CS211786B1 - Method of preparation of the /u14c/fructose-1,6-diphosphate - Google Patents

Description

The present invention provides a process for the preparation of (U-C) fructosyl-1,6-diphosphate and unsaturated. by the enzymes of phosphase glucose / alpha-D-glucoso-1,6-diphosphate: alpha-D-glucoso-1-phosphate phosphotransferase, EC 2.7.5.1/ and glucose phosphate isomerase /? -glucose-6-tosate ketol isoraerase, EC 5.3.1.9/ and phosphofructokinase / ATP: D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11/ on alpha-D-glucopyranosyl phosphate / U- ⁴ Gt of high molar activity, prepared by the action of phosphorylase enzyme on water-soluble alpha-type polysaccharide / Ί - * 4 /6/.7 glucan / A.0. no. No. 194,587] isolated from radioactive biological material, preferably from green and blue-green algae, in the process of complex processing of radioactive biomass / Ss. pat. 121 808), wherein the enzyme reaction product is separated from the reaction mixture by chromatography, yielding up to 44 Z for fructose-1,6-diphosphate, up to 23 Z for fructose-6-phosphate and up to 23 µ for glucose-6-phosphate.

Among the biochemical methods for the preparation of glucoso-6-phosphate (U-C), a procedure using D-glucose (U-C) and the enzyme nexokinase (EC 2.7.1.1) is reported, the reaction being carried out in the presence of ATP. Although the yield in this reaction is high, the disadvantage is that D-glucose is treated and not the initial source of D-glucose, water-soluble alpha / Ί -? 4/6/7 glucan (U-C).

This disadvantage is solved by the process according to the invention, wherein the starting material is a directly water-soluble polysaccharide prepared as part of a complex treatment of radioactive bioraase / Cc. pat. no. 121,808 /.

SUMMARY OF THE INVENTION The invention is based on the fact that for a branched polysaccharide of the ¢ 6 (1 - * 4) glucan type with alpha (16) linkages, 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 phosphorylase / alpha-1,4-glucan orthophosphate glucosyltransferase enzyme, EC 2.4.1.1./ in the presence of a buffered phosphate solution of pH 6 to 7 (A.0). No. 194 587), wherein the obtained

1786 al phaDg lucopyr anose 1 phophate (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 to the obtained glucose-6-phosphate (U-1 * C) is impregnated with a buffered glucose-6-phosphate isomerase solution at pH 9.0 and for the produced fructose o-6-phosphate with a buffered phosphofructokinase solution (pH 8.0 to 8.5) in the presence of adenosine triphosphate, cysteine and magnesium salts and the resulting product is separated by chromatography on paper. This procedure yields fructose-1,6-diphosphate up to 44%.

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

the whole preparation process is very simple in technical conversion and allows the completion of the reaction process at the level of individual honey products of the glycolytic pathway up to fructozo-1,6-diphosphate

4 provides another possibility of utilizing water-soluble glucan (U-C), isolated from radioactive biological material, all enzymes required to carry out the indicated procedures are commercially available, optionally prepared under laboratory conditions.

Example 1

To 0.1 ml of a 17% water-soluble solution of green algae with a total activity of 17 kBq was added 1 ml of 0.2 M phosphate buffer pH 6.0 and 1 ml of phosphorylase. Potato / AO no. 194 588) and the reaction mixture was incubated at 35 ° C for 4 hours.

After the reaction was run, the pH of the reaction 2 months 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 to 0.1 mM conc. and 10 l of phosphocytase (from yeast, 0.34 U) and the reaction mixture was incubated for a further 2 hours at 35 ° C.

After completion of the reaction with phosphoglucomutase, the pH of the reaction mixture was adjusted to 9.0 with 0.5 M glycine sodium hydroxide buffer (pH 10) and yeast glucose-6-phosphate isomerase (10 µl, 0.23 U) was added. and incubating for an additional hour at 35 ° C. After reaction with glucose-6-phosphate isomerase, the reaction mixture is adjusted to pH 8.0 with Tris-HCl buffer, adenosine intriphosphate (Na salt, 0.02 M) and phosphofructokinase (hare muscle) (20 µl) are added, 4.2 U / a and the reaction was allowed to run at 35 ° C for 3 hours.

After chromatographic separation of phosphorylated metabolites, by paper chromatography on Whatman 1 paper in isobutyric-ammonia-water system (66: 1: 3) for 100 hours, the zone corresponding to fructoso-1,6-diphosphate was excised and eluted with 50% aqueous ethanol. The yield of fructozo-1,6-diphosphate (U-C) was 3.24 kBq (44%).

The phosphorylated metabolite D-fructose is an important biochemical which, when labeled as 14 / U-C / radiochemicals, can be used to study in vitro enzymatic processes as well as histochemical studies and to monitor the course of glycolysis in cells. They can be considered as important starting materials for chemical syntheses in the area of sugar chemistry.

Table I

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

Claims (1)

A process for the preparation of (U- ¹⁾ C (1) -cructose-1,6-diphosphate from radioactive biological material, preferably branched water-soluble alpha (Ί 4 * 6/6) glucan from green or blue-green 14 algae, characterized in that a solution of a-D-glukopyranozylfosfátu / U- C / fosfog1ukomutázy They act buffer solution at pH 7.5 to 8 cisteínu presence, gluc ozonolysis 1, 6-diphosphate and magnesium salts, and the resulting / C ^1zt U / g of L in the kó The 6-phosphate is treated with a buffered solution of glucose-6-phosphate isomerase at pH 9 and the fraction formed from the 6-phosphate-1β-buffered solution of phosphorructokinase at pH 8 to 8.5 in the presence of adenosine triphosphate, cistein and magnesium salts, and the resulting product is isolated by chromatography on paper.