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Biomedical subjects

M W Duffel

Publications and source records attributed to M W Duffel.

At least 37 records · Page 2Linked to original sources
PubMed1991

Benzylic alcohols as stereospecific substrates and inhibitors for aryl sulfotransferase.

Aryl sulfotransferase IV catalyzes the 3'-phosphoadenosine-5'-phosphosulfate (PAPS)-dependent formation of sulfuric acid esters of benzylic alcohols. Since the benzylic carbon bearing the hydroxyl group can be asymmetric, the possibility of stereochemical control of substrate specificity of the sulfotransferase was investigated with benzylic alcohols. Benzylic alcohols of known stereochemistry were examined as potential substrates and inhibitors for the homogeneous enzyme purified from rat liver. For 1-phenylethanol, both the (+)-(R)- and (-)-(S)-enantiomers were substrates for the enzyme, and the kcat/Km value for the (-)-(S)-enantiomer was twice that of the (+)-(R)-enantiomer. The enzyme displayed an absolute stereospecificity with ephedrine and pseudoephedrine, and with 2-methyl-1-phenyl-1-propanol; that is, only (-)-(1R,2S)-ephedrine, (-)-(1R,2R)-pseudoephedrine, and (-)-(S)-2-methyl-1-phenyl-1-propanol were substrates for the sulfotransferase. In the case of 1,2,3,4-tetrahydro-1-naphthol, only the (-)-(R)-enantiomer was a substrate for the enzyme. Both (+)-(R)-2-methyl-1-phenyl-1-propanol and (+)-(S)-1,2,3,4-tetrahydro-1-naphthol were competitive inhibitors of the aryl sulfotransferase-catalyzed sulfation of 1-naphthalenemethanol. Thus, the configuration of the benzylic carbon bearing the hydroxyl group determined whether these benzylic alcohols were substrates or inhibitors of the rat hepatic aryl sulfotransferase IV. Furthermore, benzylic alcohols such as (+)-(S)-1,2,3,4-tetrahydro-1-naphthol represent a new class of inhibitors for the aryl sulfotransferase.

Animals↗
PubMed1991

Purification, immunochemical characterization, and immunohistochemical localization of rat hepatic aryl sulfotransferase IV.

Aryl sulfotransferases catalyze the formation of sulfuric acid esters from a diverse group of endogenous and xenobiotic organic chemicals. The isoenzyme of aryl sulfotransferase in livers of male Sprague-Dawley rats that exhibits the most varied substrate specificity is aryl sulfotransferase IV. A new method for the purification to homogeneity of aryl sulfotransferase IV was developed that, when compared with previously described procedures, provided a greater than 10-fold increase in total yield of enzyme/g of tissue. Homogeneous aryl sulfotransferase IV was used to prepare polyclonal antibodies in male New Zealand White rabbits. Results of immunochemical analyses demonstrated that these antibodies reacted with only a single protein in rat hepatic 100,000 x g supernatant fractions and, further, that the immunoreactive protein had the isoelectric point and subunit molecular mass characteristic of aryl sulfotransferase IV. Immunohistochemical analyses demonstrated that aryl sulfotransferase IV is present in hepatocytes throughout the liver, although centrilobular cells contain a significantly greater (p less than 0.01) amount of aryl sulfotransferase IV than do either midzonal or periportal cells, in which similar levels of the enzyme are found.

Animals↗
PubMed1991

Characterization of arylamine acetyltransferase in the rabbit eye.

The activity of arylamine acetyltransferase with p-aminobenzoic acid (PABA), sulfamethazine (SMZ), and aminozolamide as substrates was studied in rabbit tissue homogenates of the corneal epithelium, stroma-endothelium, iris-ciliary process, and liver. Rabbits were classified as rapid or slow acetylators with respect to their rate of hepatic acetylation of SMZ. The ocular disposition of aminozolamide in the two phenotypes was compared using a topical ocular infusion method that permitted a constant concentration to remain in contact with the intact cornea. The effect of hepatic-acetylator phenotype on the intraocular pressure (IOP) recovery rate and drug concentrations in tissues after single-dose administration of aminozolamide also was studied. In general, the rank order of arylamine acetyltransferase activity regardless of substrate was liver greater than iris-ciliary process greater than corneal epithelium greater than stroma-endothelium. The specific activity with aminozolamide as substrate was greater than that with SMZ in each tissue homogenate and greater than with PABA as substrate in all tissues except the stroma-endothelium of slow hepatic-acetylator rabbits. Very low enzyme activity ratios for ocular acetylation between rapid and slow hepatic-acetylating rabbits indicated that acetylation in the ocular tissues did not correspond with the acetylation phenotype. At various times during and after topical infusion to the anesthetized rabbit, assay determinations of drug and metabolite in ocular tissues indicated that there were no significant differences between phenotypes in the disposition of either drug or metabolite. These results correlate with the IOP measurements after topical infusion; they also showed no difference in the effect of aminozolamide between hepatic-acetylator phenotypes. These results indicate that the ocular disposition and the decrease in IOP from topical application of aminozolamide is independent of the hepatic-acetylation phenotype in the rabbit. There are significant amounts of acetyltransferase activity in the ocular tissues of the rabbit with these three substrates, indicating that acetylation may be occurring for other arylamine drugs used in the eye.

4-Aminobenzoic Acid↗
PubMed1989

Peroxidase as a model for reduction of tertiary amine oxides catalyzed by rat hepatic supernatant and microsomal fractions.

Rat hepatic microsomal and 100,000 g supernatant fractions catalyzed an NADH- and FMN-dependent reduction of amine oxides. Horseradish peroxidase (HRP) served as a model for the amine oxide reductase located in rat hepatic 100,000 g supernatant fraction. The HRP-catalyzed reaction displayed saturation kinetics with respect to NADH and the amine oxide substrate; however, there was an optimum concentration for FMN after which inhibition was observed at increased concentrations of FMN. The reductase in the 100,000 g hepatic supernatant fraction closely paralleled HRP-catalyzed amine oxide reduction in coenzyme requirements, sensitivity to inhibitors, and substrate specificity. Moreover, the peroxidase activity of HRP and microsomal and 100,000 g supernatant fractions correlated with the NADH- and FMN-dependent amine oxide reductase activities of these enzyme preparations. The NADH- and FMN-dependent amine oxide reductase activity in 100,000 g supernatant fractions, however, did not parallel the aldehyde oxidase activity. Thus, the results indicate that there is an amine oxide reductase in rat hepatic 100,000 g supernatant fraction with catalytic properties that are modeled well by horseradish peroxidase.

Amines↗
PubMed1989

Assay of purified aryl sulfotransferase suitable for reactions yielding unstable sulfuric acid esters.

An assay procedure for purified aryl sulfotransferase is described. The method utilizes isocratic paired-ion reverse-phase HPLC analysis of adenosine-3',5'-diphosphate formed in the reaction. Evaluation of the assay procedure was carried out with 1-naphthalene-methanol as a model substrate for purified rat hepatic aryl sulfotransferase IV. Kinetic constants for sulfation of 1-naphthalenemethanol determined by this method compared favorably with those determined using thin-layer chromatographic assays of 35S incorporation. These results indicate that the method will be suitable for determination of kinetic constants in sulfotransferase-catalyzed reactions where the product sulfuric acid ester may be chemically unstable.

Adenosine Diphosphate↗
PubMed1989

In vitro metabolic transformations of vinblastine: oxidations catalyzed by peroxidase.

Vinblastine is converted to a single major metabolite during in vitro enzymatic oxidations catalyzed by horseradish peroxidase in the presence of hydrogen peroxide. Preparative-scale enzyme incubation permitted the isolation of sufficient amount of the transformation product for complete structural identification and biological evaluation. The metabolite was identified as catharinine (also known as vinamidine) by 1H and 13C NMR and by mass spectrometry. Incubations conducted in H2(18)O-enriched water gave catharinine in which a single atom of 18O was incorporated into the metabolite structure. The labeling experiment provided evidence for an unusual ring-fission pathway by which peroxidase transforms vinblastine to catharinine. Catharinine is 77 times less active than vinblastine when tested in vitro against the human T-cell leukemic cell line (CRFF-CEM).

Animals↗
PubMed1989

In vitro metabolic transformations of vinblastine: oxidations catalyzed by human ceruloplasmin.

The dimeric Vinca alkaloid vinblastine (VLB) undergoes metabolic transformation to three products in a reaction catalyzed by the human serum copper oxidase ceruloplasmin. The enzyme reaction requires chlorpromazine as a shuttle oxidant, and the course of the oxidation reaction appears to be subject to the nature of the shuttle oxidant used. Preparative-scale incubations have resulted in the isolation of three products, which were characterized by chemical and spectral analyses. The metabolites were identified as the ring fission product catharinine, obtained by oxidation of the Iboga ring system; an enamine/ether derivative obtained by oxidation of the Aspidosperma portion of VLB; and a metabolite embodying the same structural changes in both parts of the vinblastine dimeric structure. Catharinine is identical with the product of VLB oxidation obtained by peroxidase oxidation. The other two products are new metabolites and are derivatives of VLB. All of the metabolites are less active than VLB when tested in vitro vs the human T-cell leukemic cell line (CRFF-CEM).

Biotransformation↗
PubMed1988

Sulfation of benzylic alcohols catalyzed by aryl sulfotransferase IV.

Aryl sulfotransferase (AST) IV catalyzes the 3'-phosphoadenosine 5'-phosphosulfate-dependent sulfation of a variety of benzylic alcohols. Several molecular characteristics of benzylic alcohols were investigated for their ability to influence the catalytic efficiency of a homogeneous preparation of rat hepatic AST IV. The results of these studies indicated that lipophilicity of the benzylic alcohol was a major factor in determining catalytic efficiency, as represented by the values of kcat/Km. Furthermore, this effect was primarily mediated by a decrease in the apparent Km as a function of increasing lipophilicity of the molecules. This effect of lipophilicity was documented by a linear correlation between the apparent Km values of the benzylic alcohols and the logarithms of their octanol/water partition coefficients. In contrast to previously observed effects of para substituents on phenols, electronic effects of substituents on the phenyl ring had no effect on the catalytic efficiency of the enzyme with benzylic alcohols. In a further difference between phenol and benzylic alcohol substrates for AST IV, 2-naphthol exhibited pronounced substrate inhibition at pH 7.0, whereas the analogous benzylic alcohol, 2-naphthalenemethanol, did not. Stereochemistry at the benzylic carbon also had an effect on the catalytic efficiency of the AST IV; kcat/Km values for S-(-)-1-phenylethanol were approximately 3-fold higher than for R-(+)-1-phenylethanol.

Animals↗
PubMed1987

Ocular disposition of aminozolamide in the rabbit eye.

We have previously determined that 6-amino-2-benzothiazolesulfonamide (aminozolamide) significantly lowers IOP in rabbits and, more importantly, in ocular hypertensive human subjects. Results from in vitro experiments established that both the inhibitory activity of aminozolamide against carbonic anhydrase B as well as the penetration rate across excised rabbit corneas were equivalent to ethoxzolamide. Consequently, we have investigated the ocular disposition of aminozolamide to explain its activity when instilled topically to the eye. A constant concentration of 67.4 micrograms/ml of drug was applied for 90 min to the left eye of anesthetized rabbits. Drug and metabolite were measured in both aqueous humor and iris/ciliary body over time. The metabolite was collected and purified. Identification using mass spectroscopy, high pressure liquid chromatography (HPLC) and fluorescence scans indicated that the metabolite was 6-acetamido-2-benzothiazolesulfonamide. Relatively high levels of metabolite were identified in the cornea and iris/ciliary body but were much lower in aqueous humor. Tissue concentrations over time for the metabolite in iris/ciliary body were approximately 2-fold higher than levels of metabolite measured in aqueous humor. When compared to drug levels measured in either aqueous humor or iris/ciliary body, metabolite levels in these respective tissues were much higher. It is hypothesized that topical activity is a consequence of both metabolite retention in the iris/ciliary body as well as inhibition of 99+% of carbonic anhydrase. Both of these events must occur over a sufficient time period to effect a significant lowering of IOP.

Animals↗
PubMed1986

N-Substituted sulfonamide carbonic anhydrase inhibitors with topical effects on intraocular pressure.

N-Methylacetazolamide was shown to be active topically in reducing intraocular pressure (IOP) to a small but statistically significant level in the normotensive rabbit eye. In vivo experiments with N-methylacetazolamide suggest that ocular metabolism to acetazolamide was responsible for the observed topical activity. Examination of initial rate kinetics of carbonic anhydrase catalyzed p-nitrophenyl acetate hydrolysis showed that N-methylacetazolamide was a competitive inhibitor, in contrast to noncompetitive inhibition seen with acetazolamide and other primary sulfonamide inhibitors. N-Substituted and unsubstituted 4-chlorobenzene- and 4-nitrobenzenesulfonamides were also synthesized, and their biochemical characteristics and in vivo ability to lower IOP when applied topically were determined. The primary sulfonamides were reversible noncompetitive inhibitors of carbonic anhydrase, with no effect on IOP after topical administration. 4-Nitrobenzene- and 4-chlorobenzenesulfonamides containing both N-hydroxy and N-methyl substituents were model irreversible inhibitors of carbonic anhydrase and exhibited a trend toward topical activity in reducing IOP in normotensive rabbit eyes. Therefore, this paper describes the synthesis and characterization of two types of carbonic anhydrase inhibitors; the N-methyl-substituted sulfonamides are reversible competitive inhibitors of carbonic anhydrase, while the N-hydroxy-N-methyl-substituted sulfonamides are irreversible inhibitors.

Administration, Topical↗
PubMed1985

One-electron oxidation of vindoline and 16-O-acetylvindoline catalyzed by peroxidase.

The mechanism of oxidation of the alkaloids vindoline (1) and 16-O-acetylvindoline (1a) was examined by use of the reversible redox cycle of horseradish peroxidase (HRP). Oxidation of 1 by HRP resulted in the formation of the enamine dimer 5. The highly reactive radical cation species 2 is an implied intermediate in the oxidation process. During the reaction, HRP-I was reduced to HRP-II by abstraction of an electron from vindoline. The vindoline radical thus formed eliminates a second electron and a proton to produce a highly reactive iminium derivative which undergoes intramolecular etherification and dimerization. Oxidation of 16-O-acetylvindoline (1a) by HRP-I results in the production of an iminium derivative 3a concomitant with the formation of HRP-II. The iminium 3a was isolated and characterized and was converted into monodeuterated 1a by reduction with NaBD4. The stoichiometry (HRP-II)/(substrate) was determined to be 4.77 +/- 0.17 for vindoline and 2.27 +/- 0.20 for 16-O-acetylvindoline. The enamine dimer also reduced HRP-I to form HRP-II, but the stoichiometry of this reaction was variable.

Catalysis↗
PubMed1984

Microsomal flavin-containing monooxygenase activity in rat corpus striatum.

Microsomal fractions isolated from rat corpus striatum catalyze the oxidation of thiobenzamide to the sulfoxide. The rate of thiobenzamide sulfoxidation is 6.9 +/- 4.8 nmol(min)-1 (mg microsomal protein)-1. The reaction is inhibited by an excess of sulfur- and nitrogen-containing substrates for the microsomal flavin-containing monooxygenase. These inhibitors of thiobenzamide sulfoxidation include methimazole, cysteamine, and trimethylamine. Enzyme activity is also destroyed by treatment of the microsomal preparation at 60 degrees for 1 min. In parallel experiments, rat liver microsomes exhibit similar inhibition characteristics. The data indicate the presence in corpus striatum of a microsomal monooxygenase with catalytic properties of the hepatic microsomal flavin-containing monooxygenase.

Animals↗
PubMed1982

Cysteine S-conjugate N-acetyltransferase from rat kidney microsomes.

An acetyltransferase from rat kidney microsomes that catalyzes the N-acetylation of thioethers of L-cysteine has been solubilized, stabilized, and separated from hydrolytic enzymes active against both the acetylated product, a mercapturic acid, and acetyl coenzyme A. Efficiency of catalysis varies with the lipophilicity of the substituent at sulfur in the order, ethyl less than propyl less than benzyl less than butyl, as predicted by the Hansch pi constants. Although L-tryptophan is acetylated at a very low rate, acetylation is not detectable for L-cysteine, L-methionine, L-serine, L-leucine, L-phenylalanine, or L-glutamic acid. The properties and substrate specificity reported here, along with previous studies on enzyme distribution, suggest that cysteine S-conjugate N-acetyltransferase is responsible for the final step in mercapturic acid biosynthesis.

Acetyltransferases↗