Wolfram Christ

Comparison of catechol-O-methyltransferase from rat brain, erythrocytes and liver

Abstract Catechol-O-methyltransferase activity from supernatant of brain, red blood cells and liver of the rat were characterized by the following items: 1) substrate specificity with respect to five catechol substrates, 2) meta/para ratio of O-methylation, 3) apparent Michaelis constants, 4) pH dependence of reaction rate. Under identical conditions, a progressive decrease in the rate of O-methylation by rat liver supernatant can be noticed in the sequence of 3.4-dihydroxy-acetophenone > 3.4-dihydroxybenzoic acid > 3.4-dihydroxyphenylacetic acid. Using 3.4-dihydroxybenzaldehyde as substrate, the meta/para ratio of O-methylation and the apparent Michaelis constants are similar for catechol-O-methyltransferase from brain and red blood cells and differ from the values obtained with liver supernatant. In phosphate buffer, the pH curves are almost identical for catechol-O-methyltransferase from brain, red blood cells and liver, in glycine/NaOH buffer, however, the liver enzyme shows a broad peak. From our experiments it is obvious that the biochemical characteristics selected are similar for soluble catechol-O-methyltransferase from brain and erythrocytes which differ from that obtained with liver enzyme.

Formation of chlorpromazine sulphoxide and monodesmethylchlorpromazine by microsomes of small intestine

SummaryThe metabolism of chlorpromazine by microsomal preparations of the small intestine from guinea pig and rat was studied. 35S-chlorpromazine was incubated with these preparations in Krebs-Ringer bicarbonate buffer at 37°C. Control values were obtained by performing the assay at 0°C. The metabolites were extracted with dichloroethane and separated by TLC. In incubations with intestinal microsomes from guinea pigs chlorpromazine sulphoxide and monodesmethyl chlorpromazine were identified as main metabolites. The apparent Michaelis constant for sulphoxidation of chlorpromazine is approximately 20–30 μM and for N-demethylation in the range of 30–100 μM.Using microsomal preparations from rat intestine, however, noteworthy formation of chlorpromazine metabolites could not be found. This observation can be explained by the fact that the cytochrome P450 content of rat intestinal microsomes was extremely low compared with that determined in guinea pig microsomes.

A Simple and Sensitive Spectrophotometric Determination of Monoamine Oxidase Activity

is based on the high extinction coefficient of 4-hydroxy-3-methoxy-benzaldehyde (vanillin) at 345 nm at pH ~ 11.7. Vanillin is formed by inonoamine oxidase when 4-hydroxy-3-methoxy-benzylamine is used as substrate. The primary oxidation product, 4-hydroxy-3-methoxybenzaldehyde, is not further metabolized by aldehyde reductase or aldehyde dehydrogenase. The apparent Km value of 4-hydroxy-3-methoxy-benzylamine for monoamine oxidase of rat brain is 150 to 180 /^mol/1 (pH 7.0). The pH optimum of the rat brain monoamine oxidase with this substrate was found to be at about 9.0. Inhibition of monoamine oxidase of rat brain after pretreatment of rats with iproniazid, pargyline and tranylcypromine was investigated with this method. The data were analyzed according to LINEWEAVER and BUR . Eine einfache, empfindliche und schnelle spektrophotometrische Bestimmungsmethode der Monoamin-Oxidase-Aktivitat in Gewebehomogenaten wird beschrieben. Die Methode beruht auf dem hohen Extinktionskoeffizienten von 4-Hydroxy-3-methoxy-benzaldehyd (Vanillin) bei 345 nm und bei einem pH von 11,7. Dieser Aldehyd entsteht, wenn man der Monoamin-Oxidase 4-Hydroxy-3-methoxybenzylamin als Substrat anbietet. Vanillin wird weder durch die Aldehyd-Reduktase noch durch die Aldehyd-Dehydrogenase weiter metabolisiert. Der scheinbare Km-Wert des 4-Hydroxy-3-rhethoxy-benzylamins liegt fur die Monoamin-Oxidase aus Rattengehirn bei 150—180 //mol/1 (pH 7,0) und das pH Optimum liegt fur dieses Substrat bei etwa 9,0. Es wurde die Hemmung der Monoamin-Oxidase-Aktivitat des Rattengehirns nach in vitro-Vorbeha ndlung der Ratten mit MonoaminOxidase-Inhibitoren wie Iproniazid, Pargylin und Tranylcypromin mit dieser Methode untersucht. Die Ergebnisse wurden in LINEWEAVER-BuRK-Diagrammen ausgewertet.

Critical evaluation of measurement of platelet monoamine oxidase in man.

Some biochemical characteristics such as substrate specificity, substrate affinity and inhibitor sensitivity of monoamine oxidase of human blood platelets were investigated. Tyramine, tryptamine and beta-phenylethylamine were used as substrates. The apparent Michaelis constants, maximal velocity rates and I50 for the inhibitor tranylcypromine were determined. The data were analyzed according to Lineweaver-Burk and Dixon. The influence of amitriptyline, a prototype of tricyclic antidepressants, on the selected variables (Km, V, I50), was studied. The parameters investigated showed remarkably low interindividual differences when healthy volunteers were tested. The inhibitor activity of amitriptyline towards platelet monoamine oxidase depends on the substrate used. Amitriptyline concentrations which showed a pronounced effect on the enzyme characteristics are significantly higher than plasma levels of the drug found under therapeutic conditions.

[10] Preparation and purification of nicotinamide mononucleotide analogs

Publisher Summary This chapter discusses the methods for the preparation and purification of nicotinamide mononucleotide analogs. Many nicotinamide mononucleotide derivatives have been prepared chemically as intermediary products in different syntheses of NAD + and NAD + analogs. The preparative methods for the chemical synthesis of NMN molecules become complicated when labile pyridine derivatives, such as thionicotinamide and selenonicotinamide, are used as starting material. It has been shown that thionicotinamide cannot be directly N 1 -alkylated and that selenonicotinamide is stable in aqueous solutions for only a few hours. A simple enzymic method has been developed, for obtaining nicotinamide mononucleotide (NMN) analogs, with alterations to the pyridine moiety of the molecule, starting with the available NAD(P) + analogs. In this method, the crude nucleotide pyrophosphatase from Crotalus venom was used. No attempt was made to purify the pyrophosphatase, by separating the contaminating enzymes, notably the 5′-nucleotidase. The pyrophosphate linkages of NAD(P) + and the NAD(P) + analogs investigated are hydrolyzed, except in 6-AN-ADP + and INH-ADP + , because of low substrate specificity of this nucleotide pyrophosphatase. It was found that only NMN, either obtained commercially or prepared from NADP + , is condensed enzymically with adenosine triphosphate (ATP). Under the conditions of the standard assay selected, none of the NMN analogs (thio-NMN, 3-AcPyr-MN, or 3-OHPyr-MN) reacts with ATP.

Convenient method for preparation and purification of nicotinamide mononucleotide analogs

Abstract 1. Nucleotide pyrophosphatase from Crotalus adamanteus venom cleaves the pyrophosphate linkage of NADP + , thio-NADP + , 3-AcPyr-ADP + , seleno-NADP + , thio-NAD + , 3-AcPyr-AD + and 3-OHCPyr-AD + . 2. The nicotinamide mononucleotide (NMN) analogs can be obtained from reaction mixtures in good yield and high purity by chromatography on Whatman DE 52 cellulose. 3. The uv spectra and specific extinction coefficients of the NMN analogs were almost identical with those of the corresponding 1-methylpyridinium derivatives. 4. Thio-NMN, 3-AcPyr-MN and 3-OHCPyr-MN can be condensed with AMP to the corresponding NAD + analogs using dicyclohexylcarbodiimide.

Phase I metabolism of imipramine by microsomes of small intestine in comparison with metabolism by liver microsomes.

SummaryThe metabolism of imipramine was investigated by the incubation of C-14 labelled compound in Krebs-Ringer bicarbonate buffer with microsomes of small intestine and of liver from guinea pigs.Imipramine and its metabolites were extracted with chloroform, separated by TLC and determined quantitatively by direct scanning with a TLC Linear Analyzer.With intestinal microsomes, the following metabolites could be identified: DMI, 2-OH-IMP, IMP-N-oxide. DMI is the main metabolite. The same metabolites appeared after incubation with liver microsomes, but the proportions were different: the N-oxide formation was predominant, followed by N-demethylation and hydroxylation.The formation of DMI and 2-OH-IMP seems to follow Michaelis-Menten kinetics, both in assays with intestinal and with liver microsomes.The liver/intestine ratio of DMI and 2-OH-IMP formation is proportional to the cytochrome P-450 ratio in the microsomes, in contrast to N-oxide formation.

Pharmakologische und biochemische Eigenschaften des Thionicotinamids (Thio-NA) und seiner NAD(P)-Analogen

wir aufgrund ihres Amingehaltes (843 :L 149ng Noradrenalin/mg Protein: n = 6) und ihrer geringen Fumaraseaktiviti~t ( ~ 0,1 ~Mol Fumara t /mg Protein/rain) als spezifisch Noradrenalin speichernde Granulafraktion ansehen. Bei Inkubat ion in Trispuffer pH 7,4 und 37°C spalten diese Granula in Anwesenheit yon 2,5 mM Ca++ 169 :J: 20~g P/mg Protein/30 rain (n -~ 5) und yon 2,5 mM Mg ++ 173 :J: 48 ~g P/rag Protein/30 rain (n ----5) aus 5 mM ATP. Zus~tzliche Gabe yon 30 mM Na + und 20 mM K + vermag diese ATPase nicht zu stimulieren. Gleichzeitige Anwesenheit yon Ca ++ und Mg ++ verursacht cine additive Wirkung auf die ATP-Spaltung. Nach 20 rain Inkubat ion bei 37°C in Trispuffer p i t 7,4 mit 5 • 10 -5 M Noradrenalin steigt durch Zugabe yon 5 mM ATP und 2,5 mM Mg++ der Amingehalt der Mflznervengranula auf 190 :L 17°/o (n ---6) gegenfiber Versuchen mit Noradrenalin allein. Durch 6 . 1 0 -5 M Reserpin wird die Zunahme der Aminaufnahme aufgehoben (104 :L 20o/0; n----3) und durch 6 . 1 0 5 M Prenylamin stark reduziert (136 ± 14°/0; n ~ 3). Gleiche Konzentrationen Reserpin und Prenylamin vermindert die ATP-Spaltung auf 76 und 740/0. Unter gleichen Bedingungen bewirken 5 mM ATP mit 2,5 mM Ca ++ keine Steigerung der Aminaufnahme (90 ~: 12°/o; n ~ 5). Der Amingehalt ist weder durch Reserpin noeh durch Prenylamin zu beeinfiussen (86 4-20o/0; n ~ 4 und 86 ~: 11°/o; n ~ 3), obwohl die ATP-Spaltung dutch diese Hemmstoffe auf 77 und 61°/0 vermindert wird. Demnach stimuliert Ca ++ wahrscheinlich die Amin-Anfnahme und -Abgabe im gleiehen Umfang, w~hrend Mg ++ vorwiegend die Aufnahme steigert. Der Mg ++und Ca ++abhiingigen ATPase aus Milznervengranula schreiben wit eine Bedeutung ffir den Amintransport zu.

Interactions of Different Drugs with the N-Demethylation of Pefloxacin in the Rat as Determined by Analysis of 14CO2-Exhalation

In man, at least 20% of an oral pefloxacin dose is converted to norfloxacin (Montay et a1. 1984). Demethylation reactions are catalysed by the mixed function oxidase system involving cytochrome P-450 enzymes. For investigation of pefloxacinN-demethylation in rats in vivo, we synthesised [N14CH3]-pefloxacin by N-methylation of norfloxacin using 14CH3J. [N-14CH3]-pefloxacin was administered orally to female Wistar rats and 14C02exhalation was continuously monitored by use of the breath test. We studied the interaction of different drugs with the N-demethylation of pefloxacin. The parameters evaluated, such as maximum exhalation rate and c).lmulative exhalation, reflect the effects of different drugs on the mixed function oxidase system.