CS211785B1 - Method of preparation of the /u-14c/glucose -6-phosphate - Google Patents

Method of preparation of the /u-14c/glucose -6-phosphate Download PDF

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CS211785B1
CS211785B1 CS268680A CS268680A CS211785B1 CS 211785 B1 CS211785 B1 CS 211785B1 CS 268680 A CS268680 A CS 268680A CS 268680 A CS268680 A CS 268680A CS 211785 B1 CS211785 B1 CS 211785B1
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phosphate
glucose
preparation
alpha
green
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CS268680A
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Czech (cs)
Slovak (sk)
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Juraj Zemek
Stefan Kucar
Jozef Kolina
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Juraj Zemek
Stefan Kucar
Jozef Kolina
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Description

Vynález rieši sposob přípravy /U- C/ glukozo-6-fosfátu účinkom enzýmu fosfoglukomutázy /oí -D-glukózo-1 , 6d ifo sf á t :©(-D-glukó zo-1 -f osf át fosfotransferáza, EC 2.7.5.1/ na^-D-glukopyranozylfosfát /U-^C/ o vysokej mólovej aktivitě, připravený účinkom enzýmu fosforylázy na vodorozpustný polysacharid typu alfa£l 4/6/]glukánu podía /A.O. Č. 194 587/ izolovaný z rádioaktívného biochemického materiálu, s výhodou zo zelených a modrozelených rias, v procese komplexného spracovania radioaktivněj biomasy /Čs. pat. 121 808pričom produkt enzymovej reakcie sa separuje z reakčnej zmesi chromatograficky, o výtažku až 23 % pre glukozo-614. , , *,The invention solves a process for the preparation of (U-C) glucose-6-phosphate by the action of the enzyme phosphoglucomutase / α-D-glucose-1,6d ifphosphate: (-D-glucose-1-phosphate phosphotransferase, EC 2.7. 5.1 (.alpha.-D-glucopyranosyl phosphate (U-C)) of high molar activity, prepared by the action of the phosphorylase enzyme on the water-soluble alpha-14 (6)] glucane polysaccharide according to (AO No. 194 587) isolated from radioactive biochemical material; preferably from green and blue-green algae, in the process of complex processing of radioactive biomass / US Pat. No. 121,808, wherein the enzyme reaction product is separated from the reaction mixture by chromatography, up to 23% yield for glucoso-614.

-fosfát. Z biochemických metod přípravy glukozo-6-f osf átu /U- C/ je uvádzaný postup využívájúci D-glukózu a enzym hexokinázu /EC 2.7.1.1/, pričom reakcia prebieha za přítomnostiphosphate. Among the biochemical methods for the preparation of glucoso-6-phosphate (U-C), a process using D-glucose and the enzyme hexokinase (EC 2.7.1.1) is reported, wherein the reaction proceeds in the presence of

ATP. Aj ked výtažok v tejto reakcii je vysoký, nevýhodou je, že aa spracováva D-glukóza a nie východzí zdroj D-glukózy, vodorozpustný alfa/l 4/6/J glukán /U-^C/. Táto nevýhoda je riešená postupom podía vynálezu, kedy východzou látkou přípravy je priamo vodorozpustný polysacharid, připravený v rámci komplexného spracovania rádioaktívnej biomasy /Cs. pat. Č. 1 21 808/,ATP. Although the yield in this reaction is high, the disadvantage is that aa processes D-glucose and not the starting D-glucose source, the water-soluble alpha (14/6) J glucan (U-C). This disadvantage is solved by the process according to the invention, wherein the starting material of the preparation is a directly water-soluble polysaccharide prepared in the framework of the complex treatment of radioactive biomass / Cs. pat. No. 1 21 808

Podstata vynálezu spočívá v tom, že na vetvený polysacharid typu alfa /1 *> 4/ glukánu s větvením alfa/1—>6/ vazbami, izolovaný z rádioaktívného biologického materiálu, s výhodou so zelených,resp. modrozelených rias, v procese komplexného spracovania rádioaktívnej biomasy, sa posobí enzýmom fosforylázou /alfa-1,4-glukán ortofosfát glukozyltransferáza, EC 2.4.1.1/ v přítomnosti pufrového roztoku fosfátu o pH 6 až 7 /A.O. č. 194 587/, pričom na získaný alfa-D-glukopyranozylfosfát /U-^C/ sa posobí pufrovaným roztokom fosfoglukomutázy v přítomnosti cysteinu, síranu horečnatého a glukózo-1,6-difosfátu /pH 7,5 až 8,0/ a získaný glukózo-6-fosfát /U-^C/ sa oddělí jednoducho chromatografiou na papieri. Uvedeným postupem možno získat gLukózo-6-fosfát /O-^C/ o vysokej mólovej aktivitě a výtažku až 95 Z, vzhladom na alfa-D-glu14 kopyranozylfosfát /U- C/.SUMMARY OF THE INVENTION It is the object of the invention to provide a branched polysaccharide of the alpha (1 * 4) glucan type with alpha (1 → 6) branching isolated from radioactive biological material, preferably green or green, respectively. Blue-green algae, in the process of complex processing of radioactive biomass, is treated with the enzyme phosphorylase / alpha-1,4-glucan orthophosphate glucosyltransferase, EC 2.4.1.1/ in the presence of a phosphate buffer solution of pH 6 to 7 / A.O. no. 194 587), wherein the obtained alpha-D-glucopyranosyl phosphate (U-C) is treated with a buffered solution of phosphoglucomutase in the presence of cysteine, magnesium sulfate and glucose-1,6-diphosphate (pH 7.5 to 8.0) and the glucose obtained The 6-phosphate (U-C) is separated simply by chromatography on paper. Glucose-6-phosphate (O-C) having a high molar activity and a yield of up to 95 Z can be obtained by this procedure, based on alpha-D-gluc14-copranosyl phosphate (U-C).

17851785

44

Výhodou navrhovaného spósobu prípravy /U- C/ glukozo-6-fosfátu je, že:An advantage of the proposed process for the preparation of (U-C) glucose-6-phosphate is that:

- celý postup prípravy je velmi jednoduchý v technickom převedení- the whole preparation process is very simple in technical conversion

- poskytuje ďalšiu možnost využitia vodorozpustného glukánu /U-^C/, izolovaného z rádioaktívneho biologického materiálu- provides another possibility of using water-soluble glucan (U-C) isolated from radioactive biological material

- všetky enzýmy potřebné k prevedeniu naznačených postupov sú komerčně dostupné, připadne pripravitelné v laboratórnych podmienkach.- all the enzymes required to carry out the indicated procedures are commercially available or can be prepared under laboratory conditions.

Příklad 1Example 1

K 0,1 ml 1Z roztoku vodorozpustného polysacharidů /U-^C/ zo zelených rias o celkovej aktivitě 47 kBq sa přidal 1 ml 0,2 M fosfátového pufru o pH 6,0 a 1 ml fosforylázy zo zemiakov /A.O. 194 588/ a reakčná zmes sa inkubovala pri 35 °C po dobu 4 hodin. Po přeběhnutí reakcie sa nastavilo pH reakčnej zmesi na hodnotu 7,5 pomocou 0,5 M fosfát, pufru /pH 8,0/ a přidal sa cystein na výslednú konc. 50 mM, síran horečnatý na 6 raM konc. a glukózo-1,6-difosfát na 0,1 raM konc. a 10 fosfoglukomutázy /z kvasíniek, 0,34 U/ a reakčná zmes sa inkubovala ďalšie 2 hodiny pri 35 °C. Po ukončení reakcie sa reakčná zmes naniesla na papier Whatraan 1 a separácia glukózo-6-fosfátu sa previedla v systéme kyselina izomaslová-čpavok-voda /66:1:33/ po dobu 100 hodin. Výťažok glukózo-6-fosfátu /U- C/ 7,2 kBq, to je 95 % na množstvo vytvořeného alfá-D-glukopyranozylfosfátu /7,44 kBq/,To 0.1 ml of a 1 Z solution of water-soluble polysaccharides (U-C) from green algae with a total activity of 47 kBq was added 1 ml of 0.2 M phosphate buffer pH 6.0 and 1 ml of potato phosphorylase / A.O. 194 588) and the reaction mixture was incubated at 35 ° C for 4 hours. After the reaction was run, the pH of the reaction mixture was adjusted to 7.5 with 0.5 M phosphate buffer (pH 8.0) and cysteine was added to the resulting conc. 50mM, magnesium sulfate at 6mM conc. and glucose-1,6-diphosphate per 0.1mA conc. and 10 phosphoglucomutase (from yeast, 0.34 U) and the reaction mixture was incubated for another 2 hours at 35 ° C. After completion of the reaction, the reaction mixture was loaded onto Whatraan 1 paper and the separation of glucose-6-phosphate was carried out in isobutyric-ammonia-water system (66: 1: 33) for 100 hours. Glucose-6-phosphate yield (U-C) of 7.2 kBq, i.e. 95% based on the amount of alpha-D-glucopyranosyl phosphate formed (7.44 kBq),

Příklad 2Example 2

Tak ako je uvedené v příklade 1, s tým rozdielom, že po ukončení reakcie sa zvyškový alfa-D-glukopyranozylf09fát /U~ C/ rozloží prídavkom HC1 na výslednú koncentráciu 0,1 N k reakčnej zraesi, tá sa povarí po dobu 3 minút a kyselina chlorovodíková sa odpaří na vákuovej odparke. Chromatografická separácia sa prevedie tak, ako je uvedené v příklade 1.As in Example 1, except that after completion of the reaction, the residual alpha-D-glucopyranosyl phosphate (U-C) is decomposed by adding HCl to a final concentration of 0.1 N to the reaction precipitate, which is boiled for 3 minutes and the hydrochloric acid was evaporated in a vacuum evaporator. Chromatographic separation was performed as described in Example 1.

G l'ukózo-6-f osf át je dóležitá biochemikália, ktorá v znacenom stave ako /U-^C/ rádíocheraikália sa móže použit k štúdiu enzymových procesov prebiehajúcich ín vitro, ako aj k histochemíckým Štúdiara a sledovaniu priebehu glykolýzy v buňkách. Možno ho považovat aj za dóležitú východziu látku k chemickým syntézám v oblasti chemie cukrov.Glucose-6-phosphate is an important biochemical which, in the form of (U-C) radocheraemia, can be used to study enzymatic processes running in vitro as well as the histochemical investigator and the monitoring of the course of glycolysis in cells. It can also be considered as an important starting point for chemical syntheses in the field of sugar chemistry.

T a b u Ϊ k a 1T a b u Ϊ k a 1

Hodnoty chromatografických pohyblivostí niektorých fosfátov D-glukózy a D-fruktózy, vztahované na glukózo-6-fosfát v systéme kyselina izomaselná-čpavok-voda /66:1:33/.Chromatographic mobility values of some D-glucose and D-fructose phosphates referenced to glucose-6-phosphate in isobutyric-ammonia-water system (66: 1: 33).

Glukozo-6-fosfát 1,0Glucoso-6-phosphate 1.0

Fruktozo-6-fosfát 1,31Fructozo-6-phosphate 1.31

Fruktózo-1,6-difo8fát 0,51 íC-D-g lukopyr ano zy lf o sf á t 1,11Fructose-1,6-diphosphate 0.51C-D-g lucopyrylphosphate 1.11

Claims (2)

PREDMET VYNALEZUOBJECT OF THE INVENTION 14 Spósob prípravy /11- C/ glukozo-6-f osf átu z rad ioaktívneho biologického materiálu, s výhodou větveného vodorozpustného alfa/Ί ** 4/6/.7g lukánu zo zelených alebo modrozelených rias, vyznačujúci sa tým, že na roztok získaného a 1 f a -D-g lu ko pyr ano zy 1 f o s f á tu /U-^C/ sa pósobí pufrovaným roztokom fosfoglukomutázy pri pH 7,5 až 8,0 v přítomnosti cysteinu, glukozo-1,6-difosfátu a horečnatých solí a vzniknutý produkt sa izoluje chromatograficky na papieri.14 A process for the preparation of (11-C) glucoso-6-phosphate from a series of ioactive biological material, preferably branched water-soluble alpha (Ί ** 4/6) .7g green or blue-green algae luana, characterized in that the obtained α 1 -Dg luicycyclosyl phosphate (U-C) is treated with a buffered phosphoglucomutase solution at pH 7.5 to 8.0 in the presence of cysteine, glucose-1,6-diphosphate and magnesium salts; and the resulting product is isolated by chromatography on paper. Severografia, n. p.. závod 7, MostSeverography, n. p. plant 7, Most
CS268680A 1980-04-17 1980-04-17 Method of preparation of the /u-14c/glucose -6-phosphate CS211785B1 (en)

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