SU1146605A1 - Method of determination of thrombocyte monoaminoeoxidase activity - Google Patents

Abstract

METHOD OF MECHANICAL OPTIONS the fraction of 0.1 M phosphate buffer at pH 7.4, in the resulting eluate is determined using a substrate, the form of myoaminoxoxidase activity of 0.02 ± 0t nmol / mg protein per minute, then The same phosphate buffer is added with addition of Triton X-100 in a concentration gradient of 0.001-0.25%, and the form of monoamine oxidase with an activity of 1.910.2 nmol / mg protein is determined in the resulting eluate. per minute, then elute with 0.4 M phosphate buffer at pH 7.4 with the addition of (L of 0.25% Triton X-100), the monoamine oxidase form of 1j7 tOj2 nmol / mg protein per minute is determined in the eluate.

Description

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The invention relates to the field of biochemistry, biotechnology and medicine, and names: to methods for determining the activity of an enzyme in the body, specifically to methods for determining monoamine oxide (MAO) activity, and can be used in medical and laboratory practice, as well as for scientific purposes.

The aim of the invention is to improve the accuracy of the method by determining the multiple forms of monoamine oxidase.

An embodiment of the method, 500 MP of donor blood containing 0.38% sodium citrate, is centrifuged for 10 minutes. The erythrocyte sediment is washed with 0.15 M NaCl twice and separated by centrifugation. The combined supernatants are centrifuged under the same conditions, discarded, the supernatant is centrifuged for 20 minutes at 650 g. Platelets are suspended in 0.01 M K - On phosphate buffer at

pH 7.4. The platelet mixture is centrifuged for 10 min at 30 TMC.g, the supernatant is discarded, and the precipitate is homogenized in a glass homogenizer in a solubilizing mixture consisting of 1.3 M urea, 1.3% Triton X-100 in O, of M phosphate buffer at pH 7.4. After 30 min, the mixture is centrifuged for 30 min at 30 TMC.g, desalted on a Sephadex G-50 column (23-1.2 cm) and concentrated on an Amicon PM-30 ultrafilter 7 times. The solution is applied to an AN-Sepharose 4B column with affinity sorbent (13-1.2 cm. Washed with 0.01 M phosphate buffer at pH 7.4; multiple forms of MAO are degraded with phosphate buffer of various concentrations and a gradient of triton X-100. Determined protein and the activity of monoamine oxidase (for example, with benzyl amine as a substrate) in samples, an elution profile is obtained that characterizes the content (isoenzyme composition) and the amount of activity of three multiple forms of monoamine oxidase.

Claims (1)

METHOD FOR DETERMINING MONOAMINE-OXZZASE ACTIVITY OF THROMBOCYTES by incubating a solubilizer of platelet membranes with benzylamine as a substrate, characterized in that, in order to increase the accuracy of the cell by determining the multiple forms of monoamine oxidase, the membrane is solubilized with an amylose 0.1 M phosphate buffer at pH 7.4, in the obtained eluate, the form of monoamine oxidase with an activity of 0.02 ± 0.01 nmol / mg protein per minute is determined using a substrate, then elute they are removed with the same phosphate buffer with the addition of Triton X-100 in a concentration gradient of 0.001-0.25%, the form of monoamine oxidase with an activity of 1.9 ± 0, ^ nmol / mg protein per minute is determined in the obtained eluate, then elute with 0.4 M phosphate buffer at pH 7.4 with the addition of 0.25% triton X-100, in the resulting eluate determine the form of monoamine oxidase activity of 1.7 t +0.2 nmol / mg protein per minute.