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Solubilization and some properties of a semicarbazide-sensitive amine oxidase in brown adipose tissue of the rat.

ABSTRACT: A semicarbazide-sensitive clorgyline-resistant amine oxidase (SSAO) was solubilized from membrane fractions of rat brown adipose tissue by the non-ionic detergent, Triton X-100. Alteration of ionic strength or addition of chelating agents alone failed to release the enzyme from its membrane. Lipid-depletion led to loss of enzyme activity and alteration of substrate affinity. Over 80% of the activity of the solubilized enzyme was found in gel filtration fractions corresponding to an Mr of between 160 000 and 180 000. The glycoprotein nature of SSAO was established from affinity chromatography with either immobilized concanavalin A or Lens culinaris lectin. Elution of over 50% SSAO activity from the lentil lectin was achieved with 0.25M-alpha-methyl D-mannoside to give 80-90-fold purification of the enzyme. Irradiation inactivation gave a value for Mr of around 183 000 for both soluble and membrane-bound SSAO. Substrate affinity and inhibitor sensitivity of the enzyme were unaltered by solubilization. The copper-chelating agent, diethyldithiocarbamate, did not affect the enzyme, shedding doubt on the suggestion that SSAO is a copper-requiring enzyme. The significance of these findings in relation to the nature of SSAO and to its disposition within the cell membrane is discussed.

SUBMITTER: Barrand MA

PROVIDER: S-EPMC1144201 | biostudies-other | 1984 Sep

REPOSITORIES: biostudies-other

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Project description:Background: Methylxanthines including caffeine and theobromine are widely consumed compounds and were recently shown to interact with bovine copper-containing amine oxidase. To the best of our knowledge, no direct demonstration of any interplay between these phytochemicals and human primary amine oxidase (PrAO) has been reported to date. We took advantage of the coexistence of PrAO and monoamine oxidase (MAO) activities in human subcutaneous adipose tissue (hScAT) to test the interaction between several methylxanthines and these enzymes, which are involved in many key pathophysiological processes. Methods: Benzylamine, methylamine, and tyramine were used as substrates for PrAO and MAO in homogenates of subcutaneous adipose depots obtained from overweight women undergoing plastic surgery. Methylxanthines were tested as substrates or inhibitors by fluorimetric determination of hydrogen peroxide, an end-product of amine oxidation. Results: Semicarbazide-sensitive PrAO activity was inhibited by theobromine, caffeine, and isobutylmethylxanthine (IBMX) while theophylline, paraxanthine, and 7-methylxanthine had little effect. Theobromine inhibited PrAO activity by 54% at 2.5 mM. Overall, the relationship between methylxanthine structure and the degree of inhibition was similar to that seen with bovine PrAO, although higher concentrations (mM) were required for inhibition. Theobromine also inhibited oxidation of tyramine by MAO, at the limits of its solubility in a DMSO vehicle. At doses higher than 12 % v/v, DMSO impaired MAO activity. MAO was also inhibited by millimolar doses of IBMX, caffeine and by other methylxanthines to a lesser extent. Conclusions: This preclinical study extrapolates previous findings with bovine PrAO to human tissues. Given that PrAO is a potential target for anti-inflammatory drugs, it indicates that alongside phosphodiesterase inhibition and adenosine receptor antagonism, PrAO and MAO inhibition could contribute to the health benefits of methylxanthines, especially their anti-inflammatory effects.

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Solubilization and some properties of a semicarbazide-sensitive amine oxidase in brown adipose tissue of the rat.

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