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A Y Lu

Publications and source records attributed to A Y Lu.

At least 55 records · Page 3Linked to original sources
PubMed1987

Rat liver NAD(P)H: quinone reductase nucleotide sequence analysis of a quinone reductase cDNA clone and prediction of the amino acid sequence of the corresponding protein.

We have determined the nucleotide sequence of a cDNA clone, pDTD55, complementary to rat liver quinone reductase mRNA (Williams, J.B., Lu, A.Y.H., Cameron, R.G., and Pickett, C.B. (1986) J. Biol. Chem. 261, 5524-5528). The cDNA clone contains an open reading frame of 759 nucleotides encoding a polypeptide comprised of 253 amino acids with a Mr = 28,564. To verify the predicted amino acid sequence of quinone reductase, we have been able to align the amino acid sequences of a cyanogen bromide digest of the purified enzyme to the sequence deduced from the cDNA clone. A comparison of the quinone reductase sequence with other known flavoenzymes did not reveal a significant degree of amino acid sequence homology. These data suggest that the quinone reductase gene has evolved independently from genes encoding other flavoenzymes.

Amino Acid Sequence↗
PubMed1986

Rat liver glutathione S-transferases. DNA sequence analysis of a Yb2 cDNA clone and regulation of the Yb1 and Yb2 mRNAs by phenobarbital.

We have constructed a cDNA clone, pGTA/C48, which is complementary to the rat liver glutathione S-transferase Yb2 mRNA. Recombinant clone pGTA/C48 contains a cDNA insert of 845 base pairs which overlaps nucleotides 108-952 of the Yb1 cDNA clone, pGTA/C44, described previously by our laboratory (Ding, G. J.-F., Lu, A. Y. H., and Pickett, C. B. (1985) J. Biol. Chem. 260, 13268-13271). Over the protein coding region of the Yb1 and Yb2 cDNA clones there is an 84% nucleotide sequence homology, whereas the 3' untranslated regions are only 32% homologous. The complete amino acid sequence of the Yb2 subunit has been determined from a combination of DNA sequence analysis of pGTA/C48 and conventional protein sequence analysis of the glutathione S-transferase Yb1 Yb2 heterodimer. The Yb2 subunit is comprised of 218 amino acids with a molecular weight of 25,705 and has an amino acid sequence which is 79% homologous to the sequence of the Yb1 subunit. We have utilized the divergent 3' untranslated regions of three rat liver glutathione S-transferase cDNA clones as specific probes to determine the effect of phenobarbital on the level of Yb1, Yb2, and Yc mRNAs. Our results clearly show that the Yb1 and Yb2 mRNAs are elevated approximately 5-6-fold by phenobarbital administration; whereas the Yc mRNA is only modestly elevated by this xenobiotic. Finally, our data suggest that the Yb2 subunit is encoded by a gene(s) which is distinct from the Yb1 gene(s) and provides direct evidence for the existence of multiple glutathione S-transferase Yb genes in the rat.

Amino Acid Sequence↗
PubMed1986

Rat liver NAD(P)H:quinone reductase. Construction of a quinone reductase cDNA clone and regulation of quinone reductase mRNA by 3-methylcholanthrene and in persistent hepatocyte nodules induced by chemical carcinogens.

We have used polysomal immunoabsorption techniques to purify rat liver quinone reductase mRNA (NAD(P)H:quinone oxidoreductase, EC 1.6.99.2, formerly called DT-diaphorase). Using the purified mRNA as template, cDNA clones complementary to quinone reductase mRNA have been constructed. One cDNA clone, pDTD55, has a 1900-base pair insert which has been demonstrated by hybrid-select translation experiments to be complementary to quinone reductase mRNA. Clone pDTD55 has been used in RNA and DNA blot hybridizations to show that quinone reductase mRNA is approximately 1900 nucleotides in length and is encoded by a gene which spans approximately 7000-8000 base pairs. We have also shown that quinone reductase mRNA is markedly elevated by 3-methylcholanthrene administration and in persistent hepatocyte nodules induced by chemical carcinogens. The elevation of quinone reductase mRNA in persistent hepatocyte nodules is not due to either gene amplification of DNA rearrangement. Rather, the quinone reductase gene is hypomethylated in persistent hepatocyte nodules compared to the gene in either liver tissue surrounding the nodule or normal liver. These data suggest that hypomethylation of specific gene sequences occurs at early stages during chemical carcinogenesis.

Animals↗
PubMed1986

Studies on the mechanism of activation and the mutagenicity of ronidazole, a 5-nitroimidazole.

Substantial evidence implicates the obligatory nucleophilic attack by water at C4 for the elimination of the carbamate and subsequent immobilization by electrophilic attack on protein thiols. Consequently, the strong correlation between the structural requirements for protein alkylation and for mutagenicity in TA100 suggests a possible role of nucleophilic addition at C4 or at the 2-methylene carbon for the expression of mutagenicity. Further studies directed at evaluating this possibility are currently in progress.

Alkylation↗
PubMed1986

Multiple Ya subunits of glutathione S-transferase detected by monoclonal antibodies.

Monoclonal antibodies to ligandin (YaYa) and glutathione (GSH) S-transferase B (YaYc) were produced by hybridomas derived from the fusion of mouse myeloma cells and spleen cells of mice immunized with the YaYa or YaYc proteins, respectively. Enzyme-linked immunosorbent assay was used to screen for antibody-producing clones. Immunoblotting of the subunits of transferase B, ligandin, and another GSH S-transferase containing Yb subunits showed that the monoclonal antibodies produced by two anti-YaYa subclones recognized the Ya subunits of both ligandin and transferase B, but they did not bind Yc or Yb subunits. It was also revealed that antibodies produced by several anti-YaYc subclones recognized the Yc subunit, but not the Ya subunit of the antigen which was used for the immunization of the mice. However, these monoclonal antibodies did bind the Ya subunit of ligandin. These results indicate that the Ya subunits of GSH S-transferase B and of ligandin do share at least one common determinant. However, these two Ya subunits are structurally distinct as evidenced by their differences in binding by monoclonal anti-YaYc antibodies.

Animals↗
PubMed1986

Acetone-inducible cytochrome P-450: purification, catalytic activity, and interaction with cytochrome b5.

A procedure was developed for the purification of an acetone-inducible form of cytochrome P-450 (P-450ac) to electrophoretical homogeneity from liver microsomes of acetone-treated rats. The P-450ac preparation containing 16.0 to 16.5 nmol P-450/mg protein moved as a single protein band with an estimated molecular weight of 52,000 upon gel electrophoresis in the presence of sodium dodecyl sulfate. The ferric P-450ac showed an absorption maximum at 394 nm at 25 degrees C, suggesting that it exists mainly in the high-spin form. It also existed in the low-spin form, especially at lower temperatures, as indicated by the absorption maximum in the 412-nm region. Upon reconstitution with NADPH: cytochrome P-450 reductase and phospholipid, P-450ac efficiently catalyzed both the demethylation and denitrosation of N-nitrosodimethylamine (NDMA) showing Vmax values of 23.8 and 2.3 nmol min-1 nmol P-450-1, respectively. The catalytic activity of P-450ac was greatly affected by cytochrome b5 which decreased the Km values of these reactions by a factor of 10 and increased the Vmax values. Cytochrome b5 appeared to interact with P-450 at a molar ratio of 1:1 and an intact cytochrome b5 structure was required for such interaction. Among the substrates studied, the demethylation of NDMA was affected the most by cytochrome b5 and showed the highest rate. P-450ac also catalyzed the oxygenation of N-nitrosomethylethylamine and aniline and the activity was enhanced slightly by cytochrome b5. Cytochrome b5 did not enhance the P-450ac-catalyzed metabolism of other drug substrates such as benzphetamine, aminopyrine, and ethylmorphine. P-450ac appeared to be similar in property to the previously studied rat P-450et (ethanol-inducible), rat P-450j (isoniazid-inducible), and rabbit P-450LM3a (ethanol-inducible). These P-450 species represent a new class of P-450 isozymes that are important in the metabolism of many endobiotics and xenobiotics.

Acetone↗
PubMed1986

Tumorigenicity of nitrated derivatives of pyrene, benz[a]anthracene, chrysene and benzo[a]pyrene in the newborn mouse assay.

Eight nitropolycyclic aromatic hydrocarbons (PAHs), including 1- and 4-nitropyrene, 1,3-, 1,6- and 1,8-dinitropyrene, 7-nitrobenz[a]anthracene, 6-nitrochrysene and 6-nitrobenzo-[a]pyrene and their parent PAHs were tested fro tumorigenicity in the newborn mouse model by i.p. administration at 1, 8, and 15 days after birth. Both pyrene and 1-nitropyrene induced similar incidences of hepatic tumors in males, yielding a 12-15% and a 21-28% tumor incidence at total doses of 700 and 2800 nmol per mouse, respectively. Liver tumors did not occur in females and the 3-10% lung tumor yield in both sexes was similar to that found in solvent-treated controls. The presumed proximate carcinogen, 1-nitrosopyrene, administered at 700 nmol per mouse, caused liver tumors in 45% of the males and in 9% of the females. 4-Nitropyrene was more tumorigenic than pyrene or 1-nitropyrene; at a dose of 2800 nmol, it induced liver tumors in 83% of the males and 7% of the females, with a lung tumor yield of 38 and 31%, respectively. Female mice treated with 200 nmol of 1,3-, 1,6- or 1,8-dinitropyrene did not develop liver tumors but the hepatic tumor incidence in males was 20, 32 and 16%, respectively, which was significantly greater than that found in mice treated with pyrene. In male mice administered 2800 nmol of benz[a]anthracene, the hepatic tumor incidence was 79%, while treatment with 7-nitrobenz[a]anthracene showed an incidence of only 28%. Similarly, 560 nmol of benzo[a]pyrene caused a 49% liver tumor yield in males while those given 6-nitrobenzo[a]pyrene had a 28% incidence. Treatment with benzo[a]pyrene also induced a 35 and 48% lung tumor incidence in males and females while the comparable values in 6-nitrobenzo[a]pyrene-treated mice were 14 and 2%. Chrysene administered at 2800 nmol per mouse induced hepatic and lung tumors in 41% and 21% of the males, respectively; at the 700-nmol dose, it induced only liver tumors in 29% of the males and in none of the females. In contrast, treatment with 6-nitrochrysene at 700 nmol per mouse resulted in a 76 and 23% hepatic tumor incidence in males and females, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Rat liver glutathione S-transferases. Nucleotide sequence analysis of a Yb1 cDNA clone and prediction of the complete amino acid sequence of the Yb1 subunit.

We have constructed a nearly full length cDNA clone, pGTA/C44, complementary to the rat liver glutathione S-transferase Yb1 mRNA. The nucleotide sequence of pGTA/C44 has been determined, and the complete amino acid sequence of the Yb1 subunit has been deduced. The cDNA clone contains an open reading frame of 654 nucleotides encoding a polypeptide comprising 218 amino acids with Mr = 25,919. The NH2-terminal sequence deduced from DNA sequence analysis of pGTA/C44 is in agreement with the first 19 amino acids determined for purified glutathione S-transferase A, a Yb1 homodimer, by Frey et al. (Frey, A. B., Friedberg, T., Oesch, F., and Kreibich, G. (1983) J. Biol. Chem. 258, 11321-11325). The DNA sequence of pGTA/C44 shares significant sequence homology with a cDNA clone, pGT55, which is complementary to a mouse liver glutathione S-transferase (Pearson, W. R., Windle, J. J., Morrow, J. F., Benson, A. M., and Talalay, P. (1983) J. Biol. Chem. 258, 2052-2062). We have also determined 37 nucleotides of the 5'-untranslated region and 348 nucleotides of the 3'-untranslated region of the Yb1 mRNA. The Yb1 mRNA and subunit do not share any sequence homology with the rat liver glutathione S-transferase Ya or Yc mRNAs or their corresponding subunits. These data provide the first direct evidence that the Yb1 subunit is derived from a gene or gene family which is distinct from the Ya-Yc gene family.

Amino Acid Sequence↗
PubMed1985

Kinetic isotope effects on cytochrome P-450-catalyzed oxidation reactions: full expression of the intrinsic isotope effect during the O-deethylation of 7-ethoxycoumarin by liver microsomes from 3-methylcholanthrene-induced hamsters.

The intrinsic primary deuterium isotope effect for the O-deethylation of 7-ethoxycoumarin has been estimated by the Northrop method [D. B. Northrop (1977) in Isotope Effects on Enzyme-Catalyzed Reactions (Cleland, W. W., O'Leary, M. H., and Northrop, D. B., eds.), pp. 122-152, University Park Press, Baltimore] for the microsomal cytochrome P-448 system from 3-methylcholanthrene-induced hamster livers. The intrinsic isotope effect (Dk = 5.5) was found to be equivalent to the observed deuterium isotope effect, demonstrating that the isotope effect for this reaction was fully expressed by this cytochrome P-448 system. These data unequivocally demonstrate that C-H bond cleavage is the rate-limiting step in the overall reaction catalyzed by this system. The decrease in the rate of product formation, occurring as a consequence of deuterium substitution, resulted in a reduction in the quantity of substrate metabolized but was not accompanied by the change in regiospecificity observed in previous studies with a hepatic cytochrome P-448 isozyme purified from 3-methylcholanthrene-induced rats. These data demonstrate that the catalytic site of the hamster isozyme(s) offers more constraints to 7-ethoxycoumarin reorientation than does the catalytic site of the rat liver isozyme.

Animals↗
PubMed1985

Rat liver glutathione S-transferases. Construction of a cDNA clone complementary to a Yc mRNA and prediction of the complete amino acid sequence of a Yc subunit.

Using polysomal immunoselected rat liver glutathione S-transferase mRNAs, we have constructed cDNA clones using DNA polymerase I, RNase H, and Escherichia coli ligase (NAD+)-mediated second strand cDNA synthesis as described by Gubler and Hoffman (Gubler, U., and Hoffman, B. S. (1983) Gene 25, 263-269). Recombinant clone, pGTB42, contained a cDNA insert of 900 base pairs whose 3' end showed specificity for the Yc mRNA in hybrid-select translation experiments. The nucleotide sequence of pGTB42 has been determined, and the complete amino acid sequence of a Yc subunit has been deduced. The cDNA clone contains an open reading frame of 663 nucleotides encoding a polypeptide comprising 221 amino acids with a molecular weight of 25,322. The NH2-terminal sequence deduced from pGTB42 is in agreement with the first 39 amino acids determined for a Ya-Yc heterodimer by conventional protein-sequencing techniques. A comparison of the nucleotide sequence of pGTB42 with the sequence of a Ya clone, pGTB38, described previously by our laboratory (Pickett, C. B., Telakowski-Hopkins, C. A., Ding, G. J.-F., Argenbright, L., and Lu, A.Y.H. (1984) J. Biol. Chem. 259, 5182-5188) reveals a sequence homology of 66% over the same regions of both clones; however, the 5'- and 3'-untranslated regions of the Ya and Yc mRNAs are totally divergent in their sequences. The overall amino acid sequence homology between the Ya and Yc subunits is 68%, however, the NH2-terminal domain is more highly conserved than the middle or carboxyl-terminal domains. Our data suggest that the Ya and Yc subunits of the rat liver glutathione S-transferases are products of two different mRNAs which are derived from two related yet different genes.

Amino Acid Sequence↗
PubMed1985

2-[3-(1,1-Dimethylethyl)-5-methoxyphenyloxazolo[4,5-b]pyridine, a new topical antiinflammatory and analgesic compound lacking systemic activity and gastric side effects.

The substituted oxazolopyridine 2-[3-(1,1-dimethylethyl)-5-methoxyphenyl]oxazolo[4,5-b]pyridine (OZP) inhibits phorbol myristate acetate-induced increases in vascular permeability and neutrophil accumulation in rat ears with ED50 of 253 and 200 micrograms, respectively. This compound is as potent as indomethacin to inhibit UV-induced erythema in guinea pig skin and is an effective analgesic when applied topically to the rat footpad in the yeast hyperalgesia model. OZP is a cyclooxygenase inhibitor with an IC50 of 0.06 mumol/l and inhibits prostaglandin E2, but not leukotriene C4 synthesis, by mouse peritoneal macrophages. This compound is inactive in the carrageenan paw edema assay at 90 mg/kg when administered orally or intraperitoneally, but is effective when injected into the paw. OZP is not a contact allergen and does not cause gastric irritation in rats at doses up to 180 mg/kg orally. OZP is rapidly metabolized by rat liver microsomes in a concentration and time dependent manner. Furthermore, when administered orally, OZP is cleared rapidly in rats with plasma levels being detected only at 5 and 30 min following a 2 mg/kg dose. There was no drug in the gastrointestinal tract of rats 3 h after an oral dose. Thus, this compound appears to be a new, potent and safe topical antiinflammatory and an analgesic agent lacking systemic effects.

Analgesics↗
PubMed1984

Rat liver glutathione S-transferases. Complete nucleotide sequence of a glutathione S-transferase mRNA and the regulation of the Ya, Yb, and Yc mRNAs by 3-methylcholanthrene and phenobarbital.

With the use of cDNA probes reverse transcribed from purified glutathione S-transferase mRNA templates, four cDNA clones complementary to transferase mRNAs have been identified and characterized. Two clones, pGTB38 and pGTB34, have cDNA inserts of approximately 950 and 900 base pairs, respectively, and hybridize to a mRNA(s) whose size is approximately 980 nucleotides. In hybrid-select translation experiments, pGTB38 and pGTB34 select mRNAs specific for the Ya and Yc subunits of rat liver glutathione S-transferases. Clone pGTB33, which harbors a truncated cDNA insert, hybrid-selects only the Ya mRNA. All of the clones, pGTB38, pGTB34, and pGTB33, hybrid-select another mRNA which is specific for a polypeptide with an electrophoretic mobility slightly greater than the Ya subunit. The entire nucleotide sequence of the full length clone, pGTB38, has been determined and the complete amino acid sequence of the corresponding polypeptide has been deduced. The mRNA codes for a protein comprising 222 amino acids with Mr = 25,547. We have also identified a cDNA clone complementary to a Yb mRNA of the rat liver glutathione S-transferases. This clone, pGTA/C36, hybrid-selects only Yb mRNA(s) and hybridizes to a mRNA(s) whose size is approximately 1200 nucleotides. Although the Ya, Yb, and Yc mRNAs are elevated coordinately by phenobarbital and 3-methylcholanthrene, the Ya-Yc mRNAs are induced to a much greater extent compared to the Yb mRNA(s). These data suggest that the mRNAs for each transferase isozyme are regulated independently.

Amino Acid Sequence↗
PubMed1984

Kinetic isotope effects on cytochrome P-450-catalyzed oxidation reactions. The oxidative O-dealkylation of 7-ethoxycoumarin.

The primary deuterium and tritium isotope effects on Vm/Km and on Vm have been measured for the O-deethylation of 7-ethoxycoumarin catalyzed by two purified isozymes of cytochrome P-450. From these data the intrinsic isotope effects have been calculated as described by D. B. Northrop (Biochemistry (1975) 14, 2644-2651). The observed deuterium isotope effects on Vm/Km are 3.79 and 1.90 for the isozymes isolated from the livers of rats induced by phenobarbital and 3-methylcholanthrene, respectively. The calculated intrinsic isotope effects, however, are similar and much larger (kH/kD = 12.8 to 14.0) than the observed isotope effects on Vm/Km for the two enzymes. This demonstrates that the intrinsic isotope effects are attenuated by various steps preceding the isotopically sensitive C-H bond cleavage step resulting in the low values for the observed isotope effects. Thus, the observed isotope effects do not accurately reflect the magnitude of the intrinsic isotope effect associated with this reaction. No incorporation of 18O into the 7-hydroxycoumarin product was observed in studies employing H218O or 18O2 demonstrating that the phenolic oxygen arises exclusively from the substrate. Taken together, these data provide compelling evidence that both cytochrome P-450 isozymes catalyze the O-dealkylation of this substrate by an identical radical recombination mechanism during the obligatory formation of a hemiacetal intermediate.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1984

Kinetic isotope effects on cytochrome P-450-catalyzed oxidation reactions. Evidence for the irreversible formation of an activated oxygen intermediate of cytochrome P-448.

Substitution of deuterium for hydrogen on the alpha-carbon of 7-ethoxycoumarin results in an intrinsic isotope effect of approximately 14 during the cytochrome P-448-catalyzed O-deethylation of this substrate (G. T. Miwa, J. S. Walsh, and A. Y. H. Lu (1984) J. Biol. Chem. 259, 3000-3004). This dramatic decrease in the C-H bond cleavage rate does not, however, alter the rate of 7-ethoxycoumarin disappearance or the rates of NADPH and oxygen consumption indicating that the catalytic turnover of this enzyme is unaffected. Moreover, hydrogen peroxide formation and the concentrations of an oxycytochrome P-448 complex (lambda max = 440 nm) are also unchanged demonstrating that the steady state concentrations of various oxy intermediates of cytochrome P-448 are also unchanged. An inescapable conclusion from these data is that an irreversible step exists between the formation of these intermediates and the oxidation of the substrate. These data are in agreement with the view that an irreversible cleavage of the dioxygen bond precedes substrate oxidation. Moreover, the oxidatively competent form of the cytochrome must then be committed to substrate oxidation. The latter conclusion is substantiated from high performance liquid chromatography studies which demonstrate the formation of a second metabolite, 6-hydroxy-7-ethoxycoumarin, from the deuterated substrate arising from the metabolic switching away from the O-ethyl group to the aromatic ring of this substrate.

Animals↗