Heterogeneity of Hepatic Microsomal UDP-Glucuronosyltransferase(s) Activities: A New Kinetic Approach for the Study of Induction and Specificity

Boutin, Jean A.; Antoine, B; Siest, Gérard

doi:10.1002/jps.2600830430

Cited by 3 publications

(1 citation statement)

References 38 publications

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“…Basically, the affinity data have been collected using the classical process described by Segel (38) for bisubstrate enzymes as applied on this transferase (30,31) or on other ones (39). For the apparent K m value, the substrate concentrations were varied from 0.06 to 4 mM, whereas the cosubstrate (acetyl-CoA) concentration was 1 mM.…”

Section: Kinetic Studiesmentioning

confidence: 99%

Substrate Specificity and Inhibition Studies of Human SerotoninN-Acetyltransferase

Ferry¹,

Loynel²,

Kucharczyk³

et al. 2000

Journal of Biological Chemistry

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Arylalkylamine N-acetyltransferase (AANAT) catalyzes the reaction of serotonin with acetyl-CoA to form N-acetylserotonin and plays a major role in the regulation of the melatonin circadian rhythm in vertebrates. In the present study, the human cloned enzyme has been expressed in bacteria, purified, cleaved, and characterized. The specificity of the human enzyme toward substrates (natural as well as synthetic arylethylamines) and cosubstrates (essentially acyl homologs of acetylCoA) has been investigated. Peptide combinatorial libraries of tri-, tetra-, and pentapeptides with various amino acid compositions were also screened as potential sources of inhibitors. We report the findings of several peptides with low micromolar inhibitory potency. For activity measurement as well as for specificity studies, an original and rapid method of analysis was developed. The assay was based on the separation and detection of N-[ 3 H]acetylarylethylamine formed from various arylethylamines and tritiated acetyl-CoA, by means of high performance liquid chromatography with radiochemical detection. The assay proved to be robust and flexible, could accommodate the use of numerous synthetic substrates, and was successfully used throughout this study. We also screened a large number of pharmacological bioamines among which only one, tranylcypromine, behaved as a substrate. The synthesis and survey of simple arylethylamines also showed that AANAT has a large recognition pattern, including compounds as different as phenyl-, naphthyl-, benzothienyl-, or benzofuranyl-ethylamine derivatives. An extensive enzymatic study allowed us to pinpoint the amino acid residue of the pentapeptide inhibitor, S 34461, which interacts with the cosubstrate-binding site area, in agreement with an in silico study based on the available coordinates of the hAANAT crystal.Melatonin (5-methoxy-N-acetyltryptamine) is a pineal hormone that modulates a variety of endocrinological, neurophysiological, and behavioral functions in vertebrates (1). It is involved in the regulation of circadian rhythms and in the reproduction of photoperiodic species (2). The chronobiotic effects of melatonin in humans have been mainly studied in circadian rhythm sleep disorders (3). Moreover, alterations of the melatonin profiles have been reported in other biological rhythm disorders (3). Melatonin exerts its effects through at least three targets: 2 receptor subtypes, mt 1 and MT 2 , and a binding site, MT 3 (4). The two first ones have been cloned (5-6) and their pharmacological effects largely studied, and several specific and potent ligands (7-9) discovered. The MT 3 subtype is still a putative binding site under intensive research from purification attempts to pharmacological characterizations (10 -11). Since melatonin is implicated in several types of mild to severe pathologies, including mood disorders (3, 12), it is considered a valuable therapeutic target. Beside the classical search for agonists and antagonists of the melatonin receptors, a series of programs was launched t...

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Section: Kinetic Studiesmentioning

confidence: 99%

Substrate Specificity and Inhibition Studies of Human SerotoninN-Acetyltransferase

Ferry¹,

Loynel²,

Kucharczyk³

et al. 2000

Journal of Biological Chemistry

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Physico-chemical and biological analysis of true combinatorial libraries

Lambert¹,

Bertin²,

Volland³

et al. 1999

Journal of Chromatography B: Biomedical Sciences and Applicatio

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Induction and inhibition of cicletanine metabolism in cultured hepatocytes and liver microsomes from rats

Menard¹,

Lamiable

Vistelle

et al. 2000

Fundamemntal Clinical Pharma

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Cicletanine, a racemic furopyridine derivative synthesized as racemate, is used as an antihypertensive agent. Its two enantiomers are involved in the pharmacological effects of the drug. Cicletanine is metabolized by conjugation enzyme systems (phase II) into sulfoconjugated or glucuroconjugated enantiomers. This study reports on the use of both the induction with 3-methylcholanthrene (3-MC) or phenobarbital (PB) and inhibition with selective compounds to determine and identify UGT isoenzymes involved in the metabolism of cicletanine enantiomers. PB and 3-MC both enhanced the cicletanine enantiomer glucuronidation. These two compounds being known as inducing agents of UGT2B1 and UGTIA6 isoforms, respectively, this suggests an implication of UGT2B1 and UGT1A6 isoforms in the metabolism of the two cicletanine enantiomers: ( + )-cicletanine and ( - )-cicletanine. The use of selective compounds for inhibition study evidenced, in addition to UGT2B1 and UGT1A6 isoforms, the involvement of other UGT isoforms such as UGT1A1, UGT2B7 and UGT2B15 in cicletanine metabolism.

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Heterogeneity of Hepatic Microsomal UDP-Glucuronosyltransferase(s) Activities: A New Kinetic Approach for the Study of Induction and Specificity

Cited by 3 publications

References 38 publications

Substrate Specificity and Inhibition Studies of Human SerotoninN-Acetyltransferase

Substrate Specificity and Inhibition Studies of Human SerotoninN-Acetyltransferase

Physico-chemical and biological analysis of true combinatorial libraries

Induction and inhibition of cicletanine metabolism in cultured hepatocytes and liver microsomes from rats

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