Biomedical subjects

M W Duffel

Publications and source records attributed to M W Duffel.

At least 19 recordsLinked to original sources

PubMed2001

Mechanism of lower genotoxicity of toremifene compared with tamoxifen.

An increased incidence of endometrial cancer has been reported in breast cancer patients taking tamoxifen (TAM) and in healthy women participating in the TAM chemoprevention trials. Because TAM-DNA adducts are mutagenic and detected in the endometrium of women treated with TAM, TAM adducts are suspected to initiate the development of endometrial cancer. Treatment with TAM has been known to promote hepatocarcinoma in rats, but toremifene (TOR), a chlorinated TAM analogue, did not. TAM adducts are primarily formed via sulfonation of the alpha-hydroxylated TAM metabolites. To explore the mechanism of the lower genotoxicity of TOR, the formation of DNA adducts induced by TOR metabolites was measured using (32)P-postlabeling/ high-performance liquid chromatography analysis and compared with that of TAM metabolites. When alpha-hydroxytoremifene was incubated with DNA, 3'-phosphoadenosine 5'-phosphosulfate, and either rat or human hydroxysteroid sulfotransferase, the formation of DNA adducts was two orders of magnitude lower than that of alpha-hydroxytamoxifen. alpha-hydroxytoremifene was a poor substrate for rat and human hydroxysteroid sulfotransferases. In addition, the reactivity of alpha-acetoxytoremifene, a model activated form of TOR, with DNA was much lower than that of alpha-acetoxytamoxifen. Thus, TOR is likely to have lower genotoxicity than TAM. TOR may be a safer alternative by avoiding the development of endometrial cancer.

Animals↗

PubMed2001

Bacterial expression, purification, and characterization of rat hydroxysteroid sulfotransferase STa.

Hydroxysteroid (alcohol) sulfotransferase catalyzes numerous reactions that are important to our understanding of the metabolism of both endogenous steroids and exogenous alcohols. Here we report a method for prokaryotic expression and rapid purification of the recombinant hydroxysteroid sulfotransferase STa, a major isoform of hydroxysteroid sulfotransferase in the rat. The cDNA encoding STa was cloned into a pET-3c vector and expressed in Escherichia coli BL21 cells. After disruption of the cells by sonication, the enzyme was purified in one step by affinity chromatography on adenosine 3',5'-diphosphate-agarose. The purified recombinant STa had a relative molecular mass on SDS-PAGE that was identical with the native hepatic STa in rat liver. The expressed enzyme displayed similar substrate inhibition characteristics with dehydroepiandrosterone as have been noted previously with the native enzyme purified from rat liver. Furthermore, the catalytic efficiency in sulfation of 7-hydroxymethyl-12-methylbenz[a]anthracene, as well as the stereoselectivity of sulfation of the enantiomers of 1-phenyl-1-heptanol and 1-naphthyl-1-ethanol, catalyzed by the recombinant STa were consistent with characteristics of the STa isolated from rat liver.

Animals↗

PubMed2000

Structure-function modeling of the interactions of N-alkyl-N-hydroxyanilines with rat hepatic aryl sulfotransferase IV.

Although previous investigations have clearly shown that N-hydroxy arylamines and N-hydroxy heterocyclic amines are substrates for sulfotransferases, relatively little is known about which structural features of the N-hydroxy arylamines are important for sulfation to occur. The purpose of this investigation was to determine the extent to which secondary N-alkyl-N-hydroxy arylamines interact with aryl sulfotransferase (AST) IV (also known as tyrosine-ester sulfotransferase or ST1A1) and to evaluate these interactions using molecular modeling techniques. AST IV is a major cytosolic sulfotransferase in the rat, and it catalyzes the sulfation of various phenols, benzylic alcohols, arylhydroxamic acids, oximes, and primary N-hydroxy arylamines. In this study, three secondary N-hydroxy arylamines, N-hydroxy-N-methylaniline, N-ethyl-N-hydroxyaniline, and N-hydroxy-N-n-propylaniline, were found to be substrates for the purified rat hepatic AST IV. However, when the N-alkyl substituent was an n-butyl group (i.e., N-n-butyl-N-hydroxyaniline), the interaction with the enzyme changed from that of a substrate to competitive inhibition. This change in specificity was further explored through the construction and use of a model for AST IV based on mouse estrogen sulfotransferase, an enzyme whose crystal structure has been previously determined to high resolution. Molecular modeling techniques were used to dock each of the above N-hydroxy arylamines into the active site of the homology model of AST IV and determine optimum ligand geometries. The results of these experiments indicated that steric constraints on the orientation of binding of secondary N-alkyl-N-hydroxy arylamines at the active site of AST IV play a significant role in determining the nature of the interaction of the enzyme with these compounds.

Aminophenols↗

PubMed2000

In vitro and ex vivo hydrolysis rates of ethacrynate esters and their relationship to intraocular pressure in the rabbit eye.

Esters of ethacrynic acid and partial structural analogs were synthesized and evaluated for topical antiglaucoma activity in rabbits. Maximum activity was shown by analogs 2 and 6 (34% and 30% reduction in intraocular pressure recovery rate, respectively). Among the esters, only the ethyl ester (2) was found to be active; the methyl and n-propyl esters (1 and 3) were inactive. Analogs 1-3 were subjected to an estimation of physicochemical properties and chemical stability. However, no correlation was found to exist between the biological activity/inactivity and the physicochemical properties of the analogs. The analogs were evaluated for ex vivo hydrolysis using rabbit aqueous humor (AH), corneal (C) homogenate and iris-ciliary body (ICB) homogenate. For all tissues, the rate of enzymatic hydrolysis increased significantly with an increasing ester chain length. The ICB-mediated hydrolysis was the fastest among the three tissues for all of the analogs. The relationship between the rate constants for the tissue-mediated hydrolyses were: analog 1, ICB>C>AH; analog 2, ICB>C=AH and analog 3, ICB>AH>C. Apparent Michaelis-Menten kinetic parameters were determined for the three analogs using corneal homogenate. Analog 2 showed the highest v0 for all substrate concentrations studied. The conventional Michaelis-Menten equation did not fit the data as well as a sigmoidal model. Both fits of the data showed the fastest enzyme-mediated hydrolysis for analog 2. The parameters of the sigmoidal fit of the data correlated with the activity/inactivity of the analogs. The data indicate that the major factors responsible for the observed activity/inactivity are the differences in the corneal enzymatic hydrolysis of the esters in conjunction with the rapid dynamics of ocular prodrug absorption.

Administration, Topical↗

PubMed1999

Importance of peri-interactions on the stereospecificity of rat hydroxysteroid sulfotransferase STa with 1-arylethanols.

Hydroxysteroid (alcohol) sulfotransferases catalyze the sulfation of polycyclic aromatic hydrocarbons (PAHs) that contain benzylic hydroxyl functional groups. This metabolic reaction is often a critical step in the activation of a hydroxyalkyl-substituted PAH to form an electrophilic metabolite that is capable of forming covalent bonds at nucleophilic sites on DNA, RNA, and proteins. Since hydroxyalkyl-substituted PAHs are often metabolically formed by the stereoselective enzymatic hydroxylation of a benzylic position on an alkyl-substituted PAH, we have investigated the possibility that the sulfation of hydroxyalkyl aromatic hydrocarbons is also stereoselective. Homogeneous preparations of rat hepatic hydroxysteroid (alcohol) sulfotransferase STa were utilized to investigate the stereoselectivity of its catalytic function with the enantiomers of model 1-arylethanols. While only minimal stereoselectivity was observed for the catalytic efficiency of STa with the enantiomers of 1-(2-naphthyl)ethanol and 1-acenaphthenol, the enzyme was stereospecific for (R)-(+)-1-(1-naphthyl)ethanol, (R)-(+)-1-(1-pyrenyl)ethanol, and (R)-(+)-1-(9-phenanthryl)ethanol as substrates. Moreover, (S)-(-)-1-(1-naphthyl)ethanol, (S)-(-)-1-(1-pyrenyl)ethanol, and (S)-(-)- 1-(9-phenanthryl)ethanol were competitive inhibitors of STa. Structural and conformational analyses of these 1-arylethanols indicated that steric interactions between the substituents on the benzylic carbon and the hydrogen in the peri-position on the aromatic ring system were important determinants of the stereospecificity of the enzyme with these molecules. The findings presented here have implications for the more accurate prediction of the ability of hydroxyalkyl-substituted PAHs to be activated via metabolic formation of electrophilic sulfuric acid esters.

Algorithms↗

PubMed1998

Studies on an affinity label for the sulfuryl acceptor binding site in an aryl sulfotransferase.

Active site-directed affinity labeling was utilized to elucidate peptide sequences at the binding site for sulfuryl acceptors in rat hepatic aryl sulfotransferase (AST) IV (also known as tyrosine-ester sulfotransferase, EC 2.8.2.9). The affinity labeling reagent, N-bromoacetyl-4-hydroxyphenylamine, was designed on the basis of substrate specificity studies with para-substituted phenols, utilization of a bromoacetamido group for reactivity with active site amino acid residues and its similarity to acetaminophen, a known substrate for aryl (phenol) sulfotransferases. AST IV utilized N-bromoacetyl-4-hydroxyphenylamine as a substrate with kinetic constants that compared favorably to those obtained with acetaminophen. Incubation of AST IV with N-bromoacetyl-4-hydroxyphenylamine at pH 7.0 in the absence of PAPS and other substrates resulted in an irreversible inactivation of the enzyme that was both time- and concentration-dependent. [14C]-N-bromoacetyl-4-hydroxyphenylamine was synthesized and used to analyze the regions of protein sequence that were involved in the binding of the affinity label. AST IV was incubated with [14C]-N-bromoacetyl-4-hydroxyphenylamine, hydrolyzed with endoproteinase Lys-C and the labeled peptides were purified by HPLC. Control incubations of AST IV with the affinity label in the presence of 4-propylphenol and PAP were utilized to ascertain the specificity of the interaction. Sequence analysis of the labeled peptides, carried out by automated Edman degradation, revealed labeling sites on cysteine (Cys-232, Cys-283 and Cys-289) and lysine (Lys-286) residues near the C-terminus of the protein. The locations of these labeling sites were further evaluated both by sequence-alignment with other sulfotransferases and by theoretical calculations on predicted secondary structure.

Affinity Labels↗

PubMed1998

Alpha-hydroxytamoxifen is a substrate of hydroxysteroid (alcohol) sulfotransferase, resulting in tamoxifen DNA adducts.

When alpha-hydroxytamoxifen (alpha-OHTAM) was incubated with rat liver hydroxysteroid (alcohol) sulfotransferase a (STa) and 3'-phosphoadenosine 5'-phosphosulfate, (E)-alpha-OHTAM was found to be a better substrate for STa than (Z)-alpha-OHTAM. To explore the formation of tamoxifen (TAM)-derived DNA adducts, DNA was incubated with STa and either (E)-alpha-OHTAM or (Z)-alpha-OHTAM in the presence of 3'-phosphoadenosine 5'-phosphosulfate. Using 32P-postlabeling analysis, the amount of TAM-DNA adducts resulting from (E)-alpha-OHTAM was 29 times higher than that observed with (E)-alpha-OHTAM alone. Using (Z)-alpha-OHTAM and STa, some TAM-DNA adducts were also detected but at levels 6.5 times lower than that observed with (E)-alpha-OHTAM and STa. When compared with standards of stereoisomers of 2'-deoxyguanosine 3'-monophosphate-N2-tamoxifen, the major tamoxifen adduct was identified chromatographically as an epimer of the trans form of alpha-(N2-deoxyguanosinyl)tamoxifen, and the minor adduct was identified as an epimer of the cis form. In the reaction mixture, a conversion from (E)-alpha-OHTAM to (Z)-alpha-OHTAM through the carbocation intermediate was also detected. These results show that sulfation of alpha-OHTAM catalyzed by STa results in the formation of TAM-DNA adducts.

Animals↗

PubMed1998

Sulfation of alpha-hydroxytamoxifen catalyzed by human hydroxysteroid sulfotransferase results in tamoxifen-DNA adducts.

The formation of tamoxifen (TAM)-derived DNA adducts was investigated by incubation of DNA with (E)-alpha-hydroxytamoxifen [(E)-alpha-OHTAM], 3'-phosphoadenosine 5'-phosphosulfate (PAPS), and human recombinant sulfotransferase. Using 32P-post-labeling and HPLC analysis, two TAM-DNA adducts were detected in incubations that included the human hydroxysteroid sulfotransferase SULT2A1 (hHST). When compared with standards of stereoisomers of alpha-(N2-deoxyguanosinyl)tamoxifen 3'-monophosphate (dG3'P-N2-TAM), the major adduct was identified chromatographically as an epimer of the transform of dG-N2-TAM, and the minor adduct was identified as an epimer of the cis-form. The amount of TAM adducts formed by hHST was approximately three times less than that formed by an equivalent amount of rat hydroxysteroid (alcohol) sulfotransferase a. These results indicate that sulfation of alpha-OHTAM catalyzed by hHST results in the formation of dG-N2-TAMs, highly miscoding lesions, in human tissues.

Animals↗

PubMed1997

Oxidations of vincristine catalyzed by peroxidase and ceruloplasmin.

The dimeric Catharanthus alkaloid vincristine (1) is oxidized to the same ring fission product in incubations with either horseradish peroxidase or the human serum copper oxidase ceruloplasmin. Horseradish peroxidase-catalyzed oxidation of vincristine requires hydrogen peroxide, whereas ceruloplasmin-catalyzed oxidation of vincristine requires chlorpromazine as a "shuttle oxidant". Preparative-scale incubations allowed for the production, isolation, structural characterization, and biological evaluation of the metabolite. The metabolite was identified as the heterocyclic ring cleavage product N-formylcatharinine (5). N-Formylcatharinine was 118 times less active than vincristine in an in vitro test against a human T-cell leukemic cell line. Therefore, these enzyme-catalyzed reactions lead to bioinactivation of vincristine.

Antineoplastic Agents, Phytogenic↗

PubMed1997

Oxidation-dependent inactivation of aryl sulfotransferase IV by primary N-hydroxy arylamines during in vitro assays.

The sulfation of primary N-hydroxy arylamines is a critical intermediate step in the bioactivation of many carcinogenic arylamines, arylamides and nitroaromatics. However, the study of this reaction in vitro is often complicated by the chemical instability of these molecules. We have examined the stability of two highly purified N-hydroxy arylamines, N-hydroxyaniline and N-hydroxy-2-aminofluorene, under different oxidative reaction conditions pertinent to the assay of sulfotransferases. Furthermore, these compounds, as well as the products of their oxidative degradation, were examined for their interactions with homogeneous aryl sulfotransferase (AST) IV. Under reaction conditions where oxidative degradation of the N-hydroxy arylamines occurred, N-hydroxyaniline and N-hydroxy-2-aminofluorene produced time-dependent and irreversible inhibition of AST IV. While this inhibition was not dependent upon the presence of 3'-phosphoadenosine 5'-phosphosulfate in the reaction mixture, analysis of the N-hydroxy arylamines by UV spectroscopy showed that the inhibition of AST IV did require non-enzymatic oxidation of the N-hydroxy arylamine. Under reaction conditions that prevented the oxidative degradation of N-hydroxyaniline, this N-hydroxy arylamine was a substrate for AST IV. Likewise, under similar conditions, 4-chloro-N-hydroxyaniline was also a substrate for the enzyme. In contrast, no AST IV catalyzed sulfation of N-hydroxy-2-aminofluorene was detected under conditions that prevented the oxidation of N-hydroxy-2-aminofluorene. Adequate protection of these N-hydroxy arylamines from oxidative degradation required the addition of L-ascorbic acid to reaction mixtures that had also been degassed and purged with argon. The irreversible inhibition of AST IV exhibited by these N-hydroxy arylamines, even in reaction mixtures where attempts were made to limit oxidative degradation by degassing and purging with argon, emphasized the importance of completely preventing such degradation when utilizing in vitro assays to assess the potential for an N-hydroxy arylamine to serve as a substrate for a specific sulfotransferase.

Amines↗

PubMed1997

Studies on the interactions of chiral secondary alcohols with rat hydroxysteroid sulfotransferase STa.

Hydroxysteroid (alcohol) sulfotransferase STa catalyzes the 3'-phosphoadenosine 5'-phosphosulfate-dependent O-sulfonation of a diverse array of alcohols including neutral hydroxysteroids. Many of the secondary alcohols that interact with this sulfotransferase are the metabolic products of stereoselective oxidation or reduction reactions. The role that the stereochemistry of secondary alcohol substrates plays in the catalytic efficiency of STa was investigated with a series of chiral benzylic alcohols and the enantiomeric 3-hydroxyl-containing steroids, androsterone and epiandrosterone. In the case of (R)-(+)- and (S)-(-)-enantiomers of 2-methyl-1-phenyl-1-propanol and 1-phenyl-1-butanol, the effect of stereochemistry on the catalytic efficiency of STa was small (less than 2-fold in favor of (R)-(+)-enantiomers). However, as the number of carbons in the alpha-alkyl chain increased, the stereoselectivity for the sulfation of enantiomers increased as well. The (R)-(+)-enantiomers of 1-phenyl-1-pentanol, 1-phenyl-1-hexanol, and 1-phenyl-1-heptanol were preferred as substrates over the (S)-(-)-enantiomers with a 3-fold difference in catalytic efficiency. STa showed absolute stereospecificity in the sulfation of the enantiomers of 1-phenyl-1-cyclohexylmethanol; (R)-(+)-1-phenyl-1-cyclohexylmethanol was a substrate for STa, while the (S)-(-)-enantiomer was a competitive inhibitor of the enzyme. Although a lower degree of stereoselectivity was observed with the 3-hydroxyl-containing steroids, androsterone and epiandrosterone, results with these substrates were also consistent with the conclusion that the stereochemistry of secondary alcohols is an important factor in the catalytic efficiency of STa.

Alcohols↗

PubMed1995

Enzyme- and sex-specific differences in the intralobular localizations and distributions of aryl sulfotransferase IV (tyrosine-ester sulfotransferase) and alcohol (hydroxysteroid) sulfotransferase a in rat liver.

Aryl sulfotransferase (AST) IV and alcohol (hydroxysteroid) sulfotransferase a (STa) catalyze the formation of sulfuric acid esters from a diverse array of xenobiotic and endogenous molecules in the liver. Despite the fact that many studies have addressed the metabolic importance and catalytic characteristics of these two sulfotransferases, relatively little is known about their comparative in situ localizations and intralobular distributions in liver. The present investigation utilized specific rabbit antisera prepared against AST IV and STa for immunoperoxidase staining of serial sections from livers of male and female Sprague-Dawley rats and computer-assisted image analysis of immunohistochemical staining intensity by means of microdensitometry. The overall concentration of AST IV was greater in males than in females, although the intralobular distribution of the enzyme was similar in the livers of both male and female rats, wherein centrilobular hepatocytes contained a greater level of AST IV than did midzonal cells, and midzonal hepatocytes had a greater concentration of AST IV than did periportal hepatocytes. In marked contrast, STa was present in livers of female rats at a much greater overall concentration than in livers of male rats. Furthermore, whereas the intralobular distribution of the enzyme was similar in both males and females, STa was present at greater concentrations in periportal hepatocytes than in midzonal hepatocytes and at greater concentrations in midzonal cells than in centrilobular hepatocytes. Significant intrazonal heterogeneity in STa levels within hepatocytes was also observed, particularly in livers of female rats. These results indicate that, whereas the overall hepatic concentrations of these enzymes are clearly sex-dependent, the intralobular distributions of AST IV and STa are characteristic of each particular sulfotransferase.

Amino Acid Sequence↗

PubMed1994

Affinity labeling of aryl sulfotransferase IV. Identification of a peptide sequence at the binding site for 3'-phosphoadenosine-5'-phosphosulfate.

2'-O-[(R)-Formyl(adenin-9-yl)-methyl]-(S)-glyceraldehyde 3'-triphosphate (also designated as ATP dialdehyde or ATPDA) was utilized as an affinity label for the 3'-phosphoadenosine 5'-phosphosulfate (PAPS) binding site of an aryl sulfotransferase. The sulfotransferase employed in these studies was rat hepatic aryl sulfotransferase (AST) IV (also known as tyrosine-ester sulfotransferase, EC 2.8.2.9), for which a cDNA had been previously cloned and expressed in Escherichia coli and the resulting enzyme purified to homogeneity. ATPDA was a time-dependent irreversible inhibitor of the recombinant AST IV, and this inhibition was prevented by including either PAPS or adenosine 3',5'-diphosphate (PAP) in the incubation of AST IV with ATPDA. Experiments relating covalent binding of [2,8-3H]ATPDA with catalytic activity indicated that 1 nmol of the affinity label was bound per nmol of AST IV subunit. Incubation of [2,8-3H]ATPDA with the enzyme followed by reduction with sodium cyanoborohydride, proteolysis with trypsin, and separation of the resulting peptides by high pressure liquid chromatography yielded two labeled peptide fractions. Automated sequence analysis showed that both modified peptide fractions were derived from the same sequence in AST IV: 63-Leu-Glu-Lys-Cys-Gly-Arg-68. Both the sequencing results and examination of the two peptide fractions by matrix-assisted laser desorption ionization mass spectrometry indicated that the ATPDA affinity label was bound to the hexapeptide at both lysine 65 and cysteine 66. These affinity labeled amino acids are located within a region of sequence in AST IV that shows considerable homology with various sulfotransferases that possess diverse specificities for acceptor substrates, and this may provide insight into PAPS binding in other sulfotransferases.

Adenosine Triphosphate↗

PubMed1994

Evidence of two separate mechanisms for the decrease in aryl sulfotransferase activity in rat liver during early stages of 2-acetylaminofluorene-induced hepatocarcinogenesis.

Enzymatic and immunohistochemical experiments were conducted to evaluate the mechanistic basis for the downregulation of the important detoxication/bioactivation enzyme aryl sulfotransferase IV (AST IV) during 2-acetylaminofluorene (2AAF)-induced hepatocarcinogenesis. To distinguish between possible genotoxic and cytotoxic actions of 2AAF, three different dietary protocols were used in these experiments: group 1 received 2AAF for 12 wk, group 2 received 2AAF for 3 or 6 wk and then a control diet lacking xenobiotics for 3 or 6 wk, and group 3 received 2AAF for 3 or 6 wk and then phenobarbital for 3 or 6 wk. When hepatic AST IV activity was assessed, N-hydroxy-2AAF sulfotransferase activity was found to decrease 80-90% in response to 2AAF feeding, but activity recovered to essentially normal levels in the livers of rats subsequently placed on either control diets or diets with phenobarbital, suggesting a reversible cytotoxic mechanism for loss of AST IV activity. However, when liver sections from the rats were evaluated immunohistochemically, two distinct patterns were detected for the downregulation of AST IV activity. In the livers of rats administered only 2AAF (group 1), a general pattern of overall downregulation of AST IV expression was observed throughout the liver and among most but not all newly developed nodules. In tissue sections from rats initially fed 2AAF and then placed on a control diet (group 2) or a diet with phenobarbital (group 3), the nodules continued to show low levels of AST IV expression, while expression in the areas surrounding nodules returned to the normal, high levels. In addition, among those rats fed 2AAF for just 3 wk and then control diet or diet containing phenobarbital for 6 wk, only rats fed phenobarbital developed altered foci that stained weakly for AST IV expression. These results show that there were two kinds of 2AAF-mediated decrease in hepatic AST IV activity: a general overall loss of AST IV expression dependent on administration of 2AAF and reversible upon removal of 2AAF from the diet and a loss of AST IV expression among newly developed liver foci and nodules that persisted in the absence of 2AAF administration and appeared to be a property of 2AAF-induced subpopulations of cells. These patterns may correspond, respectively, to cytotoxic and genotoxic mechanisms of 2AAF action.

2-Acetylaminofluorene↗

PubMed1994

Molecular specificity of aryl sulfotransferase IV (tyrosine-ester sulfotransferase) for xenobiotic substrates and inhibitors.

Studies on the interactions of benzylic alcohols, aldehydes, and carboxylic acids with homogeneous preparations of aryl sulfotransferase (AST) IV have yielded information about the nature of the active site of the enzyme. Lipophilicity and stereochemical configuration of benzylic alcohols are key factors in determining their interaction with the active site of AST IV. Furthermore, aldehydes and carboxylic acids corresponding to the subsequent oxidation states derived from benzylic alcohols are inhibitors of the enzyme. Additional investigations on the catalytic specificity of AST IV indicate that both primary and secondary N-hydroxy arylamines can serve as substrates for the enzyme. These results with benzylic alcohols, aldehydes, carboxylic acids, and N-hydroxy arylamines have yielded insight into some of the parameters important in recognition of substrates and inhibitors by the active site of the enzyme and should be useful both in understanding in vivo metabolic interactions and in designing appropriate new inhibitors to use as selective probes for the role of sulfation in metabolism of specific xenobiotics.

Aldehydes↗

PubMed1992

Tyrosine-ester sulfotransferase from rat liver: bacterial expression and identification.

A nucleotide sequence that had been proposed for, but not identified as, rat liver aryl sulfotransferase (EC 2.8.2.1) was prepared in an appropriate vector and transformed into Escherichia coli. The protein, expressed in large amounts, was not aryl sulfotransferase (EC 2.8.2.1) but rather tyrosine-ester sulfotransferase (EC 2.8.2.9), a sulfotransferase also active with phenols but having a much wider substrate range that includes hydroxylamines and esters of tyrosine. The recombinant tyrosine-ester sulfotransferase was identified by its unique substrate spectrum, by comparison with three peptides that were sequenced from homogeneous tyrosine-ester sulfotransferase isolated directly from rat liver, and by the specificity of antibody raised to the rat liver enzyme. Two isoforms were obtained, each of which was difficult to solubilize upon sonication of E. coli. Both forms were solubilized with a solution of polyols (glycerol and sucrose) and subsequently purified to homogeneity.

Amino Acid Sequence↗

PubMed1992

Metabolism of the Catharanthus alkaloids: from Streptomyces griseus to monoamine oxidase B.

More than three decades after their discovery and implementation in medicine, essentially nothing is known about the metabolism or the implications of metabolism in mechanism of action or toxicity of the Catharanthus alkaloids. The frustrating paucity of information about pathways of metabolism has limited a major source of structure-activity relationship information and has blocked a critical avenue necessary for the logical development of new and more useful Catharanthus alkaloids. Microbial transformations, peroxidases, copper oxidases, mouse and rat cytochrome P-450 systems, and mouse brain and bovine liver monoamine oxidase (MAO) preparations have been explored in the study of Catharanthus alkaloid metabolism. In this report, we present results which have clarified the involvement of enzymatic and chemically catalyzed one-electron oxidations that yield nitrogen-centered cation radicals, iminium, and carbinolamine intermediates, all of which explain how new carbon-carbon and carbon-oxygen bonds form, or break and rearrange. The dimeric Catharanthus alkaloids are recalcitrant to oxidations catalyzed by monoamine oxidases and to both normal and induced P-450 rat microsomal preparations. However, the Catharanthus alkaloids appear to be selective reversible inhibitors of MAO-B. Chemical and biochemical aspects of the metabolic transformations of dimeric Catharanthus alkaloids are reviewed together with the implications of our findings.

Animals↗

PubMed1992

Inhibition of rat hepatic aryl sulphotransferase IV by dihydrodiol derivatives of benzo[a]pyrene and naphthalene.

1. Although neither the (+)- nor (-)-enantiomer of trans-benzo[a]pyrene-7,8-dihydrodiol was a substrate for aryl sulphotransferase IV from rat liver, both enantiomers inhibited the enzyme-catalysed sulphation of 1-naphthalene-methanol with Ki values of 3.7 +/- 0.4 microM for the (+)-enantiomer, and 4.4 +/- 0.3 microM for the (-)-enantiomer. 2. Based on the magnitude of the Ki values, the binding affinity of these dihydrodiols for the aryl sulphotransferase was significantly greater than that for the corresponding phenolic derivatives of benzo[a]pyrene. That is 7-hydroxybenzo[a]pyrene and 8-hydroxybenzo[a]pyrene were both substrates for aryl sulphotransferase IV, with apparent Km values of 280 +/- 41 microM and 370 +/- 72 microM, respectively. 3. Both (+)- and (-)-trans-naphthalene-1,2-dihydrodiols were also inhibitors of aryl sulphotransferase IV, but with higher Ki values than would be expected from previously determined apparent Km and Ki values for (R)-(-)- and (S)-(+)-1,2,3,4-tetrahydro-1-naphthols, respectively.

Animals↗