Effect of diethylnitrosamine on monoamine oxidase in rat liver

The effect of diethylnitrosamine (DEN), a well-known experimental carcinogen, toward MAO-A and MAO-B activity of rat liver was investigated. The oxidations of both beta-PEA (MAO-B) and 5-HT (MAO-A) were inhibited by DEN. The K1 values of DEN in the inhibition of rat liver MAO-A and MAO-B activity were determined. The kinetic data show that DEN is a competitive, MAO-B selective inhibitor and its inhibitory effect on MAO-B is about 4-fold more potent than that on MAO-A. DEN might change the proportions of the multiple forms of MAO activity in tumor cells.     

Differential effects of triton X-100 on benzodiazepine and GABA binding in a frozen-thawed synaptosomal fraction of rat brain

The effect of Triton X-100 on 3H-GABA and 3H-diazepam binding was measured in a frozen-thawed synaptosomal fraction of rat brain. Specific binding activity (amount bound per mg protein) of both ligands was increased by the treatment. Diazepam binding capacity in the pellet was progressively decreased, while GABA binding was increased, then decreased by increasing Triton X-100. Diazepam binding affinity was unchanged, while GABA binding affinity increased. Triton X-100 appears to preferentially solubilize benzodiazepine binding sites, indicating GABA and benzodiazepine binding sites are on separate macromolecules.     

Studies of monoamine oxidases: Effect of Triton X-100 and bile salts on monoamine oxidase in brain mitochondria

Triton X-100 and the bile salts, cholate and deoxycholate, detergents often used in the solubilization of monoamine oxidase (MAO) from mitochondria, have been found to cause an inhibition of the enzyme activity. With beef brain mitochondria, it was found that there was a differential effect of Triton X-100 on the putative MAO types A and B, with MAO-A being more susceptible to inhibition by Triton X-100. This was indicated by the greater loss of serotonin-deaminating than of phenyl ethylamine-deaminating activity in the presence of Triton X-100. Although the bile salts also caused substantial inactivation at concentrations above 0.1%, no differentiation between MAO types could be made. Kinetic studies of the inhibition by Triton X-100 indicated two different mechanisms were occurring with the two MAO types. The inhibition was competitive for MAO-A, but uncompetitive for MAO-B. Removal of Triton X-100 by co-polymer beads restored some, but not all of the activity for both MAO-A and MAO-B types. This suggests that the activity loss may have been due in part to inactivation when the enzyme was separated from the mitochondrial membrane.     

Effect of intrabiliary administration of triton X-100 on biliary excretory function in the rat

Intrabiliary administration of Triton X-100 is of interest in producing effects on biliary tree permeability and canalicular biliary excretory function. Treatment with 0.4% Triton (40 μl) was shown to increase the biliary excretion of intraportably administered [³H]sucrose. It also decreased recovery of [³H]sucrose given into the biliary tree. Thus, we concluded that Triton treatment increased biliary tree permeability. Using a different set of marker compounds, canalicular transport of bromphenol blue, [¹⁴C]morphine glucuronide and [³H]ouabain was found to be decreased. The fact that [³H] taurocholate excretion into bile was not affected whereas that of [³H]ouabain was lends support to the concept that taurocholate and ouabain are not transported by a common pathway.     

Action of khimcoccid on monoamine oxidase of rat liver mitochondria

Pharmaceutical Chemistry Journal -     

Kinetic studies of membrane-bound rat liver monoamine oxidase.

(1) Competition experiments have becn carried out with membrane-bound rat liver monoamine oxidase using tyramine, serotonin, dopamine, tryptamine, phenylethylamine, benzylamine and noradrenaline. Simple competitive kinetics were exhibited by most pairs of substrates. (2) Replots of the slopes of Lineweaver-Burk plots against inhibiting substrate concentration were linear in most cases but were hyperbolic for serotonin and α-methyl-tryptamine inhibition of tyramine and dopamine oxidation, and parabolic for noradrenaline and dopamine inhibition of tryptamine oxidation. (3) Simulation using an enzyme model having two independent catalytic sites was only partially successful in producing non-linear replots of the types obtained experimentally. (4) The nature of the catalytic sites of membrane-bound monoamine oxidase is discussed in the light of these results.     

Observations on the inhibition of rat liver monoamine oxidase by clorgyline

Two forms of monoamine oxidase (MAO) denned as MAO A and B by others differ in their specificities to substrates and their sensitivities to the irreversible inhibitor clorgyline. From studies using the substrates 5-HT, tyramine and benzylamine, the presence of both MAO forms in rat liver mitochondria has been confirmed and some characteristics of their inhibition by varying concentrations of clorgyline investigated. Although both MAO forms showed time-dependent inhibition, this process occurred, in general, at a qualitatively slower rate for MAO B, despite the fact that this enzyme form requires higher concentrations of clorgyline than MAO A for inhibition of its activity. However, factors such as the concentration of enzyme, the concentration of clorgyline and the enzyme: drug ratio employed in the assay all influence the resultant time-course and the final degree of the inhibition observed. The possible importance of the lipid environment of the outer mitochondrial membrane in generating multiple MAO forms and in regulating the inhibition kinetics of these forms is discussed. The results indicate that the effects of pre-incubation time and the enzyme: drug ratio on inhibition of MAO by clorgyline should be fully recognized when using the drug to indicate multiple forms in animal tissues.     

The effect of tris buffers on rat liver mitochondrial monoamine oxidase

The effect of tris buffers upon the monoamine oxidase (MAO) activity in rat liver mitochondria has been investigated. Tris buffer was shown to inhibit MAO in a non-competitive manner with a Ki of 15–25 mm. Tyramine, 5-hydroxytryptamine and β-phenethylamine but not benzylamine oxidations were all inhibited by tris buffer. All inhibitions, except that of 5-HT, were completely reversible. It is suggested that these effects are produced by conformational changes in the structure of the MAO. The significance of these results is discussed with respect to the use of tris buffers in the extraction and estimation of the activity of MAO.     

Thyroid hormone-inducible monoamine oxidase inhibitor in rat liver cytosol

An endogenous inhibitor of monoamine oxidase (MAO) was separated by gel-filtration from 105,000 g supernate of T4-treated rat liver cytosol. The inhibition by this inhibitor was concentration-dependent and more potent for A-form MAO than for B-form MAO. The mode of inhibition was competitive either with 5-hydroxytryptamine or beta-phenylethylamine. The molecular weight of this inhibitor was estimated to be 600-700 by gel filtration. The pI value was determined to be 3.0 by isoelectric focusing. This inhibitor was proved to be heat-stable and resistant to protease treatment. MAO inhibition activity was much lower in the cytosol of thyroidectomized, non-T4-treated rats than T4-treated rats, suggesting that this inhibitor is induced by thyroid hormone T4. MAO activity in rat liver might be regulated by the level of this inhibitor.     

Studies on the selective inhibition of membrane-bound rat liver monoamine oxidase.

Serotonin and benzylamine oxidising activities of membrane-bound rat liver monoamine oxidase have been distinguished according to their sensitivities towards 5-phenyl-3-(N-cyclopropyl)ethylamine-1, 2,4-oxadiazole (PCO). Tyramine, tryptamine and dopamine deamination have been shown to exhibit dual sensitivities to PCO inhibition, corresponding to these monoamines undergoing oxidation at both the PCO sensitive and insensitive sites responsible for serotonin and benzylamine oxidation respectively. The biphasic inhibition of tyramine deamination by PCO is shown to result from ‘fast’ and ‘slow’ pseudo-first order reactions with the enzyme. Both ‘fast’ and ‘slow’' reactions are shown to have two components, of which the slower is quantitatively the most important. The corresponding 3-nitrophenyl compound (3-nitro-PCO) preferentially inhibits tyramine oxidation at low concentrations. This is shown to result from a reversal of the relative rates of attack, by this inhibitor, on the two centres of deamination. PCO has been shown to be a potent instantaneous competitive inhibitor of the enzyme. With serotonin as substrate a K_i of 10^?7M was obtained. An enzyme with serotonin oxidation completely blocked by PCO or 2-chloro-PCO, but retaining approximately half the tyramine deaminating activity has been prepared. The kinetic parameters for the oxidation of tyramine, tryptamine and dopamine by such partially inhibited preparations have been determined.     

The effect of tris buffers on rat liver mitochondrial monoamine oxidase.

The effect of tris buffers upon the monoamine oxidase (MAO) activity in rat liver mitochondria has been investigated. Tris buffer was shown to inhibit MAO in a non-competitive manner with a Ki of 15-25 mM. Tyramine, 5-hydroxytryptamine and beta-phenethylamine but not benzylamine oxidations were all inhibited by tris buffer. All inhibitions, except that of 5-HT, were completely reversible. It is suggested that these effects are produced by conformational changes in the structure of the MAO. The significance of these results is discussed with respect to the use of tris buffers in the extraction and estimation of the activity of MAO.     

Effect of triton X-100 on the conjugation of tetrahydrocortisone, in vitro

The detergent, Triton X-100, increased the conjugation of estrone, estradiol and tetrahydrocortisone (THE) by uridine diphosphate glucuronyl transferase (UDPGT) in rat liver microsomes; the maximal increase of the conjugation of these substrates was measured when the concentration of Triton in the incubation mixtures was 0.05 per cent. However, the magnitude of the increase and the effect seen with varying Triton concentration were substrate dependent, which is consistent with the hypothesis that multiple forms of UDPGT may be present in hepatic microsomes. The effects of various compounds which had previously been shown to either increase or decrease the conjugation of THE in non-activated enzyme preparations were re-examined in Triton-activated preparations. Compounds such as β-diethylaminoethyldiphenylpropylacetate (SKF-525A) and 7-hydroxychlorpromazine which inhibited conjugation in non-activated preparations also inhibited conjugation in Triton-activated preparations. Alternatively, the demethylated metabolites of chlorpromazine, which increased activity in non-treated preparations, decreased activity slightly in preparations maximally stimulated by Triton. Bisubstrate kinetic analysis of the THE conjugation of UDPGT also revealed differences between the properties of the non-treated and Triton-activated enzyme preparations. Triton activation caused an increase in the V_max of the reaction in the forward direction while having an insignificant effect on the dissociation constant for THE.     

Amine competition for oxidation by rat liver mitochondrial monoamine oxidase

Mixed substrate experiments have been carried out with rat liver mitochondrial monoamine oxidase. The results of these studies are interpreted in terms of the known specificities and sensitivities to inhibition of the two kinetically distinguishable species present in this preparation. The results of this study are discussed in terms of the function of the enzyme in vivo.     

UDP-glucuronosyltransferase(s) activities towards natural substrates in rat liver microsomes. Kinetic properties and influence of triton X-100 activation

We studied the in vitro capability of hepatic microsomal UDP-glucuronosyltransferase (UDPGT) in male rats to conjugate 22 natural xenobiotics which are known to be excreted as glucuronides in vivo. We clearly demonstrated that the Vmax can range in a decreasing scale for the following families of aglycones: 7-hydroxylated coumarins greater than 2-naphthol and phenols greater than monoterpenoid alcohols greater than 4-hydroxylated coumarins. The Km app. cannot be arranged in the same scale. This suggests that the catalytic mechanism of UDPGT is dependent on the hydroxyl group reactivity rather than on the binding interaction at the active site expressed by the Km app. The effects of various concentrations of detergent (Triton X-100) were determined on specificity (apparent Km) and activity (Vmax). For the 22 aglycones we showed that activation caused a variation in the Vmax which was a function of the concentration in detergent. The maximum of this activation did not always correspond to the same detergent/protein weight ratio. The impact of activation on Km app. was less clear since the variations observed were slightly different.     

Determination of monoamine oxidase concentrations in rat liver by inhibitor binding

The concentrations of monoamine oxidase-A and -B have been determined in mitochondria, mitochondrial outer membranes and microsomes from Sprague-Dawley and Wistar rats by determining the binding of tritium-labelled pargyline. Although the amounts of each form present depended on the source and the preparation method, this was paralleled by the specific activity such that the molecular turnover number was found to remain constant. The catalytic constants, kcat/Km, which represents the apparent second-order rate constant for the combination of enzyme and substrate, were about 0.13 and 2.1 sec-1 X microM-1 for 5-hydroxytryptamine and 2-phenethylamine, respectively, regardless of the source. Estimations of the amounts of the two forms by determining the concentrations of the inhibitors clorgyline, (-)-deprenyl, J-508 or pargyline necessary to give complete inhibition were shown to give overestimates of the true values because of the non-specific binding of these inhibitors to sites other than the monoamine oxidase active site.     

The effects of thyroid hormones on monoamine oxidase in the rat heart

The administration of thyroxine to young male rats produced an increase in the specific activity of their cardiac monoamine oxidase (MAO). A reduction in the circulating concentrations of thyroid hormones, brought about by 2-thiouracil, led to a decrease. The relative change in activity produced was greater with tyramine than with benzylamine as substrate. By following the time-course of the return of enzyme activity, with tyramine as substrate, after a single injection of pargyline in vivo, it was concluded that both excess and lack of thyroid hormones cause their effects on MAO activity by changing the rate of synthesis of the enzyme and not its degradation rate constant. The degradation rate constant did change with the age of the animal. The MAO activity, which increased towards tyramine as substrate in hyperthyroid rat hearts, behaved in the same way as that of controls to heat treatment, irreversible inhibition by pargyline or by clorgyline and also in K_m determinations. The pattern for benzylamine oxidation was similar, except for the effect of the inhibitor clorgyline which shifted the plateau region of the double sigmoid inhibition curve significantly using enzyme from hyperthyroid rat hearts. The plateau region was also shown to be affected by the age of the animal. The possibility is discussed that the increased cardiac MAO activity produced by thyroid hormones and by the growth of the animal is mediated by that form of the enzyme primarily responsible for the oxidation of tyramine. Mixed substrate experiments suggested that tyramine oxidation could be inhibited competitively by benzylamine.     

Interactions of rat brain acetylcholinesterase with the detergent Triton X-100 and the organophosphate paraoxon.

Inhibition of the critical enzyme acetylcholinesterase (E.C. 3.1.1.7) with subsequent cholinergic crisis is the mechanism of acute toxicity of the organophosphorus insecticides (B. E. Mileson et al., 1998, Toxicol. Sci.41, 8-20). Consequently, measurement of acetylcholinesterase activity is important for evaluating the mammalian toxicity of this commonly used class of insecticides. While mammalian acetylcholinesterase activity has often been determined in tissue homogenates in the presence of the nondenaturing detergent Triton X-100 at a concentration of 1%, the potential actions of this detergent on the activity of this critical enzyme are not understood. In the current study, homogenization of rat brain in buffer containing 1% Triton X-100 slightly elevated the (app)V(max) for hydrolysis of acetylthiocholine, without affecting the (app)K(m) or the (app)K(ss). However, the presence of both 1% Triton X-100 and paraoxon (at concentrations of 5 nM-100 nM) resulted in complex kinetic interactions with acetylcholinesterase, as evidenced by a curvilinear secondary plot for determination of the (app)k(i). These results suggest that measurement of acetylcholinesterase activity in the presence of up to 1% Triton X-100, but in the absence of oxon, should pose no problems with regard to data interpretation, provided it is recognized that the detergent slightly elevates activity. However, measurement of acetylcholinesterase activity after enzyme was exposed simultaneously to Triton X-100 and oxon could be problematic. Caution is warranted when interpreting data where acetylcholinesterase activity was determined under such conditions since in the presence of 1% Triton X-100, the capacity of oxon to inhibit acetylcholinesterase might change as a function of oxon levels.