JP2960804B2 - Glucose transferase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a glucose transferase, and more particularly, to a glucose transferase which is important for glycolipid biosynthesis.

[0002]

2. Description of the Related Art Enzymes present in cell membranes can react not only with water-soluble substances, but also with water-insoluble lipid components, as their substrates and products. It has become a place of. In addition, the membrane enzyme is the same lipid 2
When present in the overlay, the reaction product of one enzyme immediately becomes a substrate for another enzyme, increasing the efficiency of a continuous reaction by a series of enzyme systems.

[0003] Among glycolipids synthesized by a continuous reaction, there are many compounds which are difficult to synthesize by an organic synthesis reaction.
In the biosynthesis of a series of glycolipids, which are components of biological membranes and play an important role in the function of membrane proteins and also in the function of cells, one glycosyltransferase has only one type of glycosyltransfer reaction. does not catalytic, enzymatic reaction by the number of configuration monosaccharide to synthesize a sugar chain must be made. These glycosyltransferases use a nucleotide derivative of the monosaccharide to be transferred as a substrate. The sugar nucleotide is synthesized from nucleotide 3 phosphate and sugar phosphate by pyrophosphorylase, and the nucleotide 2 phosphate generated as a result of the glycosyltransfer reaction consumes ATP and is regenerated to nucleotide 3 phosphate.

[0004] Among such complex membrane enzymes, the presence of glucose transferase that synthesizes glucosylceramide is known, and this glucose transferase is involved in the biosynthesis of glycolipids having the above physiological functions. It is considered an important enzyme that controls the first step, the reaction.

[0005]

However, glucose transferase that synthesizes the above glucosylceramide is only known to exist in some organs to date. Therefore, the object of the present invention is to
An object of the present invention is to provide a glucose transferase useful in the field of pharmacy and the like, which is useful as the first step of glycosphingolipid synthesis, and a purified product thereof.

[0006]

Means for Solving the Problems The present inventors have found that the activity of the present enzyme is suppressed by the lipid composition of the membrane in which the present enzyme is present. In the experiment of lipid addition, it was found that phospholipid is essential for this enzyme, and conversely, the presence of lipid in the membrane that inhibits the activity was found.

[0007] The present invention has been made based on the above-mentioned findings, and provides a glucose transferase having a molecular weight of 30,000 to 100,000 and an optimum pH for transferring glucose from UDP-glucose to ceramide in the range of 6 to 7. By providing the above, the above object has been achieved.

[0008]

The glucose transferase according to the present invention is obtained by solubilizing the membrane fraction of cells once with a surfactant and then removing the surfactant. In the range, the glucosylceramide biosynthesis action, that is, the action as shown in FIG. 1 is sufficiently exhibited.

[0009]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The glucose transferase according to the present invention and a purified product containing the enzyme will be described in detail below. The glucose transferase according to the present invention has a molecular weight of 30,000 to 100,000,
The optimum pH for transferring glucose from UDP-glucose to ceramide is in the range of 6 to 7.

[0010] Specifically, the present transferase is obtained from a membrane fraction of animal cells such as rat brain, mouse brain and mouse epidermis. After solubilization with a surfactant, the surfactant is removed by gel filtration or the like. Thus, the enzyme activity can be recovered by reconstitution in the lipid bilayer membrane. In the rat brain, a 3-week-old rat brain is desirable, and the enzymatic activity decreases with the growth period. The present inventors have also revealed that the present enzyme is also present in the membrane fraction of Candida and Albicans.

[0011] The glucose transferase according to the present invention comprises UDP-
Glucose: a glucose transferase specific for ceramide, having the action shown in FIG. Further, as a result of examining the optimal pH in the enzyme reaction using these substrates, as shown in FIGS. 2 to 4, the enzyme from rat brain, mouse brain, and mouse epidermis had a buffer type of 2- ( N-morpholino)
When each of ethanesulfonic acid (MES), 3- (morpholino) propanesulfonic acid (MOPS) and tris-maleic acid was replaced, the optimum pH of the activity was around 6.5.

Further, the glucose transferase according to the present invention will be described. As shown in FIG. 5, the enzyme activity of the enzyme is changed by adding lipids and the like. FIG.
As shown in the figure, cardioline pin shows an activity promoting action,
Other lipids did not affect the activity or exhibited an inhibitory effect. In particular, sphingomyelin and sphingosine have remarkable activity-suppressing effects, suggesting that the lipids in these membranes may contribute to the control of the activity of this enzyme. It is considered that expression is inhibited.

The effect on enzyme activity of phosphatidylcholine of natural origin (soy and egg yolk) and that of synthetic dimyristoylphosphatidylcholine (DMPC) are different, presumably due to the different fatty acid chain compositions. FIG. 6 shows the activity of the present enzyme in the membrane fraction treated with phospholipase. Hosuhoripase D as shown here but had little effect on the enzymatic activity, phospholipase A ₂ and phospholipase C reduced the activity of the enzyme according to the lipase activity during membrane fractions treated. Significantly suppressed particularly by phospholipase A _2, the enzyme activity by 25units / ml, 30 min treatment was reduced to 6.7% of the control. On the other hand, when the membrane fraction was treated with sphingomyelinase (1.32 units / ml), the activity was significantly increased (180%) (data not shown). This is because sphingomyelin in the membrane is degraded by sphingomyelinase to ceramide,
It is considered that the glucose enzyme activity inhibitory action of this lipid disappeared.

From the above, it is considered that the glucose transferase according to the present invention preferably has a phospholipid, and particularly preferably has a cardiopyrine and a phosphatidyl phospholipid. The substrate specificity of the glucose transferase according to the present invention is as follows. Glucuronic acid (GlcUA), N-acetylglucosamine (GlcNAc), galactose (Gal) together with UDP- [ ¹⁴ C] glucose in the reaction solution.
-Substrate specificity for UDP-glucose by adding each sugar nucleotide of N-acetylgalactosamine (GulNAc) to a final concentration of 0.1 or 1 mM and observing the activity inhibition rate of glucose transferase, As a result, as shown in Table 1, UDP-galactose and UDP-glucuronic acid showed some inhibitory activities. However, the UDP-glucose concentration in the reaction solution was 50 μM, suggesting that the present enzyme has high substrate specificity for UDP-glucose.

[0015]

[Table 1] Effect of sugar nucleotide on glucose transferase activity ────────────────────────────────── [UDP -sugar] UDP-Gal UDP-GlcUA UDP-GalNAc UDP-GlcNAc 阻 害 inhibition rate ( %) 0.1mM 2.6 7.4 -2.7 -5.8 1.0mM 9.6 25.5 -10.6 -1.7 ────────────────────────────────本 In addition, this enzyme has a requirement for metals such as manganese and magnesium, and exhibits high enzyme activity particularly when both manganese and magnesium are present.

The enzyme requires manganese and magnesium as divalent cations.
When viewed added the influence 5mM barium calcium cadmium, magnesium, manganese, zinc in the respective reaction solution, as shown in Table 2, observed accelerating effect for each ion of manganese, magnesium, barium Ka le Siumu The order of the effect was manganese>magnesium>barium> calcium. An additive effect was observed by adding both manganese and magnesium cations to the reaction solution, and the same effect was observed when barium or calcium was added to manganese. Since the activity promoting effect was the same even when the concentration of manganese in the reaction solution was 10 mM, the concentration of manganese was considered to be necessary and sufficient. Therefore, the additive effect was insufficient for magnesium and the like. It does not compensate for the manganese concentration.

[0017]

[Table 2] Metal requirement for glucose transferase activity ─────────────────────────────── Metal ion Enzyme activity ( %) ─────────────────────────────── EDTA (reference) 10 mM 100 Cd ++ 5 mM 17.1 Zn ++ 5 mM 18.1 Ba ++ 5 mM 146 Ca ++ 5 mM 126 Mg ++ 5 mM 150 Mn ++ 5 mM 206 Mn ++ 5 mM plus Mg ++ 2.5 mM 231 ──────────────── also it causes deterioration of the enzymatic activity by phospholipase a ₂ process is not only due to fracture or flow of the change of the membrane according to phospholipid degradation, generation It may be due to fatty acids that have been used.

The molecular weight of the glucose transferase according to the present invention is in the range of 30,000 to 100,000, the solubilized enzyme solution through an ultrafiltration membrane, a result of measuring the activity of Gurukosu transferase of each filtrate, minutes in the case of using an ultrafiltration membrane fraction molecular weight of 100,000 filtrate has enzymatic activity, in the case of using an ultrafiltration membrane of fractionation molecular weight of 30,000 was shown that little enzyme activity in the filtrate. The apparent molecular weight may be 300,000 or more by gel filtration in the presence of a surfactant, because the solubilized enzyme contains lipids or associates with proteins.

Next, the purified product containing glucose transferase according to the present invention is prepared by solubilizing a cell membrane fraction having glucose transferase activity with a surfactant, and then using a buffer containing phospholipids. And the solubilized product is purified by chromatography. The cell membrane fraction having the activity of glucose transferase is obtained from the precipitate fraction obtained by crushing the above-mentioned rat brain, mouse brain and its epidermis with a homogenizer, adding a buffer solution, and centrifuging.

A. Solubilization As the surfactant used for solubilization, amphoteric surfactants, particularly 3-[(3-chloramidopropyl) dimethylammonio] -1-propanesulfonate (3-
[(3-cholamidopropyl) dimethylammonio] -1-p
ropane-sulfonate, hereinafter called CHAPS. ) Is desirable. In this case, in the buffer solution for solubilization, the surfactant CHAPS is 0.5 to 10%, particularly 1 to 5%.
% Is desirable.

The buffer solution has a protein concentration of 2 to 8 mg / m 2.
1, It is desirable from the viewpoint of solubilization that magnesium chloride is added in the range of 5 to 20 mM. It is desirable that the pH of the buffer during solubilization is in the range of 6 to 8, the solubilization time is 0.5 hours or more, and the temperature is in the range of 0 to 10 degrees.
The solubilization rate under such favorable conditions is about 60% or more.
Further, even if a surfactant is present due to such solubilization, the activity of the present transferase is restored again if the surfactant is removed in the presence of the phospholipid.

B. Separation and purification Separation and purification of the solubilized product are performed by chromatography. After the measurement of the enzyme activity fractionation is performed after removing the surfactant. Examples of the chromatography used for separation and purification include ion exchange column chromatography, hydrophobic column chromatography, gel chromatography, and affinity chromatography.

In the buffer used for chromatography, a phospholipid, preferably a phosphatidyl phospholipid,
More preferably, it is important to add phosphatidylcholine, and specifically, soybean lecithin and the like. Addition of phospholipids is thought to maintain enzyme activity during purification and to contribute to enzyme stability during purification. The concentration of the phospholipid in the buffer is preferably 1 mg / ml or more.

As a specific chromatography, i-ion exchange color chromatography is carried out using a buffer of pH 7.4 (soy lecithin 2 mg / ml).
) And adsorbed on DEAE-Toyopearl, and can be eluted from the column as the salt concentration in the eluent increases. By this operation, the specific activity can be increased about 2.4 times. ii Hydrophobic column chromatography This enzyme is adsorbed to butyl-Toyopearl in the presence of 3M sodium chloride, and can be eluted from the column as the salt concentration in the eluent decreases. By this operation, the specific activity can be increased about 5-fold. iii. Gel filtration The enzyme could be separated on Sepharose 4B or Sepharose 6B in the presence of 2% FCHAPS, 2 mg / ml soybean lecithin, and the activity recovery was 80% or more. The elution pattern changed depending on the CHAPS concentration in the eluent.

[0025]

EXPERIMENTAL EXAMPLES The purification of glucose transferase according to the present invention and its activity are specifically described below. (1) Materials The brain, which was an organ from which the enzyme was extracted, was extracted from a 3-week-old male Wister rat.

Ceramide, UDP-glucose, UD
P-glucuronic acid, UDP-N-acetylglucosamine, UDP-galactose, UDP-N-acetylgalactosamine and various phospholipids, glucosylceramide, cholesterol, and ceramide were obtained from Sigma by MOP.
S, CHAPS and 2,3-dimercaptopropanol were purchased from Wako Pure Chemical Industries, Ltd., and UDP- [U- ^14C ] glucose (11.1 GBq / mmol) was purchased from Amersham. Silica gel (HPLC packing material) and DEA
E-Toyopearl and Petil-Toyopearl were purchased from Tosoh Corporation. DEAE-cellulose was purchased from Whatman, Super Rose 12, Sepharose 4B, Sepharose 6B, and EAH-Sepharose from Pharmacia.

Phospholipase A ₂ (derived from Naja Naja Venom), phospholipase C (derived from Clostridium perfringens), phospholipase D (derived from Cabbage), and sphingomyelinase (derived from Staphylococcus aureus) were purchased from Sigma. (2) Method a. Organ crushing and cell fractionation The following operations were all performed at 0 to 4 ° C. The brain was excised, minced with scissors, and three times the weight of buffer A [0.32M sucrose,
0.25 mM dithiothreitol, 1 mM ethylenediaminetetraacetic acid (pH 7.4)] and added to a potter-type glass /
The cells were crushed with a Teflon homogenizer and homogenized. This was centrifuged at 800 g for 10 minutes to remove the supernatant, and the same volume of buffer A as the supernatant was added to the precipitate, followed by crushing again. Further, the mixture was centrifuged at 800 g for 10 minutes to separate a supernatant and a precipitate.
The supernatant was used. This was centrifuged at 10,000 g for 30 minutes to obtain a 10,000 g precipitate fraction, which was frozen in liquid nitrogen and stored at -80 ° C. b. Solubilization of glucose transferase 10,000 g sedimented fraction of rat brain was added to buffer B [0.25M
Sucrose, 10 mM magnesium chloride, 1 mM dithiothreitol, 0.02% sodium azide, 0.5 mM
Phenylmethylsulfonyl fluoride, 50 mM
Tris-hydrochloric acid (PH7.4)] and the surfactant CHA
PS was added and stirred at 4 ° C. This is 100,00
After centrifugation at 0 g for 60 minutes, a supernatant and a precipitated fraction were obtained. c. Buffer separated ion exchange column chromatography DEAE- Toyopearl bar column of solubilizing glucosyltransferase C [2% CHA
PS, 2 mg / ml soybean lecithin, and the above buffer solution B containing protease inhibitors]. Buffer B and soy lecithin were added to the supernatant fraction obtained in b.
1%, soy lecithin 2 mg / ml, which was added to the column. The column was washed with buffer C and eluted with a linear gradient of 0 to 0.5 M sodium chloride, and the active fraction was collected. d. Method for measuring glucose transferase activity In the measurement of glucose transferase activity, 20 μg of ceramide was dissolved in a small amount of CHCl ₃ and 1 mg of silica gel was added. The reaction solution contained 20 μg of ceramide and UDP- [U- ¹⁴ C] glucose 5.
4 nmol (13.9 KBq), 5.5 μmol of 3- (morpholino) propanesulfonic acid (PH6.5), manganese chloride 5
50 nmol, 275 nmol of magnesium chloride, 110 nmol of reduced β-nicotinamide adenine dinucleotide,
550 nmol of 2,3-dimercaptopropanol,
CHAPS 660 µg, protein 125 µg-550 µg
Was added to make a final volume of 110 μl. The reaction was started by adding UDP- [U- ¹⁴ C] glucose and incubated at 37 ° C for 30 minutes.
The reaction was stopped by adding 100 μl of cold phosphate buffered saline (hereinafter referred to as PBS). This was centrifuged at 2000 rpm for 5 minutes, and the precipitated gel was
Washed three times with 200 μl of S. Washed gel is PBS
Suspended in 200 μl, Aquasol-2 (Packard)
Was added, and the radioactivity was measured with a liquid scintillation counter.

The radioactivity of the sample after the reaction was measured in part by using a Packard Matrix 96 direct-reading beta counter. That is, after the enzymatic reaction, the silica gel was collected on a glass filter using a 96-well cell harvester (manufactured by Packard), and the radioactivity on the filter was measured using a matrix 96.

The enzyme preparation obtained by the above ion exchange column chromatography was able to produce glucosylceramide at a rate of 5 to 10 nmol per hour per 1 mg of protein under optimal conditions.

[0030]

Industrial Applicability The glucose transferase according to the present invention is used in the fields of medicine, pharmacy and the like, and can be used as a useful substance for controlling the first step of glycosphingolipid synthesis.

[Brief description of the drawings]

FIG. 1 is a diagram showing an action mechanism of a glucose transferase according to the present invention.

FIG. 2 shows the enzyme activity of the glucose transferase according to the present invention.
It is a relation graph with PH condition.

FIG. 3 shows the enzyme activity of the glucose transferase according to the present invention.
It is a relation graph with PH condition.

FIG. 4 shows the enzyme activity of the glucose transferase according to the present invention.
It is a relation graph with PH condition.

FIG. 5 is a graph showing the relationship between lipid and enzyme activity of the glucose transferase according to the present invention.

FIG. 6 is a relationship graph showing enzyme activity when a membrane fraction was treated with phospholipase.

──────────────────────────────────────────────────続 き Continued on the front page (58) Fields surveyed (Int. Cl. ⁶ , DB name) C12N 9/10 BIOSIS (DIALOG) WPI (DIALOG)

Claims (5)

(57) [Claims] 1. A glucose transferase having a molecular weight of 30,000 to 100,000 and an optimum pH for transferring glucose from UDP-glucose to ceramide in the range of 6 to 7. 2. After solubilizing a cell membrane fraction having the activity of glucose transferase according to claim 1 with a surfactant, the solubilized product is purified by chromatography in the presence of a phospholipid. A purified product containing glucose transferase. 3. The purified product containing glucose transferase according to claim 2, wherein the phospholipid is a phosphatidyl-based phospholipid. 4. The purified product containing glucose transferase according to claim 2, wherein the surfactant is a zwitterionic surfactant. 5. The purified product containing glucose transferase according to claim 2, wherein the surfactant is 3-{((3-chloramidopropyl) dimethylammonio)} 1-propanesulfonate.