SU958976A1 - Guanilatecyclasa activity determination method - Google Patents
Guanilatecyclasa activity determination method Download PDFInfo
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- SU958976A1 SU958976A1 SU813248740A SU3248740A SU958976A1 SU 958976 A1 SU958976 A1 SU 958976A1 SU 813248740 A SU813248740 A SU 813248740A SU 3248740 A SU3248740 A SU 3248740A SU 958976 A1 SU958976 A1 SU 958976A1
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- SU
- USSR - Soviet Union
- Prior art keywords
- guanilatecyclasa
- determination method
- activity determination
- cgmp
- gtp
- Prior art date
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
Изобретение относитс к медицине, а именно энзимологии. This invention relates to medicine, namely enzymology.
Известен способ определени активности гуаиилатциклазы в биологическом материале путем добавлени к исследуемой пробе гуанозинтрифосфата (ГТФ) с последующей инкубацией полученной смеси, выделени циклического 3,5-гуанозинмоноФосфата (цГМФ) хрсматографическим путем и последующего определением его концентрации фотометрически 1 .A method is known for determining the activity of guaiylate cyclase in biological material by adding guanosine triphosphate (GTP) to the test sample, followed by incubating the mixture, isolating cyclic 3,5-guanosine mono-phosphate (cGMP) by chromatographic method and then determining it photometrically 1.
Однако известный способ сложен и имеет высокую токсичность, что ограничивает область его применени .However, the known method is complex and has high toxicity, which limits its scope.
Целью изобретени вл етс упрощение способа.The aim of the invention is to simplify the method.
Эта цель достигаетс тем, что при осуществлении.определений активности туанилатциклазы в биологическом материале путем добавлени к исследуемой пробе гуанозмнтрифосфата (ГТФ) с последующей инкубацией полученной смеси, выделени цикличес -КОГО 3,5-гуанозинмонофосфата (цГМФ) хроматографйческим путем и определением его концентрации фотсжётрйчески , пробу после инкубации с ГТФ обрабатывают фосфатом хршлатографируют на ионообменной ; колонке , заполненной полистирольным анионитом , далее в пробу добавл ют спир товый раствор нитрата терби и по величине интен.сивности флуоресценции при длине волны 545 нм определ ют активность гуанилатциклазы.This goal is achieved by the implementation of the definitions of the activity of tuanylate cyclase in biological material by adding guanosine triphosphate (GTP) to the test sample, followed by incubating the resulting mixture, isolating the cyclic 3.5-guanosine monophosphate (cGMP) cyclographically and determining its concentration of photo-hygrothermophosphate (cGMP) by chromatographic methods and determining its concentration of photo-hygrothermal the sample after incubation with GTP is treated with phosphate hrshlatografiruut on ion exchange; a column filled with polystyrene anion exchanger, then an alcohol solution of terbi nitrate is added to the sample and the guanylate cyclase activity is determined by the magnitude of the intensity of fluorescence at a wavelength of 545 nm.
Способ осуществл ют следующим образом.The method is carried out as follows.
После инкубации ферментного пре10 парата, гуанилатциклазную реакцию останавливают прогревом на кип щей вод ной бане, пробы охлаждают и обрабатывают эквивалентными коли- , чествами растворов хлорида (или суль15 фата) кадми и фосфата кали , осадок фосфата кадми удал ют центрифугированием . В этих услови х до 95% ГТФ соосаждаетс с фосфате кад мн , в то врем как 95-96% цГМФ After incubation of the enzyme preparation, the guanylate cyclase reaction is stopped by heating in a boiling water bath, the samples are cooled and treated with equivalent amounts of solutions of cadmium chloride and potassium phosphate, and the precipitate of cadmium phosphate is removed by centrifugation. Under these conditions, up to 95% of GTP is co-precipitated with phosphate cad mn, while 95-96% of cGMP
20 остаетс в растворе. Дл полного удалени следов ГтФ и других неш клических гуанилрвых нуклеотидов (последние также усиливают нехарак теристическую флуоресценцию иона 25 .терби ) полученную надосадочную жид-. kocjTb хроматографируют на колонке с прлистирольнЁЛМ анионитом (типа Дауэкс и т.п.), причем, цГМФ элюируют 0,06 М нитратом амиони в 0,033 М аиетатйом буфере (рН 5,0), пригото г20 remains in solution. To completely remove traces of GTP and other non-clinical guaniline nucleotides (the latter also enhance the non-characteristic fluorescence of T-25 ion), the resulting supernatant liquid. kocjTb is chromatographed on a column with a polystyrene anion exchange resin (such as Dowex, etc.), and cGMP is eluted with 0.06 M amionic nitrate in 0.033 M aietta buffer (pH 5.0), prepared
30thirty
Claims (1)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SU813248740A SU958976A1 (en) | 1981-02-20 | 1981-02-20 | Guanilatecyclasa activity determination method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SU813248740A SU958976A1 (en) | 1981-02-20 | 1981-02-20 | Guanilatecyclasa activity determination method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| SU958976A1 true SU958976A1 (en) | 1982-09-15 |
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ID=20943347
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| SU813248740A SU958976A1 (en) | 1981-02-20 | 1981-02-20 | Guanilatecyclasa activity determination method |
Country Status (1)
| Country | Link |
|---|---|
| SU (1) | SU958976A1 (en) |
-
1981
- 1981-02-20 SU SU813248740A patent/SU958976A1/en active
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