CS211784B1 - Method of preparation of the /u-14c/fructose-6-phosphate - Google Patents

Description

(54) Process for the preparation of (U- ^{* 14} C) fructose-6-phosphate

The present invention provides a process for the preparation of (U-C) fructoso-6-phosphate by the following action of the enzymes phosphoglucomutase / alpha-D-glucoso-1,6-diphosphate: alpha-D-glucoso-1-phosphate phosphotransferase, EG 2.7.5.1/ and glucose phosphate isomerase (D-glucose-6-phosphate ketol isomerase, EC 5.3.1.9) to alpha-D-glucopyrrosis anosylfolate (U-C) of high molar activity, prepared by the action of the phosphorylase enzyme on a water-soluble alpha-pol polysaccharide 4/6 /} g of the alkane under 1a / AO no. 194 587 (isolated from radioactive biological material, preferably from green and blue-green algae) in the process of complex processing of radioactive biomass (Cs. pat. 121 808), wherein the enzyme reaction product is separated from the reaction mixture by chromatography, up to 44% for fructose-1,6-diphosphate and 23% for fructose-6-phosphate and up to 23% for glucose-6-phosphate.

£ biochemical method for the preparation of a glucose - ^ - phosphate / ^U-1Z * C / the declared method using D-glucose / U- ^ C /, and the enzyme hexokinase / EC 2.7.1.1/, the reaction taking place in the presence of ATP. Although the extract in this reaction is high, the disadvantage is that D-glucose is treated and not the starting source of D-glucose, the water-soluble alpha (Ί → 4/6) glucane (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 as part of a complex treatment of radioactive bioraase / Cs. pat. no. 121,808 /.

SUMMARY OF THE INVENTION The invention is based on the fact that on a branched polysaccharide of the alpha (1 → 4) -glucan type with alpha (1 → 4) -branching, isolated from radioactive biological material, preferably from green and 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 glucose1 transferase, EC 2.4.1.1./ in the presence of buffered phosphate solution pH 6 to 7 (AO no. 194 587), wherein the obtained alpha-D-glucopyranosyl phosphate (U- ^q C) is treated with a buffered solution of phosphoglucomutase in the presence of 7 8 4

1784 of cysteine, magnesium sulphate and glucose-1,6-diphosphate (pH 7.5 to 8.0) and to the obtained glu14 goat-6-phosphate (U-C) are treated with a buffered glucose-6-phosphate isomerase solution at pH 9.0 and the fructozo-6-phosphate formed is separated by paper chromatography. With respect to alpha-D-glucopyrene and 1-phophate (U- ^Zt C) titers, the yield of fractions of z-6-phosphate is up to 76%.

An advantage of the proposed process for the preparation of (U-C) labeled phosphorylated D-glucose and D-fructose derivatives is that:

the whole preparation process is very simple in technical conversion and allows completion of the reaction process at the level of individual intermediates of the glycolytic pathway, provides another possibility of using water-soluble glucane / U active biological material,

C 1, isolated from the order of the enzymes required to carry out the above-described procedures, are commercially available, and may be prepared under laboratory conditions.

Example

K 0, activate metal / A. 0.

ml of 0.2 ml of 0.2 M phosphate buffer, pH 6.0 c, was added from the water-soluble polysaeharide / U-C / 17 kBq solution. 194 588) and the reaction mixture was incubated at 35 ° C for green algae and 1 ml phosphorylase for 5 hours.

After completion of the reaction, 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. 50 mM, magnesium sulfate to 6 mM conc. and glucose 1,6-diphosphate per 0.1 mM conc. and 10 L phosphoglucomutase (from yeast, 0.34 U) and the reaction mixture was incubated for an additional 2 hours at 35 ° C.

After the phosphoglucination reaction is completed, the pH of the reaction mixture is adjusted to 9.0 with 0.5 M glycine-sodium hydroxide buffer (pH 10) and yeast glucose-6o is added.

-phosphate isomerase (10 µL, 0.23) and incubated for an additional hour at 35 ° C.

After working up the reaction mixture, the separation of the phosphorylated derivatives is carried out in an isobutyric-ammonia-water system (66: 1: 32) by paper chromatography for 100 hours. Fructose-6-phosphate was obtained with a yield (U-C of 5.9 kBq) of 76f.

Phosphorylated D-fructose metabolites are important biochemicals that, when labeled as (U-1C) radiochemicals, can be used to study in vitro enzymatic processes, as well as histochemical studies and monitoring of the course of cell glycolysis. They can also be considered as important starting materials for chemical syntheses in the field of sugar chemistry.

Table I

Chromatographic mobility values of some D-glucose and D-fructose phosphates relative to glucose-6-phosphate in the isobutyric acid-ammonia-water system (66: 1: 33).

Glucose-6-phosphate 1.0

Fro k thozo-6-phos ph 1.31

Fructose 1,6-diphosphate 0.51

Alpha-D-glucopyranosyl phosphate-1, T1

Claims (1)

OBJECT OF THE INVENTION 14. Process for the preparation of (U-C) fruk Co 20-6-phosphate from radioactive biological material, preferably branched, water-soluble alpha / Ί -> 4/6 / Zglucan from green or blue-green algae, characterized by: of the obtained .alpha.-D-glucopyranosyl phosphate (U- ¹⁴ 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 to the obtained (.alpha.-C). The glucose of o-6-phosphate is treated with a buffered glucose-6-phosphate isomerase solution at pH 9.0 and the resulting product is isolated by chromatography on paper.