CS195756B1 - Method of determination of the efficacy of the glycogenphosphorylasis in the blood - Google Patents
Method of determination of the efficacy of the glycogenphosphorylasis in the blood Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 23
- 210000004369 blood Anatomy 0.000 title claims abstract description 6
- 239000008280 blood Substances 0.000 title claims abstract description 6
- 102000009097 Phosphorylases Human genes 0.000 claims abstract description 19
- 108010073135 Phosphorylases Proteins 0.000 claims abstract description 19
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 claims abstract description 14
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims abstract description 14
- 229920002472 Starch Polymers 0.000 claims abstract description 13
- 239000008107 starch Substances 0.000 claims abstract description 13
- 235000019698 starch Nutrition 0.000 claims abstract description 13
- 229920002527 Glycogen Polymers 0.000 claims abstract description 12
- 229940096919 glycogen Drugs 0.000 claims abstract description 12
- 239000000203 mixture Substances 0.000 claims abstract description 11
- YMAWOPBAYDPSLA-UHFFFAOYSA-N glycylglycine Chemical compound [NH3+]CC(=O)NCC([O-])=O YMAWOPBAYDPSLA-UHFFFAOYSA-N 0.000 claims abstract description 10
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims abstract description 8
- 102000004190 Enzymes Human genes 0.000 claims abstract description 6
- 108090000790 Enzymes Proteins 0.000 claims abstract description 6
- 229910019142 PO4 Inorganic materials 0.000 claims abstract description 6
- 238000011534 incubation Methods 0.000 claims abstract description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims abstract description 6
- 239000010452 phosphate Substances 0.000 claims abstract description 6
- 239000000758 substrate Substances 0.000 claims abstract description 6
- 108010008488 Glycylglycine Proteins 0.000 claims abstract description 5
- 229940043257 glycylglycine Drugs 0.000 claims abstract description 5
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 claims abstract description 4
- 210000002966 serum Anatomy 0.000 claims description 19
- 239000007795 chemical reaction product Substances 0.000 claims description 5
- 238000005374 membrane filtration Methods 0.000 claims description 4
- 230000002255 enzymatic effect Effects 0.000 claims description 3
- AVPCPPOOQICIRJ-UHFFFAOYSA-L sodium glycerol 2-phosphate Chemical compound [Na+].[Na+].OCC(CO)OP([O-])([O-])=O AVPCPPOOQICIRJ-UHFFFAOYSA-L 0.000 claims description 3
- 239000012636 effector Substances 0.000 claims description 2
- 229960002901 sodium glycerophosphate Drugs 0.000 claims description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims 2
- 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 claims 1
- 239000008103 glucose Substances 0.000 claims 1
- 239000011780 sodium chloride Substances 0.000 claims 1
- REULQIKBNNDNDX-UHFFFAOYSA-M sodium;2,3-dihydroxypropyl hydrogen phosphate Chemical compound [Na+].OCC(O)COP(O)([O-])=O REULQIKBNNDNDX-UHFFFAOYSA-M 0.000 claims 1
- 239000000243 solution Substances 0.000 abstract description 32
- PUZPDOWCWNUUKD-UHFFFAOYSA-M sodium fluoride Chemical compound [F-].[Na+] PUZPDOWCWNUUKD-UHFFFAOYSA-M 0.000 abstract description 10
- 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 abstract description 7
- 229950010772 glucose-1-phosphate Drugs 0.000 abstract description 7
- UDMBCSSLTHHNCD-UHFFFAOYSA-N Coenzym Q(11) Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(O)=O)C(O)C1O UDMBCSSLTHHNCD-UHFFFAOYSA-N 0.000 abstract description 5
- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 abstract description 5
- LNQVTSROQXJCDD-UHFFFAOYSA-N adenosine monophosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(CO)C(OP(O)(O)=O)C1O LNQVTSROQXJCDD-UHFFFAOYSA-N 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 5
- 229910052708 sodium Inorganic materials 0.000 abstract description 5
- 239000011734 sodium Substances 0.000 abstract description 5
- 239000011775 sodium fluoride Substances 0.000 abstract description 5
- 235000013024 sodium fluoride Nutrition 0.000 abstract description 5
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 abstract description 4
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 abstract description 2
- DKNPRRRKHAEUMW-UHFFFAOYSA-N Iodine aqueous Chemical compound [K+].I[I-]I DKNPRRRKHAEUMW-UHFFFAOYSA-N 0.000 abstract description 2
- 229930006000 Sucrose Natural products 0.000 abstract description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 abstract description 2
- 239000002253 acid Substances 0.000 abstract description 2
- 230000015572 biosynthetic process Effects 0.000 abstract description 2
- 229910052816 inorganic phosphate Inorganic materials 0.000 abstract description 2
- 238000000926 separation method Methods 0.000 abstract description 2
- 210000001124 body fluid Anatomy 0.000 abstract 6
- 239000010839 body fluid Substances 0.000 abstract 6
- 229960001484 edetic acid Drugs 0.000 abstract 4
- 239000007864 aqueous solution Substances 0.000 abstract 1
- 238000001514 detection method Methods 0.000 abstract 1
- 235000013681 dietary sucrose Nutrition 0.000 abstract 1
- 238000007865 diluting Methods 0.000 abstract 1
- 238000010828 elution Methods 0.000 abstract 1
- 239000012530 fluid Substances 0.000 abstract 1
- 235000021317 phosphate Nutrition 0.000 abstract 1
- 238000001179 sorption measurement Methods 0.000 abstract 1
- 229960004793 sucrose Drugs 0.000 abstract 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 abstract 1
- 238000005406 washing Methods 0.000 abstract 1
- 102000007390 Glycogen Phosphorylase Human genes 0.000 description 6
- 108010046163 Glycogen Phosphorylase Proteins 0.000 description 6
- WZIMSXIXZTUBSO-UHFFFAOYSA-N 2-[[bis(carboxymethyl)amino]methyl-(carboxymethyl)amino]acetic acid Chemical compound OC(=O)CN(CC(O)=O)CN(CC(O)=O)CC(O)=O WZIMSXIXZTUBSO-UHFFFAOYSA-N 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 208000010125 myocardial infarction Diseases 0.000 description 3
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 2
- 210000004165 myocardium Anatomy 0.000 description 2
- QHQZEEGNGSZBOL-UHFFFAOYSA-N 2-(aminomethyl)-2-(hydroxymethyl)propane-1,3-diol Chemical compound NCC(CO)(CO)CO QHQZEEGNGSZBOL-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- 229920000856 Amylose Polymers 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 108010044467 Isoenzymes Proteins 0.000 description 1
- 235000010716 Vigna mungo Nutrition 0.000 description 1
- 244000042295 Vigna mungo Species 0.000 description 1
- 229960003190 adenosine monophosphate Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 125000003147 glycosyl group Chemical group 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000000663 muscle cell Anatomy 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
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Abstract
Description
Vynález se týká způsobu stanovení účinnosti lsoíosforylázy v krvi. Jde zejména o glykogen fosforylázy.The invention relates to a method for determining the efficacy of isosophosphorylase in blood. These are in particular glycogen phosphorylase.
Vynález si klade za úkol zlepšit způsob stanovení glykogenfosforylázy tak, aby bylo možno stanovit jak celkovou fosforylázu, tak určité isofosforylázy.It is an object of the present invention to provide a method for determining glycogen phosphorylase so that both total phosphorylase and certain isophosphorylase can be determined.
Jsou známy způsoby kvantitativního stanovení glykogenfosforylázy, například účinnosti fosforylázy ze svalů měřením glukózo-l-fosfátu nebo orthofosfátu v homogenátech tkání.Methods are known for quantitatively determining glycogen phosphorylase, for example, the efficacy of muscle phosphorylase by measuring glucose-1-phosphate or orthophosphate in tissue homogenates.
1. Pfannemilller, B.: In: Handbuch der Physiologisch- und pethologisch-chemlschen Analyse (Hrg. Hoppe-Beyler u. Thierfelder], Springer Verlag, Berlin-Gčttingen-Heidelberg, sv. VI/B, str. 225 až 253 (1966),1. Pfannemilller, B .: In: Handbuch der Physiologisch- und Pethologisch-Chemlschen Analyze (Hrg. Hoppe-Beyler u. Thierfelder), Springer Verlag, Berlin-Getttingen-Heidelberg, Vol. VI / B, pp. 225-253 ( 1966),
2. Will, H. urid R. G. Krause: Acta biol. med. german. 28, 919 až 933 (1972).2. Will, H. urid, R.G. Krause: Acta biol. copper. german. 28, 919-933 (1972).
Tyto způsoby spočívají v tom, že se zjišťují různé reakční produkty reakce, která je katalyzována enzymem. Jde o uvolnění anorganického fosfátu, prodloužení glykosylových řetězců molekuly glykogenu, popřípadě tvorba glukózo-l-fosfátu. Ke zjištění těchto produktů je známa celá řada způsobů, které se od sebe liší svou citlivostí jen nepodstatně. Známé způsoby se více neboThese methods consist in detecting various reaction products of an enzyme-catalyzed reaction. These are the release of inorganic phosphate, the extension of the glycosyl chains of the glycogen molecule, or the formation of glucose-1-phosphate. A number of methods are known for detecting these products which differ only insignificantly in their sensitivity. The known methods are more or less
S měně hodí ke kvantitativnímu stanovení účinnosti enzymu, bez ohledu na kvalitativní rozdíly mezi různými isoenzymy fosforylázy. Stanovení účinnosti je v krevním séru spojeno s tou nevýhodou, že. je přítomno pouze velmi malé množství fosforylázy při velkém přebytku jiných bílkovin.It is suitable for the quantitative determination of enzyme activity, regardless of the qualitative differences between the various phosphorylase isoenzymes. Determination of efficacy in blood serum is associated with the disadvantage that:. only a very small amount of phosphorylase is present with a large excess of other proteins.
Zjištění účinnosti fosforylázy, zejména specifické fosforylázy pro srdeční sval v krevním séru u lidí, má velký diagnostický význam, protože po poškození buněk svalu, například při infarktu, se tento enzym objevuje v krevním séru [Krause, E. G., H. Běhm, H. Will a A. Wollenberger: Dtsch. Ges. wesen 27, 903 (1972)). Způsob zjišťování účinnosti glykofenfosforylázy v krevním séru však dosud není znám.Determining the efficacy of phosphorylase, in particular the specific serum phosphorylase in the blood serum of humans, is of great diagnostic importance since, after damage to muscle cells, for example in a heart attack, this enzyme appears in blood serum [Krause, EG, H. Behhm, H. Will and A. Wollenberger: Dtsch. Ges. Res. 27, 903 (1972)). However, the method for determining the efficacy of blood glycophosphorylase is not yet known.
Vynález si tedy klade za úkol navrhnout způsob stanovení účinnosti glykogenfosforylázy v krevním séru.SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide a method for determining the efficacy of glycogen phosphorylase in blood serum.
Předmětem vynálezu je způsob stanovení účinnosti glykogeníosforylázy v krevním séru, vyznačující se tím, že se krevní sérum po předběžném zpracování smísí se substrátem pro enzym a roztokem, obsahujícím efektor a po inkubaci se měří enzymatická účinnost podle vzniklých reakčních produktů.The present invention provides a method for determining the efficacy of glycogen phosphorylase in blood serum, characterized in that, after pretreatment, the blood serum is mixed with the enzyme substrate and the effector-containing solution and after incubation the enzymatic activity is measured according to the reaction products formed.
Krevní sérum se předběžně zpracovává koncentrací pomocí membránové filtrace v poměru 2:1 až 3:1 a popřípadě se předběžně zpracovává škrobem. К předběžnému zpracování škrobem se sérum nanese na sloupec naplněný škrobem při teplotě 0 až 5 °C. Sloupec se uvede do· rovnovážného stavu v roztoku s obsahem 1 až 103 molu/litr fluoridu sodného, 0,1 až 10“4 molu/litr j3-glycerofosfátu sodného a 0,1 až 104 molu/litr merkaptoethanolu o pH 5,5 až 7,0. Potom se roztok nechá procházet sloupcem, načež se vymývá fosforylázou při teplotě 10 až 35 °C a pH 6,0 až 7,5 dalším roztokem, který obsahuje 0,1 až 10 “4 molu/litr β-glycerofosfátu sodného, 1 až 10~4 molu/litr kyseliny ethylendiamintetraoctové a 0,1 až 10_4 molu/litr merkaptoethanolu.Blood serum is pretreated by concentration by membrane filtration in a ratio of 2: 1 to 3: 1 and optionally pretreated with starch. For starch pre-treatment, serum is applied to a starch-filled column at 0-5 ° C. The column is equilibrated in a solution containing 1 to 10 3 mol / liter sodium fluoride, 0.1 to 10 4 mol / liter sodium β-glycerophosphate and 0.1 to 10 4 mol / liter mercaptoethanol, pH 5, 5 to 7.0. Then the solution is passed through the column and then eluted phosphorylase at a temperature of 10-35 ° C and pH 6.0 to 7.5 second solution comprises 0.1 to 10 "4 mole / liter of sodium β-glycerophosphate, 1 to 10 ~ 4 mol / l of ethylenediaminetetraacetic acid and 0.1 to 10 mole _4 / l mercaptoethanol.
Sérum, koncentrované membránovou filtrací, se smísí s roztokem, který obsahuje 1 až 103 molu/litr glycylglycinu, 0,1 až 104 molu/litr merkaptoethanolu a 1 až 104 molu/litr kyseliny ethylendiamintetraoctové při pH 6 až 7,5.The serum, concentrated by membrane filtration, is mixed with a solution containing 1 to 10 3 mol / liter glycylglycine, 0.1 to 10 4 mol / liter mercaptoethanol and 1 to 10 4 mol / liter ethylenediaminetetraacetic acid at pH 6 to 7.5.
Tato směs, popřípadě eluát ze škrobového sloupce se přidá к roztoku, který obsahuje roztok glykogenu, glukózo-l-fosfátu a adenosinmonofosfátu v poměru 5:3:2 a výsledná směs se inkubuje 60 minut při teplotě 37 °C. Potom se směs denaturuje kyselinou trichloroctovou a obsah fosfátu se stanoví opticky.This mixture or starch column eluate is added to a solution containing a 5: 3: 2 solution of glycogen, glucose-1-phosphate and adenosine monophosphate, and the resulting mixture is incubated at 37 ° C for 60 minutes. The mixture is then denatured with trichloroacetic acid and the phosphate content is determined optically.
Glykogenfosíoryláza se dělí na 3 isofosforylázy elektroforézou, aby bylo možno stanovit formu, typickou pro srdeční sval. Po inkubaci za přítomnosti substrátu se isoíosforylázy barví podle reakčního produktu směsí jodu a jodidu draselného.Glycogen phosphorylase is divided into 3 isophosphorylase by electrophoresis to determine the form typical of the cardiac muscle. After incubation in the presence of the substrate, the iso-phosphorylase is stained according to the reaction product with a mixture of iodine and potassium iodide.
Způsob podle vynálezu je vhodný jako vyšetřovací metoda к diagnóze srdečního infarktu, což při použití dosavadních způsobů nebylo možné. V krevním séru zdravých osob není možno naměřit žádnou účinnost fosforylázy. Z tohoto hlediska principiálně způsobuje jakékoli stanovení fosforylázy v krevním séru podezření na infarkt.The method according to the invention is suitable as an examination method for the diagnosis of heart attack, which has not been possible with the prior art methods. No phosphorylase activity can be measured in the blood serum of healthy persons. In this regard, any determination of serum phosphorylase in principle causes a suspected heart attack.
V následujících příkladech se popisuje stanovení fosforylázy i složení činidla, které se к provádění způsobu podle vynálezu používá.The following examples describe the determination of phosphorylase as well as the composition of the reagent used to carry out the process of the invention.
PřikladlHe did
Činidlo ke stanovení fosforylázy má následující složení:The phosphorylase reagent has the following composition:
roztok 1solution 1
0,1 m fluoridu sodného0.1 m sodium fluoride
0,001 m /J-glycerofosfátu sodného o pH 6,1 0,015 m merkaptoethanolu roztok 2Sodium J-glycerophosphate 0.001 m, pH 6.1 0.015 m mercaptoethanol solution 2
0,04 m jS-glycerofosfátu sodného o pH 6,8 0,001 m kyseliny ethylendiamintetraoctové 0,015 m merkaptoethanolu roztok 30.04 m sodium s-glycerophosphate, pH 6.8 0.001 m ethylenediaminetetraacetic acid 0.015 m mercaptoethanol solution 3
0,04 m glycylglycinu o pH 6,80.04 m glycylglycine at pH 6.8
0,01 m merkaptoethanolu 0,008 m kyseliny ethylendiamintetraoctové roztok 40,01 m mercaptoethanol 0,008 m ethylenediaminetetraacetic acid solution 4
3,3 o/o glykogenu roztok 53.3 o / o glycogen solution 5
0,083 m glukózo-l-fosfátu0.083 m glucose-1-phosphate
0,005 m adenosínmonofosfátu0.005 m adenosine monophosphate
Sérum se koncentruje membránovou filtrací v poměru 2:1 až 3:1, popřípadě se předběžně zpracuje nerozpustným škrobem. V tomto případě se 3 až 4 ml séra nanesou při 4 °C na sloupec škrobu o rozměru 2X1 centimetrů, v. rovnovážném, stavu ,v...rozto.-. ku 1. Potom se sloupec promyje 3 X 3 ml roztoku 1 a vymývá se 2 ml roztoku 2 při 30 °C fosforylázou.The serum is concentrated by membrane filtration in a ratio of 2: 1 to 3: 1, optionally pretreated with insoluble starch. In this case, 3-4 ml of serum are applied at 4 ° C to a 2X1 centimeter starch column, equilibrium, in solution. Then, the column was washed with 3 X 3 mL of solution 1 and eluted with 2 mL of solution 2 at 30 ° C with phosphorylase.
díl koncentrovaného séra se smísí s 1 dílem roztoku 3 а к 5 dílům směsi, popřípadě eluátu se přidají 3 díly roztoku 4 a 2 díly roztoku 5. Po 60 minutách inkubace při 37 °C se roztok denaturuje kyselinou trichloroctovou a známým způsobem [Wěií -Melherbe, H.: In: Handbuch der Physiologisch- und pathologisch-hemischen Analyse (Hrg. Hoppe-Seyler ů. Thierfelder), Springer Verlag, Berlin-Góttgen-Heidelberg, sv. III/l, str. 457 až 538 (1955)], se provede analýza fosforečnanu, který je reakčním produktem.part of the concentrated serum is mixed with 1 part of the solution 3 and 5 parts of the mixture or eluate are added 3 parts of solution 4 and 2 parts of solution 5. After 60 minutes incubation at 37 ° C the solution is denatured with trichloroacetic acid and by known method [Wie-Melherbe , H.: Handbuch der Physiologisch- und Pathologisch-Hemischen Analyze (Hrg. Hoppe-Seyler, Thierfelder), Springer Verlag, Berlin-Götgen-Heidelberg, Vol. III / 1, pp. 457-538 (1955)], the reaction product phosphate is analyzed.
P ř í к 1 a d 2Example 1 a d 2
Nyní se provádí stanovení enzymatické účinnosti za přítomnosti specifických inhibičních látek isofosforylázy, takže se na rozdíl od stanovení podle příkladu i zjistí podíl fosforylázy specifické pro· srdeční sval.Enzyme activity is now determined in the presence of specific isophosphorylase inhibitors, so that, unlike the assay of Example 1, the proportion of cardiac muscle-specific phosphorylase is determined.
Činidlo к oddělení isofosforylázy a stanovení enzymatické účinnosti má následující složení:The reagent for separating the isophosphorylase and determining the enzymatic activity has the following composition:
roztok 1solution 1
0,042 m tris(hydroxymethyl)aminoethanu 0,001 m kyseliny ethylendiamintetraoctové 0,049 m kyseliny borité o pH 8,2 roztok 20.042 m tris (hydroxymethyl) aminoethane 0.001 m ethylenediaminetetraacetic acid 0.049 m boric acid pH 8.2 solution 2
0,03 m glukózo-l-fosfátu0.03 m glucose-1-phosphate
0,002 m adenosínmonofosfátu0.002 m adenosine monophosphate
0,002 m /?-glycerofosfátu sodného o pH 6,80.002 m @ 2 - sodium glycerophosphate, pH 6.8
0,002 m kyseliny ethylendiaminotetraoctové0.002 m ethylenediaminotetraacetic acid
0,001 m merkaptoethanolu0.001 m of mercaptoethanol
0,1 m fluoridu sodného roztok 30.1 m sodium fluoride solution 3
0,01 m jodu0.01 m iodine
0,014 m jodidu draselného0.014 m potassium iodide
Alikvotní část eluátu ze šroubového sloupce s obsahem fosforylázy se zředí roztokem 1 v poměru 2:1 2. m sacharózy a diskovou elektroforetickou technikou se oddělí iso fosforyláza. K dělení se užije gelu ve složení 5 % akrylamidu a 0,2 % N,N‘-methylbisakrylamidu s obsahem 0,1 % glykogenu a roztoku 1 jako · pufru, který je zároveň elektrodovým . pufrem. Po inkubaci tohoto · gelu v roztok 2 se nanese roztok 3, čímž se zbarví amylózové postranní řetězce vzniklé polymeraci glykogenu.An aliquot of the eluate from the phosphorylase-containing screw column is diluted with a 2: 1 solution of 2: 1 sucrose and the iso phosphorylase is separated by disk electrophoresis. A gel consisting of 5% acrylamide and 0.2% N, N‘-methylbisacrylamide containing 0.1% glycogen and solution 1 as a buffer, which is also an electrode buffer, is used for separation. buffer. After incubation of this gel in solution 2, solution 3 is applied to stain the amylose side chains resulting from glycogen polymerization.
Claims (4)
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DD16876573A DD101986A1 (en) | 1973-02-07 | 1973-02-07 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CS195756B1 true CS195756B1 (en) | 1980-02-29 |
Family
ID=5490033
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CS85674A CS195756B1 (en) | 1973-02-07 | 1974-02-07 | Method of determination of the efficacy of the glycogenphosphorylasis in the blood |
Country Status (9)
| Country | Link |
|---|---|
| BG (1) | BG20730A1 (en) |
| CS (1) | CS195756B1 (en) |
| DD (1) | DD101986A1 (en) |
| DE (1) | DE2401484A1 (en) |
| FR (1) | FR2327545A1 (en) |
| GB (1) | GB1451004A (en) |
| HU (1) | HU169957B (en) |
| PL (1) | PL94983B1 (en) |
| SU (1) | SU976958A1 (en) |
-
1973
- 1973-02-07 DD DD16876573A patent/DD101986A1/xx unknown
-
1974
- 1974-01-12 DE DE19742401484 patent/DE2401484A1/en not_active Withdrawn
- 1974-01-29 GB GB406174A patent/GB1451004A/en not_active Expired
- 1974-02-05 BG BG2570174A patent/BG20730A1/xx unknown
- 1974-02-06 SU SU741999385A patent/SU976958A1/en active
- 1974-02-06 HU HUAE000406 patent/HU169957B/hu unknown
- 1974-02-07 PL PL16863774A patent/PL94983B1/pl unknown
- 1974-02-07 FR FR7404148A patent/FR2327545A1/en active Granted
- 1974-02-07 CS CS85674A patent/CS195756B1/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| PL94983B1 (en) | 1977-09-30 |
| HU169957B (en) | 1977-03-28 |
| FR2327545B1 (en) | 1978-02-17 |
| DD101986A1 (en) | 1973-11-20 |
| SU976958A1 (en) | 1982-11-30 |
| GB1451004A (en) | 1976-09-29 |
| BG20730A1 (en) | 1975-12-20 |
| FR2327545A1 (en) | 1977-05-06 |
| DE2401484A1 (en) | 1974-08-29 |
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