⁺-dependent 3α-HSD (AKR1C17) that shares 95% sequence identity with AKR1C16. To elucidate the residues determining the substrate specificity of the enzymes, we performed site-directed mutagenesis of Tyr24, Asp128 and Phe129 of AKR1C16 with the corresponding residues (Ser, Tyr and Leu, respectively) of AKR1C17. The double mutation (Asp128/Tyr-Phe129/Leu) had few effects on the substrate specificity, while the Tyr24/Ser mutant showed only 3α-HSD activity, and the triple mutation of the three residues produced an enzyme that had almost the same properties as AKR1C17. The importance of the residue 24 for substrate recognition was verified by the mutagenesis of Ser24/Tyr of AKR1C17 which resulted in a decrease in 3α-HSD activity and appearance of 17β- and 20α-HSD activities. AKR1C16 is also 92% identical with rat NAD⁺-dependent 17β-HSD (AKR1C24), which possesses Tyr24. The replacement of Asp128, Phe129 and Ser137 of AKR1C16 with the corresponding residues (Glu, Ser and Phe, respectively) of AKR1C24 increased the catalytic efficiency for 17β- and 20α-hydroxysteroids.     

Purification and properties of the NAD+-dependent (type D) and O2-dependent (type O) forms of rat liver xanthine dehydrogenase.

The xanthine-oxidizing enzyme of rat liver has been purified as an NAD⁺-dependent dehydrogenase (type D) and as the O₂-dependent oxidase (type O). The purified D and O variants are nearly homogenous as judged by polyacrylamide discontinuous gel electrophoresis and are indistinguishable on sodium dodecyl sulfate-urea gels. The absorption spectrum of the type D enzyme is indistinguishable from that of the type O enzyme and closely resembles the spectra of xanthine-oxidizing enzymes from other sources. The types D and O enzymes have essentially the same cofactor composition. Oxidation of xanthine by type D is stimulated by NAD⁺ with concomitant NADH formation. Type D is able to utilize NADH as well as xanthine as electron donor to various acceptors, in contrast to type O that is unable to oxidize NADH. Arsenite, cyanide and methanol completely abolish xanthine oxidation by the type D enzyme while affecting the activities with NADH to varying extents. In these respects rat liver xanthine dehydrogenase closely resembles chicken liver xanthine dehydrogenase. However, in contrast to the avian enzyme, the purified rat liver enzyme is unstable as a dehydrogenase and is gradually converted to an oxidase. This conversion is accompanied by an increase in the aerobic xanthine → cytochrome c activity. The native type D enzyme in rat liver extracts is precipitable with antibody prepared against purified type O. The K_m for xanthine is not significantly different for the two forms.