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A Y Lu

Publications and source records attributed to A Y Lu.

At least 37 records · Page 2Linked to original sources
PubMed1992

Identification of a glycine-rich sequence as an NAD(P)H-binding site and tyrosine 128 as a dicumarol-binding site in rat liver NAD(P)H:quinone oxidoreductase by site-directed mutagenesis.

Site-directed mutagenesis was utilized to identify binding sites for NAD(P)H and dicumarol in rat liver NAD(P)H:quinone oxidoreductase (NQOR, EC 1.6.99.2). The mutant cDNA clones were generated by a procedure based on the polymerase chain reaction and were expressed in Escherichia coli. The mutant enzymes were purified to apparent homogeneity as judged by SDS-polyacrylamide gel electrophoresis and were found to contain 2 FADs/enzyme molecule identical with that of the wild-type NQOR. Purified mutant enzymes Y128D, G150F, G150V, S151F, and Y155D showed dramatic decreases in activities in the reduction of dichlorophenolindophenol in comparison with the activities of the wild-type enzyme, whereas the activities of F124L, T127V, T127E, Y128V, Y128F, S151A, and Y155V were similar to those of NQOR. Enzyme kinetic analysis revealed that the Km values of T127E, Y128D, G150F, G150V, S151F, and Y155D were, respectively, 4-, 2-, 13-, 5-, 26-, and 19-fold higher than the Km of NQOR for NADPH, and were, respectively, 2-, 3-, 7-, 3-, 20-, and 11-fold higher than that of NQOR for NADH. The kcat values of Y128D, G150F, and G150V were also much lower than those of NQOR, but the kcat values of other mutants were similar to those of the wild-type enzyme. The Km values of the mutants for dichlorophenolindophenol were the same or slightly higher than that of NQOR. The apparent inhibition constants (Ki) for dicumarol on Y128V and F124L were elevated 12 and 8 times, respectively. Similar, but smaller, changes on Ki for 4-hydroxycoumarin were also observed. This study demonstrated that residues Gly150, Ser151, and Tyr155 in the glycine-rich region of NQOR are essential for NADPH and NADH binding and Tyr128 is important for dicumarol binding. Based on the results of the study, it is proposed that the glycine-rich region of the enzyme, along with other residues around the region, forms a beta sheet-turn-alpha helix structure important for the binding of the pyrophosphate group of NADPH and NADH.

Amino Acid Sequence↗
PubMed1992

Site-directed mutagenesis of glutathione S-transferase YaYa. Important roles of tyrosine 9 and aspartic acid 101 in catalysis.

The roles of tyrosine 9 and aspartic acid 101 in the catalytic mechanism of rat glutathione S-transferase YaYa were studied by site-directed mutagenesis. Replacement of tyrosine 9 with phenylalanine (Y9F), threonine (Y9T), histidine (Y9H), or valine (Y9V) resulted in mutant enzymes with less than 5% catalytic activity of the wild type enzymes. Kinetic studies with purified Y9F and Y9T mutants demonstrated poor catalytic efficiencies which were largely due to a drastic decrease in kcat. The estimated pK alpha values of the sulfhydryl group of glutathione bound to Y9F and Y9T mutant enzymes were 8.5 to 8.7, similar to the chemical reaction, in contrast to the estimated pK alpha value of 6.7 to 6.8 for the glutathione enzyme complex of wild type glutathione S-transferase. These results indicate that tyrosine 9 is directly responsible for the lowering of the pKa of the sulfhydryl group of glutathione, presumably due to the stabilization of the thiolate anion through hydrogen bonding with the hydroxyl group of tyrosine. To examine the role of aspartic acid in the binding of glutathione to YaYa, 4 conserved aspartic acid residues at positions 61, 93, 101, and 157 were changed to glutamic acid and asparagine. All mutant enzymes retained either full or partial activity except D157N, which was virtually inactive. Kinetic studies with four mutant enzymes (D93E, D93N, D101E, and D101N) indicate that only D101N exhibited a 5-fold increase in Km toward glutathione. Also, the binding of this mutant to the affinity column was greatly reduced. These results demonstrate that aspartic acid 101 plays an important role in glutathione interaction to YaYa. The role of aspartic acid 157 in catalysis remains to be determined.

Animals↗
PubMed1992

Site-directed mutagenesis of glutathione S-transferase YaYa: functional studies of histidine, cysteine, and tryptophan mutants.

The rat cytosolic glutathione S-transferase Ya subunit contains three histidine residues (at positions 8, 143, and 159), two cysteine residues (at positions 18 and 112), and a single tryptophan residue (at position 21). Histidine, cysteine, and tryptophan have been proposed to be present either near or at the active site of other glutathione S-transferase subunits. The functional role of these amino acids at each of the positions was evaluated by site-directed mutagenesis in which valine or asparagine, alanine, and phenylalanine were substituted for histidine, cysteine, and tryptophan, respectively. Mutant enzymes H8V, H143V, H159N, C112A, and W21F retained either full or better catalytic efficiencies (k(cat)/Km) toward 1-chloro-2,4-dinitrobenzene and glutathione. Lower but significant k(cat)/Km values were observed for H159V and C18A toward 1-chloro-2,4-dinitrobenzene. Some mutants displayed different thermal stabilities and intrinsic fluorescence intensities, but all retained the ability to bind heme. These results indicate that histidine, cysteine, and tryptophan in the glutathione S-transferase Ya subunit are not essential for catalysis nor are they involved in the binding of heme to the YaYa homodimer.

Animals↗
PubMed1992

Site-directed mutagenesis of rat liver NAD(P)H: quinone oxidoreductase: roles of lysine 76 and cysteine 179.

We previously reported the expression of a full-length cDNA complementary to a rat liver NAD(P)H:quinone oxidoreductase (EC 1.6.99.2) mRNA in Escherichia coli (Q. Ma, R. Wang, C. S. Yang, and A. Y. H. Lu, 1990, Arch. Biochem. Biophys. 283, 311-317). Since cysteine residues have been suggested to be important for the catalysis of flavoproteins and a lysine residue at position 76 in NAD(P)H:quinone oxidoreductase has been proposed to be involved in electron transfer of the enzyme, we investigated the roles of lysine 76 and cysteine 179 of this enzyme in catalysis by site-directed mutagenesis. Mutant cDNA clones replacing lysine 76 with valine (K76V) and cysteine 179 with alanine (C179A) were generated by a procedure based on the polymerase chain reaction. The mutant enzymes were expressed in E. coli. The cytosolic activities of the K76V and C179A mutants were 50 and 25% of that of the wild type (DTD), due to lower levels of the mutant proteins as shown by immunoblot analysis. The mutant proteins were purified to apparent homogeneity. The purified K76V and C179A mutant enzymes maintained full activities of 2,6-dichlorophenolindophenol (DCIP) reduction compared with that of the wild type. The mutant enzymes exhibited kinetic parameters for DCIP, NADH, and NADPH similar to those of DTD except that, with K76V, the Km for NADPH was doubled. Both mutant proteins contained two molecules of FAD per enzyme molecule. Dicumarol inhibited K76V and C179A mutant activities to greater than 90% at a concentration of 10(-7) M. Heat stability studies showed that C179A was much more sensitive to inactivation at 37 degrees C than both the wild-type and K76V enzymes. It is concluded from this study that lysine 76 and cysteine 179 are not essential in catalysis and in the binding of FAD, DCIP, and dicumarol. However, lysine residue 76 appears to play a role in NADPH binding and cysteine residue 179 is important in maintaining the stability of the enzyme.

Animals↗
PubMed1992

In vivo covalent binding of [14C]trinitrotoluene to proteins in the rat.

When a single dose of [14C]trinitrotoluene was administered intraperitoneally (i.p.) to rats at 1, 10 or 50 mg/kg of body weight, covalently bound radioactivity was detected in globin, plasma proteins and proteins in the liver and kidney. The extent of covalent binding was dose dependent and was highest in plasma and renal proteins at all times up to 4 h after dosing. Covalent adduct levels in globin, however, decline slower than others. At a dose of 50 mg/kg of body weight, globin covalent adduct levels peaked at 1 h after dosing at 182 pmol/mg protein and subsequently decreased to approximately 50 pmol/mg protein between days 1 and 8. Of the covalent adduct levels in liver and kidney, those in the 10,000 x g and microsomal fractions were found to be higher than that in the cytosolic fraction. Radioactivity covalently bound to globin and the hepatic proteins was susceptible to dilute acid hydrolysis from which 2-amino-4,6-dinitrotoluene (2A) and 4-amino 2,6-dinitrotoluene (4A) were the major products recovered by solvent extraction. Upon acetylation, the hydrolysate gave rise to derivatives identified as the acetates of 2A and 4A on the basis of mass spectrometry and HPLC cochromatography with authentic samples. Four hours after an i.p. dose of [14C]TNT at 50 mg/kg of body weight about 0.4% of the dose was found as bound adducts to hemoglobin, of which approximately 48% was recovered as solvent extractable radioactivity after acid hydrolysis. About 2% of the radioactive dose was in the liver, of which approximately 30% was covalently bound to hepatic proteins, and approximately 49% of that was convertible to solvent extractable radioactivity upon acid hydrolysis. In vitro incubation of [14C]TNT with blood showed that there was a linear increase of covalent adducts in globin during the first 2 h of incubation; the concentration of covalent adducts was slightly higher than that with plasma proteins. The major compounds recovered from the hydrolysate of the globin adducts were also 2A and 4A as obtained from globin in the in vivo studies. On the basis of the in vitro and in vivo study results, we have confirmed the formation of protein adducts following a single i.p. administration of [14C]TNT at 1, 10 or 50 mg/kg of body weight to the rat or by in vitro incubation with blood.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Drug residue formation from ronidazole, a 5-nitroimidazole. VIII. Identification of the 2-methylene position as a site of protein alkylation.

Ronidazole protein-bound adducts were generated by the in vitro anaerobic incubation of [2-methylene-14C]ronidazole with microsomes from the livers of male rats. Acid hydrolysis of the protein adducts yielded an imidazole ring fragment bearing the radiolabel and an amino acid residue derived from the proteins. This fragment has been identified as carboxymethylcysteine by co-chromatography of the amino acid and its dansyl derivative with known standards under a variety of conditions. The carboxymethylcysteine was estimated to represent at least 15% of the radioactivity derived from the protein-bound adducts and provides unequivocal evidence that nucleophilic attack by protein cysteine thiols occurred at the 2-methylene position of ronidazole.

Alkylation↗
PubMed1991

Biotransformation of lovastatin. IV. Identification of cytochrome P450 3A proteins as the major enzymes responsible for the oxidative metabolism of lovastatin in rat and human liver microsomes.

Previous studies from our laboratories have shown that the metabolism of the cholesterol-lowering drug lovastatin by rat and human liver microsomes occurs primarily at the 6'-position, giving 6' beta-hydroxy- and 6'-exomethylene-lovastatin and that these oxidations are catalyzed by cytochrome P450-dependent monooxygenases. In the present study, the specific cytochrome P450 form involved in lovastatin oxidation was identified through immunoinhibition studies. Among several antibodies prepared against various cytochrome P450s, only anti-rat P450 3A IgG inhibited lovastatin metabolism in liver microsomes from untreated, phenobarbital-treated, and pregnenolone-16 alpha-carbonitrile-treated rats. Lovastatin metabolism at the 6'-position was markedly inhibited (6' beta-hydroxy, greater than 95%; 6'-exomethylene, 70-80%) by this antibody whereas the effect of anti-rat P450 3A on the 3"-hydroxylation was variable depending on the source of the microsomes. With human liver microsomes, both anti-rat P450 3A and anti-human P450 3A inhibited lovastatin metabolism. Correlation between lovastatin oxidation and the P450 3A content in human liver microsomes (measured by immunoblot analysis) was excellent (r2 = 0.97). In addition, preincubation of human liver microsomes with troleandomycin and NADPH inhibited metabolism by 60%. These results clearly indicate that cytochrome P450 3A enzymes are primarily responsible for the metabolism of lovastatin in rat and human liver microsomes.

Animals↗
PubMed1991

Site-directed mutagenesis of glutathione S-transferase YaYa: nonessential role of histidine in catalysis.

A cDNA encoding a rat liver glutathione S-transferase Ya subunit has been expressed in Escherichia coli and the expressed enzyme purified to homogeneity. In order to examine the catalytic role of histidine in the glutathione S-transferase Ya homodimer, site-directed mutagenesis was used to replace all three histidine residues (at positions 8, 143, and 159) by other amino acid residues. The replacement of histidine 8 or histidine 143 with valine did not affect the 1-chloro-2,4-dinitrobenzene-conjugating activity nor the isomerase activity. However, the replacement of histidine with valine at position 159 produced the mutant GST which exhibited only partial activity. A greater decrease in catalytic activity was observed by histidine----tyrosine or histidine----lysine replacement at position 159. On the other hand, the histidine 159----asparagine mutant retained full catalytic activity. Our results indicate that histidine residues in the Ya homodimer are not essential for catalytic activity. However, histidine 159 might be critical in maintaining the proper conformation of this enzyme since replacement of this amino acid by either lysine or tyrosine did result in significant loss of enzymatic activity.

Amino Acid Sequence↗
PubMed1990

Expression in Escherichia coli of rat liver cytosolic glutathione S-transferase Yc cDNA.

An expression plasmid, pKK-GTB2, containing the complete coding sequence of a rat liver glutathione S-transferase Yc subunit was constructed and expressed in Escherichia coli. The entire Yc cDNA sequence from plasmid pGTB42 (Telakowski-Hopskins et al., 1985, J. Biol. Chem. 260, 5820-5825) was amplified by the polymerase chain reaction, subcloned into modified expression vector A6316 (Schoner et al., 1986, Proc. Natl. Acad. Sci. USA 83, 8506-8510 and Linemeyer et al., 1987, Bio/Technology 5, 960-965) and transformed into E. coli strain AB1899. The colonies were screened by hybridization to pGTB42 and the production of Yc subunit was detected by immunoblot analysis. The purified recombinant Yc subunit was active in the conjugation and peroxidation reactions, and appeared homogeneous as judged by sodium dodecyl sulfate gel electrophoresis. Amino acid sequencing of the expressed Yc subunit revealed that about 40% of the expressed protein was blocked at the N-terminus. Approximately 25% of the sequenceable protein (15% of total protein) contained the initiation methionine residue at the amino terminus whereas the rest of the sequenceable protein had proline as the N-terminus. In contrast, only one molecular species with Pro as the first amino acid was identified when the inducer isopropyl-beta-D-thiogalactopyranoside was omitted in the growth medium. Our observation indicated that under certain growth conditions, the enzymes responsible for protein maturation were not able to complete the processing of the overproduced recombinant Yc in E. coli.

Amino Acid Sequence↗
PubMed1990

Biotransformation of lovastatin--III. Effect of cimetidine and famotidine on in vitro metabolism of lovastatin by rat and human liver microsomes.

The effects of the H2-receptor antagonists, cimetidine and famotidine, on the microsomal metabolism of [14C]lovastatin were investigated. Liver microsomes were prepared from control, phenobarbital- and 3-methylcholanthrene-pretreated rats and humans (male and female). Concentration-dependent inhibition of the metabolism of lovastatin (0.1 mM) was observed with cimetidine (0.1 to 1.0 mM). In contrast, famotidine at a similar concentration was a very weak inhibitor. The formation of 6'beta-hydroxy-lovastatin, the major microsomal metabolite of lovastatin, was similarly inhibited. The results suggest that in vivo metabolic interaction with concomitantly administered lovastatin is less likely with famotidine than with cimetidine. Phenobarbital pretreatment produced 58% stimulation in overall metabolism, whereas 3-methylcholanthrene pretreatment had no effect relative to control rats (5.4 nmol/mg protein/min). Liver microsomes from phenobarbital-pretreated rats produced 67% more of the 6'beta-hydroxy-lovastatin but 63-66% less of the 3''-hydroxy and 6'-exomethylene metabolites. Liver microsomes from 3-methylcholanthrene-treated rats also produced less 3"-hydroxy-lovastatin (49%) but similar quantities of the other two metabolites. 6'beta-Hydroxy-lovastatin was a major metabolite with human liver microsomes. Interestingly with these microsomes, hydroxylation at the 3''-position of the molecule was a negligible pathway and hydrolysis to the hydroxy acid form was not observed. The formation of 6'-exomethylene-lovastatin was also catalyzed by human liver microsomes (0.5 to 0.8 nmol/mg protein/min).

Animals↗
PubMed1990

Expression of mammalian DT-diaphorase in Escherichia coli: purification and characterization of the expressed protein.

A full-length cDNA clone, pKK-DTD4, complementary to rat liver cytosolic DT-diaphorase [NAD(P)H:quinone oxidoreductase (EC 1.6.99.2)] mRNA was expressed in Escherichia coli. The pKK-DTD4 cDNA was obtained by extending the 5'-end sequence of a rat liver DT-diaphorase cDNA clone, pDTD55, to include an ATG initiation codon and the NH2-terminal codons using polymerase chain reaction (PCR). Restriction sites for EcoRI and HindIII were incorporated at the 5'- and 3'-ends of the cDNA, respectively, by the PCR reaction. The resulting full-length cDNA was inserted into an expression vector, pKK2.7, at the EcoRI and HindIII restriction sites. E. coli strain AB1899 was transformed with the constructed expression plasmid, and DT-diaphorase was expressed under the control of the tac promotor. The expressed DT-diaphorase exhibited high activity of menadione reduction and was inhibited by dicumarol at a concentration of 10(-5)M. After purification by Cibacron Blue affinity chromatography, the expressed enzyme migrated as a single band on 12.5% sodium dodecyl sulfate-polyacrylamide gel with a molecular weight equivalent to that of the purified rat liver cytosolic DT-diaphorase. The purified expressed protein was recognized by polyclonal antibodies against rat liver DT-diaphorase on immunoblot analysis. It utilized either NADPH or NADH as electron donor at equal efficiency and displayed high activities in reduction of menadione, 1,4-benzoquinone, and 2,6-dichlorophenolindophenol which are typical substrates for DT-diaphorase. The expressed DT-diaphorase exhibited a typical flavoprotein spectrum with absorption peaks at 380 and 452 nm. Flavin content determination showed that it contained 2 mol of FAD per mole of the enzyme. Edman protein sequencing of the first 20 amino acid residues at the NH2 terminus of the expressed protein indicated that the expressed DT-diaphorase is not blocked at the NH2 terminus and has an alanine as the first amino acid. The remaining 19 amino acid residues at the NH2 terminus were identical with those of the DT-diaphorase purified from rat liver cytosol.

Amino Acid Sequence↗
PubMed1989

NADPH-cytochrome P-450 reductase is not a component of the liver microsomal steroid 5-alpha reductase.

Liver microsomal steroid 5-alpha-reduction is catalyzed by a NADPH-dependent enzyme system. The requirement of NADPH-cytochrome P-450 reductase to shuttle reduction equivalents from NADPH to steroid 5-alpha-reductase was investigated using an inhibitory antibody against NADPH-cytochrome P-450 reductase. This antibody preparation inhibited cytochrome c reduction in microsomes from female rat liver with an I50 of 0.75 mg antibody/mg of microsomal protein. Benzphetamine N-demethylation and testosterone 6-beta-hydroxylation, two cytochrome P-450-mediated oxidative reactions, were inhibited by the antibody. On the other hand, testosterone 5-alpha-reductase was not affected by the antibody. These results suggest that NADPH-cytochrome P-450 reductase is not an obligatory component of the liver microsomal steroid 5-alpha-reduction.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1989

Expression of a cDNA encoding a rat liver glutathione S-transferase Ya subunit in Escherichia coli.

A full length cDNA clone, pGTB38 (C. B. Pickett et al. (1984) J. Biol. Chem. 259, 5182-5188), complementary to a rat liver glutathione S-transferase Ya mRNA has been expressed in Escherichia coli. The cDNA insert was isolated from pGTB38 using MaeI endonuclease digestion and was inserted into the expression vector pKK2.7 under the control of the tac promoter. Upon transformation of the expression vector into E. coli, two protein bands with molecular weights lower than the full-length Ya subunit were detected by Western blot analysis in the cell lysate of E. coli. These lower-molecular-weight proteins most likely result from incorrect initiation of translation at internal AUG codons instead of the first AUG codon of the mRNA. In order to eliminate the problem of incorrect initiation, the glutathione S-transferase Ya cDNA was isolated from the expression vector and digested with Bal31 to remove extra nucleotides from the 5' noncoding region. The protein expressed by this expression plasmid, pKK-GTB34, comigrated with the Ya subunit on sodium dodecyl sulfate polyacrylamide gels and was recognized by antibodies against the YaYc heterodimer. The expressed Ya homodimer was purified by S-hexylglutathione affinity and ion-exchange chromatographies. Approximately 50 mg pure protein was obtained from 9 liters of E. coli culture. The expressed Ya homodimer displayed glutathione-conjugating, peroxidase, and isomerase activities, which are identical to those of the native enzyme purified from rat liver cytosol. Protein sequencing indicates that the expressed protein has a serine as the NH2 terminus whereas the NH2 terminus of the glutathione S-transferase Ya homodimer purified from rat liver cytosol is apparently blocked.

Amino Acid Sequence↗