Some parameters affecting the activity of monoamine oxidase in purified bovine brain mitochondria.

Abstract— Some parameters affecting the activity of monoamine oxidase (MAO) in purified beef brain mitochondria were investigated, and diversities in enzyme properties were found as a function of substrate. The deamination of the biogenic amines: serotonin, dopamine, tyramine, tryptamine, phenylethylamine and two non-physiological amines, kynuramine and m-iodobenzylamine, was studied. Anions in high concentrations inhibited enzyme activity with kynuramine being the substrate most affected. Among the biogenic amines, the activity with the indolalkylamines showed greater sensitivity to mono-valent anions such as chloride than to polyvalent ions such as phosphate whereas the opposite was true with the phenylalkylamines. However, pyrophosphate ion had little or no effect on MAO activity, regardless of substrate. The inhibition of kynuramine and serotonin deamination was non-competitive but mixed competitive inhibition was found with tyramine and phenylethylamine. The activity of MAO was markedly affected by pH, and it had been previously reported that the substrates showed different pH optima in their oxidation. The effect of pH on activity has been attributed in part to changes in the ionization of the substrate and the hypothesis that the true substrate is the non-protonated amine. This was reflected in kinetic studies showing high substrate inhibition with increased pH. It was calculated that phenylethylamine would have the highest percentage of un-ionized amine at pH 8.2 and 9.1. At these pHs, there was more pronounced inhibition with high substrate concentrations of phenylethylamine than with the other substrates. In contrast, there was little inhibition with high substrate concentrations of tyramine which was the most ionizable of the substrates tested. When K_m values obtained at pH 7.4, 8.2 and 9.1 were corrected for ionization of the substrate, the corrected K_m was lowest at pH 7.4 for all substrates. Less than 50% of MAO activity was lost when beef brain mitochondria was heated at 50°C for 20 min. However, there was only a slight variation with substrate in the thermal inactivation experiments. It is concluded that the mitochondrial membrane environment surrounding the enzyme imposes certain restrictions on the enzymatic activity with respect to the different substrates which, in turn, are also affected by such parameters as pH and ions. The results are discussed in terms of the relationship of these factors to the question of enzyme multiplicity.     

Influence of pH and sodium on the inhibition of guinea-pig heart (Na+ + K+)-ATPase by calcium.

The inhibition of guinea-pig heart (Na+ + K+)-ATPase (ATP phosphohydrolase EC 3.6.1.3) by calcium has been studied at pH 7.4, 6.8 and 6.4. 1. A decrease in pH reduced the threshold inhibitory concentration of calcium and the calcium concentration producing an inhibition of 50% of the enzyme activity. 2. Calcium reduced the apparent affinity of the enzyme of Na+, this effect occurred only at pH 7.4. 3. Calcium increased the apparent affinity of the enzyme for K+, this effect was enhanced at acidic pH. 4. Activation of the enzyme by Na+ for a constant Na+ : K+ ratio has been studied at pH 7.4 and at pH 6.8 in the absence and in the presence of 3.10(-4) M Ca 2+; the results of this experiment indicate that Ca2+ effect at pH 7.4 was not influenced by Na+ -- K+ competition and was probably due to a Na+ -- Ca2+ interaction. 5. At pH 7.4, the calcium inhibitory threshold concentration and the concentration producing 50% inhibition were reduced when Na+ was low; at pH 6.8, the calcium inhibition was not markedly modified by the change of Na+ concentration. 6. The Ca2+ -activated ATPase of myosin B which is related to the contractile behaviour of muscle and the Ca2+ -ATPase of the sarcoplasmic reticulum which is related to the ability of this structure to accumulate calcium were activated in a range of calcium concentration producing an inhibition of (Na2+ + K+) -ATPase. The present results indicate that the increase by acidity of the (Na2+ + K+) -ATPase sensitivity to calcium might be due to a suppression of a Na+ -Ca2+ interaction. On the basis of these observations, it is proposed that calcium might inhibit the Na+ -pump during the repolarization phase of the action potential and that, by this effect, it might control cell excitability.     

Oxidation of beta-phenylethylamine by both types of monoamine oxidase: effects of substrate concentration and pH.

β-Phenylethylamine (PEA) was characterized as substrate for both type A and type B monoamine oxidase (MAO) in rat brain mitochondria at different substrate concentrations and at different pHs of the reaction media. The experiments on sensitivity to clorygline and deprenyl showed that the inhibition patterns with PEA as substrate differed markedly at different substrate concentrations: at 10 μM, PEA acted as a specific substrate for type B MAO, but at 50–1000 μM it became a common substrate for both types of MAO. The inhibition patterns were also affected markedly by a small change in pH of the reaction medium, especially when PEA concentrations were 50 and 100 μM: the change in pH from 7.2 to 7.8 resulted in the incresse in the proportion of type A MAO by 20–30 per cent. To investigate the mechanisms of such changes in substrate specificity of PEA, kinetic analyses were carried out at pH 7.2 and 7.8 with the uninhibited, the clorgyline-treated (type B) and the deprenyl-treated (type A) enzyme. The Lineweaver-Burk plots for the uninhibited MAO showed strong substrate inhibition for both pHs, which is more marked at pH 7.8 than at pH 7.2. Pretreatment of the enzyme with 10^?7 M clorgyline resulted in generally similar K_m values for PEA to those of the uninhibited enzyme, and the substrate inhibition at pH 7.8 was also stronger than that at pH 7.2. After pretreatment with 10^?7 M deprenyl, the K_m values were higher and the V_max values were lower than those of the uninhibited or the clorgyline-treated enzyme; there was no or only slight substrate inhibition in these curves. These results suggest that the remarkable changes in substrate specificity observed at different PEA concentrations and at different pHs may be due to the strong substrate inhibition of type B MAO.     

Possible mechanism of selective inhibition of rat liver mitochondrial monoamine oxidase by chlorgiline and deprenyl]

The inhibition of the deamination of serotonin (the main substrate of monoamine oxidase (MAO) type A) by chlorgiline and deprenyl and of beta-phenylethylamine (the main substrate of the B type MAO) by fragments of rat liver mitochondrial membrane as well as the influence of 4-ethylpyridine on this process were studied. It was shown that the MAO activity of the mitochondrial membrane fragments was highly sensitive to chlorgiline, when serotonin was used as substrate, whereas a high sensitivity toward deprenyl was observed with beta-phenylethylamine as substrate. 4-Ethylpyridine (5.10(-3) M), a competitive and reversible inhibitor of the MAO activity, inhibited deamination of serotonin and beta-phenylethylamine by 34 and 30%, respectively. In experiments with chlorgiline (the specific inhibitor of MAO type A) 4-ethylpyridine (5.10(-3) M) introduced into the samples after preincubation of mitochondria with increasing concentrations of chlorgiline (30 min, 23 degrees C) decreased the inhibition by chlorgiline of the deamination of beta-phenylethylamine, but sharply increased the inhibitory effect of chlorgiline on the oxidation of serotonin. In analogous experiments with deprenyl (the specific inhibitor of MAO type B) 4-ethylpyridine (5.10(-3) M) decreased the inhibitory effect of deprenyl not only on the deamination of serotonin (substrate of MAO A), but also on the oxidation of beta-phenylethylamine (the main substrate of MAO type B). The decrease in the inhibitory effect of deprenyl on the deamination of beta-phenylethylamine after the addition of 4-ethylpyridine may be intensified upon preincubation of deprenyl with mitochondria in the presence of 4-ethylpyridine. The data obtained demonstrate the difference in the type and mechanism of inhibition of the deamination of serotonin by chlorgiline as well as in the type and mechanism of oxidation of beta-phenylethylamine by deprenyl. The possible mechanism of selective blocking of MAO activity by chlorgiline and deprenyl was discussed in terms of our previous data on the existence in the active center of mitochondrial MAO of specific sites for substrate binding, differing in their structure-functional characteristics.     

Metabolism of acetaldehyde by human erythrocytes.

Acetaldehyde metabolism in human erythrocytes was studied using head-space gas chromatographic determination methods, and it was found that acetaldehyde is metabolized in erythrocytes by NAD dependent cytosolic enzyme having an apparent Km value for acetaldehyde approximately 0.7 mM at pH 7.4, and more than 50% of this activity was reduced by 1 μM disulfiram. So, it is suggested that erythrocytes may have an enzyme system similar to the high Km isozyme of the liver aldehyde dehydrogenase.     

The allosteric regulation of hexokinase C from amphibian liver.

A type C hexokinase (ATP:D-hexose-6-phosphotransferase EC 2.7.1.1) was partially purified from the liver of the frog Calyptocephalella caudiverbera. The enzyme is inhibited by glucose levels in the range of normal blood sugar concentrations. The extent of the inhibition by glucose depends on the concentration of ATP, being most marked between 1 and 5 mM ATP. Fructose, although a substrate, was not inhibitory of its own phosphorylation. The inhibitory effect of high glucose levels exhibited a strong, reversible pH dependence being most marked at pH 6.5. At pH 7.5 the inhibition by high glucose levels was a function of the enzyme concentration, the effect being stronger at high enzyme concentrations, whereas no inhibition was observed when assaying very diluted preparations. At all enzyme concentrations studied, high levels of glucose caused no inhibition at pH 8.5, whereas at pH 6.5 strong inhibition was always observed. Short times of photooxidation of hexokinase C as well as incubation with low concentrations of p-chloromercuribenzoate resulted in the loss of the inhibition by excess of glucose. Glucose-6-phosphate was found to be a strong inhibitor of hexokinase C but only at high glucose levels. The inhibitory effect of glucose-6-P follows sigmoidal kinetics at low (about 0.02 mM) glucose concentrations, the Hill coefficient being 2.3. The kinetics of the inhibition became hyperbolic at high (greater than 0.2 mM) glucose levels. These results suggest that the inhibition of hexokinase C by excess glucose is due to the interaction of glucose with a second, aldose-specific, regulatory site on the enzyme. The modification of the inhibitory effect by ATP, glucose-6-P, enzyme concentration, and pH, all of them at physiological levels, indicates a major role for hexokinase C in the regulation of glucose utilization by the liver.     

Effect of protein denaturing reactions on retardation of the activity of rat liver mitochondrial monoamine oxidase by clorgyline and deprenyl

The denaturating effects of urea on clorgyline-produced inhibition of serotonin and tyramine deamination and deprenyl-produced inhibition of beta-phenylethylamine and tyramine oxidation were studied. It was shown that after preincubation of mitochondria with 1 and 2 M urea the intensity of inhibition by clorgyline and deprenyl of oxidation of these amines was not changed. With urea concentration of 3 and 4 M the inhibitory effect of clorgyline on deamination of serotonin and tyramine was increased, while that of deprenyl on oxidation of beta-phenylethylamine and tyramine was decreased. As a result of mitochondria treatment with 3 and 4 M urea the selectivity in inhibition by clorgyline of serotonin and tyramine deamination typical for intact mitochondria was reduced in the case of 3 M urea and eliminated in the case of 4 M urea. In intact mitochondria the intensity of inhibition by clorgyline of tyramine deamination in the presence of benzyl alcohol (competitive reversible MAO inhibitor) was increased, but the additive effect was not achieved. However, after preincubation of mitochondria with 3 M urea the summation of the inhibitory effects of clorgyline and benzyl alcohol was observed. The data obtained provide further evidence for the important role of spatial configuration of the monoamine oxidase molecule; the data are discussed in terms of arrangement on the protein molecule surface of the essential groups involved in the binding and deamination of amines for the inhibitory effects of clorgyline and deprenyl.     

Evidence for a calcium-sensitive factor which alters the alkaline pH sensitivity of sarcoplasmic reticulum calcium transport

Oxalase-supported, ATP-dependent Ca2+ uptake by cardiac and skeletal muscle sarcoplasmic reticulum (SR) exhibits a pH profile with the maximal rate of Ca2+ uptake at pH 6.6-6.8 and marked inhibition (90-95%) at pH 7.4-7.6, a point at which Ca2+-dependent ATPase activity is optimal. These observations are noted when the SR is first preincubated in media containing no added Ca2+. This alkaline pH inhibition is not caused by an irreversible perturbation since the Ca2+ uptake rate is fully restored by changing the alkaline pH preincubation medium to pH 6.8. When SR is preincubated with added Ca2+, Ca2+ uptake at alkaline pH (7.4-7.6) is only inhibited by 10-30%. Ca2+ uptake at pH 6.8 is the same regardless of preincubation conditions. A depressed oxalate permeability is not a factor in the observed alkaline pH inhibition of Ca2+ uptake. At alkaline pH, the relationship between the preincubation Ca2+ concentration and the rate of Ca2+ uptake is hyperbolic; the half-maximal free Ca2+ concentration for stabilization of Ca2+ uptake is 8-15 microM with a Vmax equal to the velocity at the optimal pH. The Hill coefficient is 1.0, implying a single class of Ca2+-requiring sites for stabilization at alkaline pH. In contrast to its effect on Ca2+ uptake, the presence of Ca2+ during preincubation does not alter the pH sensitivity of Ca2+-dependent ATPase activity. Thus, the presence of Ca2+ during preincubation may stabilize a state of the CaATPase, conducive to the coupling of net Ca2+ translocation to Ca2+-dependent ATPase activity, which is ordinarily opposed by alkaline pH. The data suggest a single class of Ca2+-requiring sites which favors this coupled state.     

Essential sulfhydryl groups of rat liver monoamine oxidase.

The inhibition by some thiol reagents of partly purified mitochondrial monoamine oxidase (MAO) (EC 1.4.3.4) from rat liver was studied, and the molar content of sulfhydryl groups in the enzyme determined. Sodium nitroprusside and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) inhibited the enzyme, apparently reversibly, while sodium arsenite was not inhibitory. Concentrations of the respective inhibitors causing 50% inhibition after 15 min of preincubation with the enzyme at pH 7.0 and 37 degrees C are 5.80 times 10(-4) M and 4.35 times 10(-5) M. The thiol compounds cysteine, dithiothreitol, and 2-mercaptoethanol did not inhibit MAO. The average number of sulfhydryl groups per mole of enzyme, determined by reaction with DTNB, increased from 3.6 +/- 0.2 freely reacting sulfhydryl groups (n = 4) to 18.4 to total sulfhydryl groups (n = 2) on denaturation with 8 M urea.     

Effects of tricyclic antidepressants upon human platelet monoamine oxidase

Tricyclic antidepressant drugs were found to inhibit human platelet MAO. The I50 for the inhibition by amitriptyline was 4 × 10^?6 M, 1.6 × 10^?5 M, and 2 × 10^?4 M when phenylethylamine, tryptamine, and benzylamine were used as substrates. Amitriptyline exhibited noncompetitive inhibition with the substrates phenylethylamine and tryptamine but competitive inhibition with benzylamine. Solubilization and partial purification of platelet MAO did not alter the inhibitory effects of tricyclics. Treatment of the partially purified enzyme with the chaotropic agent sodium perchlorate produced only a slight increase in the inhibition constant for amitriptyline. Our findings suggest that selective inhibition of phenylethylamine oxidation may mediate the antidepressant actions of tricyclics. In addition, our studies provide some evidence for the existence of multiple catalytic sites of MAO activity in the human platelet.     

Pyrodoxal 5′-phosphate inhibition of lactate production in rat liver supernatant fluid

The inhibitory effect of pyridoxal 5′-phosphate was examined on lactate production in rat liver 100000 × g supernatant fluid. Inhibition by pyridoxal 5′-phosphate was dependent on the duration of preincubation with supernatant fluid; lactate production exhibited a half-life of 5.5 min in the presence of 25 μM pyridoxal 5′-phosphate. The concentration of pyridoxal 5′-phosphate for 50% inhibition of lactate production from glucose as substrate was 10 μM and from glucose 6-phosphate as substrate, 185 μM.     

Alkylation of brain corticosteroid acetyltransferase by 17-hydroxyprogesterone 17-(9-oxo-10-chlorodecanoate) and related compounds

The addition of 17-hydroxyprogesterone 17-(9-oxo-10-chlorodecanoate) (1) at 1.5 μM to a partially purified preparation of corticosteroid acetyl-transferase from the primate brain at pH 7.4, results in a 50% inhibition of enzymatic activity after 30 minutes at 37°. At this concentration the analogous 9-oxo-10-diazodecanoate, 8-carbomethyoxyoctanoate or 8-carboxyoctanoate esters show no effect on the activity of this enzyme. 9-Oxo-10-chlorodecanoate and its methyl ester are respectively 0.44 and 0.07-fold as effective inhibitors as 1. The inhibition by 1 has been shown to be non-competitive and irreversible. There is no reaction of 1 with amino acids, glutathione, or human or bovine serum mercaptalbumin in a pH 7.4 phosphate buffer at 37°, demonstrating the partial specificity of this alkylating agent.     

Effects of divalent cations and pH on amiloride-sensitive Na+ fluxes into luminal membrane vesicles from pars recta of rabbit proximal tubule.

The effect of Ca2+, Cd2+, Ba2+, Mg2+ and pH on the renal epithelial Na(+)-channel was investigated by measuring the amiloride-sensitive 22Na+ fluxes into luminal membrane vesicles from pars recta of rabbit proximal tubule. It was found that intravesicular Ca2+ as well as extravesicular Ca2+ substantially lowered the channel-mediated flux. Amiloride sensitive Na+ uptake was nearly completely blocked by 10 microM Ca2+ at pH 7.4. The inhibitory effect of Ca2+ was dependent on pH. Thus, 10 microM Ca2+ produced 90% inhibition of 22Na+ uptake at pH 7.4, and only 40% inhibition at pH 7.0. The tracer fluxes measured in the absence of Ca2+ were pH independent over the range from 7.0 to 7.4. All the cations Ca2+, Cd2+, Ba2+ except Mg2+ inhibited the 22Na+ influx drastically when added extravesicularly in millimolar concentrations. The cations Cd2+, Ba2+ and Mg2+ in the same concentrations intravesicularly inhibited the 22Na+ influx only slightly. A millimolar concentration of Ca2+ intravesicularly blocked the amiloride-sensitive 22Na+ flux completely. The data indicate that Ca2+ inhibits Na+ influx specifically by binding to sites composed of one or several deprotonated groups on the channel proteins.     

Interrelationship between sodium cyanate and pH in the regulation of tumor cell division

Previous studies have suggested that the selective inhibitory effects of sodium cyanate on tumor metabolism in vivo may be related to a lower interstitial pH in tumors. In the present work, the influence of extracellular pH on the actions of sodium cyanate was studied with one rat hepatoma cell line (HTC) and two human colon tumor cell lines (HT29 and LS174T) and with rat hepatocytes to determine if the effects are accompanied by changes in intracellular pH. With some tumor cells, an inhibition of cell proliferation was observed when the cells were exposed to an acidic medium (pH 6.6). However, the LS174T line of human tumor cells divided at pH 6.6 essentially as fast as at pH 7.4. In the concentration range of 0.02-0.1 mg/ml, a greater inhibitory effect of cyanate on cell proliferation was observed at the lower pH. Intracellular pH was found to be influenced by the sodium ion concentration of the medium to a similar degree in the three tumor lines that were examined. The intracellular pH was found to be significantly affected by cyanate in rat hepatocytes and in two of the tumor cell lines (HT29 and LS174T). The data suggested that not only does extracellular pH influence the inhibitory effect of cyanate on tumor cell proliferation but also that cyanate can affect the regulation of intracellular pH in normal and neoplastic cells.     

Inhibition of monoamine oxidase activity by antidepressants and mood stabilizers

OBJECTIVE: Monoamine oxidase (MAO), the enzyme responsible for metabolism of monoamine neurotransmitters, has an important role in the brain development and function, and MAO inhibitors have a range of potential therapeutic uses. We investigated systematically in vitro effects of pharmacologically different antidepressants and mood stabilizers on MAO activity. Methods: Effects of drugs on the activity of MAO were measured in crude mitochondrial fraction isolated from cortex of pig brain, when radiolabeled serotonin (for MAO-A) or phenylethylamine (for MAO-B) was used as substrate. The several antidepressants and mood stabilizers were compared with effects of well known MAO inhibitors such as moclobemide, iproniazid, pargyline, and clorgyline. Results: In general, the effect of tested drugs was found to be inhibitory. The half maximal inhibitory concentration, parameters of enzyme kinetic, and mechanism of inhibition were determined. MAO-A was inhibited by the following drugs: pargyline > clorgyline > iproniazid > fluoxetine > desipramine > amitriptyline > imipramine > citalopram > venlafaxine > reboxetine > olanzapine > mirtazapine > tianeptine > moclobemide, cocaine > lithium, valproate. MAO-B was inhibited by the following drugs: pargyline > clorgyline > iproniazid > fluoxetine > venlafaxine > amitriptyline > olanzapine > citalopram > desipramine > reboxetine > imipramine > tianeptine > mirtazapine, cocaine > moclobemide, lithium, valproate. The mechanism of inhibition of MAOs by several antidepressants was found various. Conclusions: It was concluded that MAO activity is acutely affected by pharmacologically different antidepressants at relatively high drug concentrations; this effect is inhibitory. There are differences both in inhibitory potency and in mechanism of inhibition between both several drugs and the two MAO isoforms. While MAO inhibition is not primary biochemical effect related to their therapeutic action, it can be supposed that decrease of MAO activity may be concerned in some effects of these drugs on serotonergic, noradrenergic, and dopaminergic neurotransmission.     

Effect of substrates and effectors on the reversible inactivation of pig spleen phosphofructokinase by adenosine triphosphate

1. To investigate the mechanism of the reversible inactivation of pig spleen phosphofructokinase by ATP, the effect of order of addition of reactants (substrates, effectors and enzyme solution) was studied by preincubating the enzyme before assay with various combinations of its substrates and effectors. 2. Preincubation of the enzyme with MgATP or ATP at pH7.0 before addition of fructose 6-phosphate caused a rapid and much greater inhibition of activity than that observed when the reaction (carried out at identical substrate concentrations) was initiated with enzyme. 3. The rapid inhibition caused by preincubation with ATP, together with the sigmoidal response to fructose 6-phosphate and activation by AMP, were all blocked by prior photo-oxidation of the enzyme with Methylene Blue, which selectively destroys the inhibitory binding site for ATP [Ahlfors & Mansour (1969) J. Biol. Chem.244, 1247-1251]. 4. Fructose 6-phosphate, but not Mg(2+), protected phosphofructokinase from inhibition during preincubation with ATP in a manner that was sigmoidally dependent on the fructose 6-phosphate concentration. 5. Mg(2+), by protecting the enzyme from the inhibitory effect of preincubation at low pH (7.0) and by preventing its activation during preincubation with fructose 6-phosphate, demonstrated both a weak activating effect in the absence of the other substrates and a stronger inhibitory effect in the presence of fructose 6-phosphate. 6. Positive effectors (K(+), NH(4) (+), AMP and aspartate) protected the enzyme from inhibition during preincubation with MgATP in proportion to their potency as activators, but citrate potentiated the ATP inhibition. P(i) significantly slowed the inactivation process without itself acting as a positive effector. 7. The non-linear dependence of the initial rate of the unmodified enzyme on protein concentration (associated with increased positive homotropic co-operativity to fructose 6-phosphate) was intensified by preincubation with ATP and abolished by photo-oxidation. 8. The results are interpreted in terms of an association-dissociation model which postulates that protonation, at low pH, of a photo-oxidation-sensitive inhibitory site for ATP allows more rapid dissociation of an active tetramer to an inactive dimeric species.     

Elucidation of Growth Inhibition and Acetic Acid Production by Clostridium thermoaceticum

The production of acetic acid by Clostridium thermoaceticum was studied by using batch fermentations. In a pH-controlled fermentation with sodium hydroxide (pH 6.9), this organism was able to produce 56 g of acetic acid per liter. On the other hand, when the pH was not controlled and was decreased during fermentation to 5.4, the maximum attainable acetic acid concentration was only 15.3 g/liter. To obtain a better understanding of the end product inhibition, various salts were tested to determine their effect on the growth rate of C. thermoaceticum. An inverse linear relationship between the growth rate and the final cell concentration to the sodium acetate concentration was found. By using different concentrations of externally added sodium salts, the relative growth inhibition caused by the anion was found to be in the order of acetate > chloride > sulfate. Various externally added cations of acetate were also examined with respect to their inhibitory effects on growth. The relative magnitude of inhibition on the growth rate was found to be ammonium > potassium > sodium. The combined results have shown that the undissociated acetic acid was much more inhibitory than the ionized acetate ion. Complete growth inhibition resulted when the undissociated acetic acid concentration was between 0.04 and 0.05 M and when the ionized acetate concentration was 0.8 M. Therefore, at low pH (below 6.0), undissociated acetic acid is responsible for growth inhibition, and at high pH (above 6.0), ionized acetate ion is responsible for growth inhibition.     

The interaction of pH and cyclic adenosine 3',5'-monophosphate on activation of motility in Triton X-100 extracted bull sperm

The motility of demembranated bull sperm was found to be governed by the concentrations of cyclic adenosine 3', 5'-monophosphate (cAMP) and Ca2+ at low pH (6.6-7.1), and was less sensitive to these variables at higher pH (7.4-7.8). Although motility was generally found to increase with increasing pH in the range from 6.6 to 7.8, the addition of exogenous cAMP markedly and selectively improved the motility at the lower end of the range (pH 6.6-7.1). In the presence of 10 microM cAMP, low Ca2+ (8.0 X 10(-8) M), and a high concentration of Mg-adenosine 5'-triphosphate (ATP, 8 mM), demembranated sperm at pH 6.8 and 7.1 exhibited swimming similar to that of live ejaculated sperm. At a free Ca2+ concentration of 4.4 X 10(-5) M, the motility was rapidly inhibited at pH 6.8-7.1, whereas at pH 7.4-7.8, the activity was not greatly affected. Since calcium is known to antagonize the cAMP pathway by activating Ca2+-dependent phosphodiesterase and Ca2+-dependent phosphatase, this further supports the idea that cAMP-dependent activation is crucial for motility at low pH. Our results demonstrate that the flagellar axoneme can function normally at relatively acidic pH, and produce vigorous swimming at high levels of ATP. The ATP content of live sperm was measured and found to be high enough (approximately 8 mM) to support the vigorous motility seen at pH 6.6-7.1 in the models.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of disulfiram and its reduced metabolite, diethyl-dithiocarbamate on aldehyde dehydrogenase of human erythrocytes

Inhibition of human erythrocyte aldehyde dehydrogenase (ALDH) activity was studied using disulfiram and its reduced metabolite, diethyldithiocarbamate (DDC). The enzyme was rapidly inactivated by disulfiram and the inhibition was protected by reduced glutathione (GSH), in a concentration dependent manner when the enzyme premixed with GSH was reacted with disulfiram. Higher reactivity of the thiol group of the enzyme than that of GSH to disulfiram was suggested from the observation that half of the enzyme activity was inhibited when the ratio of disulfiram to GSH was 1:10. Although DDC alone showed no inhibitory effect on the enzyme, inactivation was mediated by a low concentration of heme-containing peroxidases, but not by methemoglobin. Under this condition, the inhibition potential was not protected, even with a high concentration of GSH. The constant reoxidation system of DDC is probably directly related to the enzyme inactivation.     

Subcellular localization and kinetic properties of arginase from the liver of Genypterus maculatus

1. From the liver of the teleost fish Genypterus maculatus, a partially purified preparation of arginase was obtained and characterized. 2. The Km value for arginine was found to be 9.1 mM at pH 7.5 and 11.5 mM at the optimum pH of 9.5. At both pH values, competitive inhibition was caused by ornithine and lysine, whereas proline, leucine, valine and isoleucine caused a non-competitive inhibitory effect. Branched chain amino acids were more inhibitory than proline. 3. The enzyme was found localized in the mitochondrial matrix of the liver of Genypterus maculatus. It is suggested that this localization would be of importance in the use of arginine as an energy source.