Comparative Studies in the Characterization of Monoamine Oxidases.

Oswald, E.O.; Strittmatter, Cornelius F.

doi:10.3181/00379727-114-28765

Cited by 39 publications

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“…The K , for purified monoamine oxidase oxidizing tyramine is lower than that reported for the rat liver enzyme [23] but Oswald and Strittmatter [24] have shown that the K , values of monoamine oxidase vary not only between species but also between organs from the same species. Substrate inhibition of the enzyme by tyramine hydrochloride has been previously reported, although it has been suggested that such inhibition may be a function of the anion used rather than the tyramine molecule itself [25].…”

Section: Discussionmentioning

confidence: 64%

The Purification of Pig Brain Mitochondrial Monoamine Oxidase

Tipton

1968

European Journal of Biochemistry

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A simple procedure for the extraction and purification of monoamine oxidase from pig brain mitochondria is described. The enzyme purified in this way appears to be homogeneous by cellulose acetate electrophoresis and the molecular weight was estimated to be approximately 102,000 by gel-filtration. The purified enzyme is inhibited by iproniazid, chelating agents and a sulphydryl reagent. The K , value for tyramine has been determined as has its Kg value for high substrate inhibition.There have been several reported purification procedures for the mitochondrial enzyme monoamine oxidase, but they have had only limited success due to difficulties in solubilizing the enzyme and keeping it soluble throughout the purification procedure. have used sonication in the presence of a detergent and a monoamine oxidase substrate to obtain a successful extraction of the enzyme from rat liver mitochondria and have purified the extracted enzyme some 200 fold. In the case of the enzyme from brain, little purification has previously been reported, although Nagatsu [S] has shown that the enzyme can be extracted from beef brain mitochondria by sonication in the presence of a detergent. I n this paper a simple but tedious procedure is described whereby pig brain mitochondrial monoamine oxidase can be extracted and purified about 1,000 times. MATERIALS AND METHODS Chemical MaterialsTris (hydroxymethyl) amino methane (Trizma base) was obtained from the Sigma Chemical Company (London) Ltd., TritonX 100 from Lennig Chemicals Ltd., bovine serum albumin from Armour Chemicals Inc., pepsin, muramidase and trypsin inhibitor from Worthington Inc., catalase from BioNan-Standard Abbreviations. iV-isonicotinyl-N2-isopropylhydrazide, iproniazid ; catechol3,5-disulphonic acid, tiron.Enzymes. Monoamine oxidase, or monoamine: 0, oxidoreductase (deaminating) (EC 1.4.3.4) ; catalase, or H202:H20z oxidoreductase (EC 1.11.1.6) ; muramidase, or mucopeptide N-acetylmuramylhydrolase (EC 3.2.1.17) ; pepsin (EC 3.4. 4.1).chemica Boehringer, DEAE cellulose from H Reeve Angel and Co. Ltd. and amino black (10 B) was obtained from G. T. Gurr Ltd. All other chemicals were obtained from British Drug Houses Ltd. or Hopkin and Williams Ltd., and were of the highest purity available. Distilled water was passed through a Permutit mark 11 deionizer before use. Cellulose acetate strips were obtained from 0x0 Ltd. Assay MethodsAll assays were carried out at 30" using solutions which had been saturated with air a t that temperature. Specific activity is expressed as patoms of oxygen consumed per minute per mg enzyme. The oxidation of tyramine was followed, using an oxygen electrode, by a modification of the method of Creasey [9] adapted for use with an oxygen electrode. The assay mixture contained in a total volume of 2.4 ml: 200 pmoles of sodium phosphate buffer pH 7.0, 100 units of catalase, 20 pmoles of semicarbazide, 2 pmoles of KCN and enzyme. The reaction was started by the addition of 100 pl of 0.025 M tyramine hydrochloride.Protein Concentration. This was determined...

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Section: Discussionmentioning

confidence: 64%

The Purification of Pig Brain Mitochondrial Monoamine Oxidase

Tipton

1968

European Journal of Biochemistry

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“…[9][10][11][12][13][14] indicates that this enzyme is localized in the outer membrane. The retention of malate dehydrogenase in the fraction scdimenting at 9,500 g suggests that the inner membrane remains well preserved.…”

Section: Discussionmentioning

confidence: 99%

“…Baudhuin et al (9) studied the distribution of this enzyme in rat liver homogenates by density gradient centrifugation and concluded that it is not associated with lysosomes or microbodies. Oswald and Strittmatter (10) found that the distributions of monoamine oxidase and succinate oxidase are similar in fractions of rat liver homogenates. Gorkin (11) showed that monoamine oxidase is firmly bound to sonic particles derived from rat liver mitochondria.…”

Section: Introductionmentioning

confidence: 97%

The Submitochondrial Localization of Monoamine Oxidase

Schnaitman¹,

Erwin²,

Greenawalt³

1967

The Journal of Cell Biology

814

209

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Controlled osmotic lysis (water-washing) of rat liver mitochondria results in a mixed population of small vesicles derived mainly from the outer mitochondrial membrane and of larger bodies containing a few cristae derived from the inner membrane. These elements have been separated on Ficoll and sucrose gradients. The small vesicles were rich in monoamine oxidase, and the large bodies were rich in cytochrome oxidase. Separation of the inner and outer membranes has also been accomplished by treating mitochondria with digitonin in an isotonic medium and fractionating the treated mitochondria by differential centrifugation. Treatment with low digitonin concentrations released monoamine oxidase activity from low speed mitochondrial pellets, and this release of enzymatic activity was correlated with the loss of the outer membrane as seen in the electron microscope. The low speed mitochondrial pellet contained most of the cytochrome oxidase and malate dehydrogenase activities of the intact mitochondria, while the monoamine oxidase activity could be recovered in the form of small vesicles by high speed centrifugation of the low speed supernatant. The results indicate that monoamine oxidase is found only in the outer mitochondrial membrane and that cytochrome oxidase is found only in the inner membrane. Digitonin treatment released more monoamine oxidase than cytochrome oxidase from sonic particles, thus indicating that digitonin preferentially degrades the outer mitochondrial membrane.

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“…Several authors have reported direct or indirect evidence for the existence of multiple forms of MAO, such as marked difference in the heat stabilities of the activities of MAO from rat liver mitochondria for serotonin and tyrarnine (15), and differences in the affinities for benzylamine and serotonin (16). However, in the present study, the Km value of placental MAO was found to be 0.21 mM for serotonin and 0.23 mM for tyramine and the optimum pH values with these two substrates were similar.…”

Section: Effects Of Inhibitorsmentioning

confidence: 99%

Studies on Monoamine Oxidase. Xviii. Enzymic Properties of Placental Monoamine Oxidase

Egashira

1976

Japanese Journal of Pharmacology

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Abstract-Enzymicproperties of partially purified monoamine oxidase (MAO) from human placenta were studied with tyramine, serotonin and benzylamine as substrates. The highest activity was obtained with serotonin and almost no activity was observed with benzylamine. These results are similar to those obtained with rat placental MAO, but different from those with rabbit placental MAO. The Km values for serotonin and tyramine were found to be 0.21 mM and 0.23 mM, respectively and the pH optimum was 8.1 with either substrate. The thermal inactivation curves of this enzyme with the two substrates were identical. The pt curves for inhibition of MAO activity by harmine, pargyline and iproniazid were similar and almost the same pI 50 values for the respective inhibitors were obtained with the two substrates. MAO in human placenta differs from that in other organs, such as liver, brain and plasma from the standpoint of the substrate specificity and the inhibitor sensitivity. The possibility that human placenta contains a single form of MAO is discussed on the basis of the present results.In 1948 Luschinsky and Tickner (1) reported that human placenta contained a relatively large amount of monoamine oxidase (MAO). The following year Thompson and Tickner (2) confirmed this observation in rat, rabbit and ginea pig placentas. About twenty years later Youdim and Sandler (3) obtained a partially purified preparation of the enzyme from human placenta. Their final product showed a specific activity of 3,500 and 400-fold puri fication over the original homogenate. There have been many clinical studies (4-8) on human placental MAO obtained at term and postmature deliveries, but characteristics and functional significance of this enzyme are unknown.In this work the enzymic properties of MAO from mitochondria of human placenta were compared with those of preparations from bovine liver and brain. To determine whether or not MAO in human placenta exists as a single enzyme with a broad substrate specificity or as a multiple form of the enzyme, the substrate specificities, pH optimum, sensitivity to inhibitors and effect of temperature on the enzyme were studied with serotonin and tyramine as substrates. MATERIALS AND METHODS Preparation of MAOHuman placental MAO: MAO was partially purified from human placental mito chondria by a slight modification of the method of Youdim and Sourkes (9). Mitochondria were prepared from human placenta by differential centrifugation, suspended in 0.1 M phosphate buffer (pH 7.5) and then were sonicated for 30 min at 20 kHz. Triton X-100 was

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Comparative Studies in the Characterization of Monoamine Oxidases.

Cited by 39 publications

References 0 publications

The Purification of Pig Brain Mitochondrial Monoamine Oxidase

The Purification of Pig Brain Mitochondrial Monoamine Oxidase

The Submitochondrial Localization of Monoamine Oxidase

Studies on Monoamine Oxidase. Xviii. Enzymic Properties of Placental Monoamine Oxidase

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