CS211785B1 - Method of preparation of the /u-14c/glucose -6-phosphate - Google Patents
Method of preparation of the /u-14c/glucose -6-phosphate Download PDFInfo
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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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- 238000000034 method Methods 0.000 title claims description 12
- 238000002360 preparation method Methods 0.000 title claims description 7
- NBSCHQHZLSJFNQ-GASJEMHNSA-N D-Glucose 6-phosphate Chemical compound OC1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H](O)[C@H]1O NBSCHQHZLSJFNQ-GASJEMHNSA-N 0.000 title description 9
- VFRROHXSMXFLSN-UHFFFAOYSA-N Glc6P Natural products OP(=O)(O)OCC(O)C(O)C(O)C(O)C=O VFRROHXSMXFLSN-UHFFFAOYSA-N 0.000 title description 8
- 229940045189 glucose-6-phosphate Drugs 0.000 title 1
- 229910019142 PO4 Inorganic materials 0.000 claims description 9
- 239000010452 phosphate Substances 0.000 claims description 9
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 5
- 102000009569 Phosphoglucomutase Human genes 0.000 claims description 4
- 108091000115 phosphomannomutase Proteins 0.000 claims description 4
- 241000195628 Chlorophyta Species 0.000 claims description 3
- 241000192700 Cyanobacteria Species 0.000 claims description 3
- RWHOZGRAXYWRNX-VFUOTHLCSA-N alpha-D-glucose 1,6-bisphosphate Chemical compound O[C@H]1[C@H](O)[C@@H](COP(O)(O)=O)O[C@H](OP(O)(O)=O)[C@@H]1O RWHOZGRAXYWRNX-VFUOTHLCSA-N 0.000 claims description 3
- 239000012620 biological material Substances 0.000 claims description 3
- 238000004587 chromatography analysis Methods 0.000 claims description 3
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 3
- 235000018417 cysteine Nutrition 0.000 claims description 3
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims description 2
- 240000000260 Typha latifolia Species 0.000 claims 1
- 159000000003 magnesium salts Chemical class 0.000 claims 1
- 238000006243 chemical reaction Methods 0.000 description 7
- 230000002285 radioactive effect Effects 0.000 description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 5
- 239000011541 reaction mixture Substances 0.000 description 5
- 229920001503 Glucan Polymers 0.000 description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- 150000004676 glycans Chemical class 0.000 description 4
- 229920001282 polysaccharide Polymers 0.000 description 4
- 239000005017 polysaccharide Substances 0.000 description 4
- 239000002028 Biomass Substances 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- 108010073135 Phosphorylases Proteins 0.000 description 3
- 102000009097 Phosphorylases Human genes 0.000 description 3
- HXXFSFRBOHSIMQ-VFUOTHLCSA-N alpha-D-glucose 1-phosphate Chemical compound OC[C@H]1O[C@H](OP(O)(O)=O)[C@H](O)[C@@H](O)[C@@H]1O HXXFSFRBOHSIMQ-VFUOTHLCSA-N 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 229950010772 glucose-1-phosphate Drugs 0.000 description 3
- 235000021317 phosphate Nutrition 0.000 description 3
- 102000005548 Hexokinase Human genes 0.000 description 2
- 108700040460 Hexokinases Proteins 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 235000019341 magnesium sulphate Nutrition 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- IGBWEXLCOAPIOB-BAOOBMCLSA-N (3s,4r,5r)-1,3,4,5,6-pentahydroxyhexan-2-one;phosphoric acid Chemical class OP(O)(O)=O.OC[C@@H](O)[C@@H](O)[C@H](O)C(=O)CO IGBWEXLCOAPIOB-BAOOBMCLSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 108010055629 Glucosyltransferases Proteins 0.000 description 1
- 102000000340 Glucosyltransferases Human genes 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- RNBGYGVWRKECFJ-ZXXMMSQZSA-N alpha-D-fructofuranose 1,6-bisphosphate Chemical compound O[C@H]1[C@H](O)[C@](O)(COP(O)(O)=O)O[C@@H]1COP(O)(O)=O RNBGYGVWRKECFJ-ZXXMMSQZSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 238000002306 biochemical method Methods 0.000 description 1
- 239000008366 buffered solution Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 229940025237 fructose 1,6-diphosphate Drugs 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000034659 glycolysis Effects 0.000 description 1
- 230000001744 histochemical effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
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)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CS268680A CS211785B1 (en) | 1980-04-17 | 1980-04-17 | Method of preparation of the /u-14c/glucose -6-phosphate |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CS268680A CS211785B1 (en) | 1980-04-17 | 1980-04-17 | Method of preparation of the /u-14c/glucose -6-phosphate |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CS211785B1 true CS211785B1 (en) | 1982-02-26 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CS268680A CS211785B1 (en) | 1980-04-17 | 1980-04-17 | Method of preparation of the /u-14c/glucose -6-phosphate |
Country Status (1)
| Country | Link |
|---|---|
| CS (1) | CS211785B1 (en) |
-
1980
- 1980-04-17 CS CS268680A patent/CS211785B1/en unknown
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