JP2001057882A - Sulfate group transferase preparation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new enzyme preparation capable of specifically transferring a sulfate group from a sulfate group donor to an OH group at the 6-position of N-acetylgalactosamine 4-sulfate residue of glycosaminoglycan, suitable for production of a sulfated polysaccharide useful for a medicine, etc., function analysis, etc. SOLUTION: This new sulfate group transferase preparation specifically transfers a sulfate group from a sulfate group donor to an OH group at the 6-position of N-acetylgalactosamine 4-sulfate residue of glycosaminoglycan, transfers the sulfate group to chondroitinsulfuric acid A derived from a whale cartilage, chondroitinsulfuric acid C derived from a shark cartilage and dermatan sulfate derived from-swine skin but not to chondroitinsulfuric acid E derived from a squid cartilage, keratan sulfate derived from bovine cornea, heparan sulfate derived from bovine kidney and CDSNS-heparin, increases activity in the presence of Mn2+, Mg2+, Ca2+, NaCl, KCl, protamine, etc., inhibits the sulfate group from transferring to chondroitinsulfuric acid A by chondroitinsulfuric acid E. The preparation has uniformity and is obtained by extraction from an organism body such as squid cartilage, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a sulfotransferase (sulfotransferase) preparation, a method for producing the same, and use of the sulfotransferase preparation.

[0002]

2. Description of the Related Art Chondroitin sulfate (CS-E) is one of the isomers of chondroitin sulfate. In addition to monosulfated disaccharide repeating units, chondroitin sulfate repeating units (GlcAβ1-3GalNAc (4 , 6-bisSO ₄₎ {GlcA is glucuronic acid residue, GalNAc (4,6-bisSO ₄₎ 4-position and the hydroxyl group at the 6-position are both sulfated N- acetylgalactosamine residues, β1-3 ｝) indicating β1-3 bond. CS-E
Was first found in squid cartilage and subsequently found to be distributed in various cells such as mast cells, polymorphonuclear granulocytes, monocytes and macrophages. CS-E releases histamine from mast cells, binds platelet factor 4 to neutrophils,
It is thought to be involved in the regulation of procoagulant activity and in the binding of lipoprotein lipase to macrophages. Rat glomeruli and rat mesangial cells are CS-
E and IdoAα1 found in the eel chord
It has been reported that -3GalNAc (4,6-bisSO ₄ ) unit {IdoA synthesizes glycosaminoglycans containing iduronic acid residues}. GalNAc (4,6-bisSO ₄₎ is also found in non-reducing end of chondroitin sulfate, the ratio of aggrecan nonreducing terminal GalNAc (4,6-bisSO ₄₎ is to be reduced in human osteoarthritis Have been found. It has been reported that the anticoagulant activity of thrombomodulin depends on the presence of chondroitin sulfate containing GalNAc (4,6-bisSO ₄ ) at the non-reducing end. These findings suggest that CS-E or GalNAc (4,6-bisSO ₄ ) at the non-reducing end of chondroitin sulfate plays an important role in various cell interactions.

[0003] As for sulfotransferase capable of producing GalNAc (4,6-bisSO ₄ ), squid cartilage (J. Biol.
m., 246, 7357-7365 (1971)), quail oviduct (J. Biol. Che.
m., 256, 5443-5449 (1981)) and human serum (J. Biol. Che.
m., 261, 4460-4469 (1986); J. Biol. Chem., 261, 447.
0-4475 (1986)) as an enzyme source. Sulfotransferase obtained from quail oviduct and human serum are mainly the 6-position sulfation of non-reducing terminal N- acetyl-4-sulfonyl galactosamine residue _{(GalNAc (4-SO 4)} ) and a catalyst, On the other hand, sulfotransferase partially purified from squid cartilage catalyzes the sulfation of internal GalNAc (4-SO ₄ ) at position 6. However, the exact substrate specificity of these sulfotransferases is unclear because no homogeneous preparation of these sulfotransferases has been obtained.

[0004]

Considering the importance of sulfation in the expression of the physiological activity of chondroitin sulfate, an enzyme having a sulfation property which has not been known so far and which transfers a sulfate group to chondroitin sulfate is known as chondroitin sulfate. It is very important not only for studying the functional analysis of, but also for providing chondroitin sulfate for the purpose of creating a drug having favorable physiological activity for humans. Then, in order to use the enzyme for such a purpose, the enzyme needs to be purified to such an extent that the sulfation characteristics such as substrate specificity and sulfation site are sufficiently established. It is also necessary to increase the specific activity as much as possible.

[0005] Accordingly, the present invention provides a sulfotransferase preparation exhibiting a sulfation characteristic which has not been known before, exhibiting its sulfation characteristic and having extremely high specific activity with respect to chondroitin sulfate and the like. The purpose is to do.

[0006]

The present inventors have studied the purification of squid cartilage-derived sulfotransferase, which has only been partially purified to date and whose substrate characteristics have not been clarified. By using the precipitate, the enzyme was established to have a sulfated property and was successfully purified to a sufficiently high specific activity, thereby completing the present invention.

Therefore, the present invention provides the following physicochemical properties:
With SDS-polyacrylamide gel electrophoresis
Sulfotransferase preparation showing a substantially uniform band
(Hereinafter, also referred to as “the preparation of the present invention”). (1) Action: From sulfate group donor, glycosaminoglycan
N-acetylgalactosamine 4-sulfate residue (hereinafter referred to as Ga
lNAc (4-SO_Four)), The 6-position hydroxyl group
Transfers sulfate groups differently. (2) Substrate specificity: chondroitin sulfate from whale cartilage
Acid A, chondroitin sulfate C from shark cartilage, pig skin
The sulfate group is transferred to dermatan sulfate. Squid cartilage
Conventional chondroitin sulfate E, keratan sulfate from bovine cornea
Acid, heparan sulfate from bovine kidney, CDSNS-Hepari
The sulfate group is not substantially transferred to the compound. (3) Activation: 20 mM Mn²⁺, Mg²⁺, Ca²⁺, S
r²⁺, Ba²⁺Or Co ²⁺Increases in the presence of.
0.1 M NaCl, 0.1 M KCl or 0.15
Activity increases in the presence of mg / ml protamine. (4) Inhibition: Chondroitin sulfate E causes chondroitin
Transfer of a sulfate group to itin sulfate A is inhibited. Dermata
Transfer of sulfate groups to chondroitin sulfate A
Transfer is inhibited. By Chondroitin Sulfate A, Dell
Transfer of sulfate groups to matane sulfate is inhibited.

The preparations according to the invention preferably further have the following physicochemical properties: (5) Molecular weight: about 63 kD, about 54 kD (SDS-polyacrylamide gel electrophoresis; under non-reducing conditions) (6) Optimum pH: around 6.2

The preparation of the present invention preferably further has the following physicochemical properties. (7) Optimal Ca ²⁺ concentration: about 20 mM (when chondroitin sulfate A is the substrate). About 100 mM (when dermatan sulfate is the substrate). (8) Km value: about 5 × 10 ⁻⁷ M (3′-phosphoadenosine 5′-phosphosulfate) about 1.3 × 10 ⁻⁷ M (dermatan sulfate from pig skin) about 1.1 × 10 ⁻⁶ M (chondroitin sulfate A derived from whale cartilage)

[0010] The preparation of the present invention is preferably derived from squid cartilage.

The present invention also provides a method for producing the preparation of the present invention. This manufacturing method includes at least the following steps. (Step 1) A step of obtaining an extract from an organism (preferably squid cartilage) containing a sulfotransferase contained in the preparation of the present invention. (Step 2) A step of bringing the extract into contact with protamine to form a precipitate. (Step 3) A step of removing the precipitate generated in Step 2.

[0012] The production method may further include an affinity chromatography step using heparin as a ligand and an affinity chromatography step using adenosine 3 ', 5'-diphosphate as a ligand, which is performed after the step 3. preferable.

Further, the present invention provides a method for producing chondroitin sulfate E using the preparation of the present invention. This method comprises reacting the preparation of the present invention with chondroitin sulfate A,
Isolating the resulting chondroitin sulfate E.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION First, the physicochemical properties of the preparation of the present invention will be described.

(1) Action: A sulfate group is specifically transferred from a sulfate group donor to a hydroxyl group at the 6-position of N-acetylgalactosamine 4-sulfate residue of glycosaminoglycan. Here, “specific” refers to a hydroxyl group and an amino group of a residue constituting glycosaminoglycan,
It means that groups other than the hydroxyl group at the 6-position of N-acetylgalactosamine 4-sulfate residue are not substantially sulfated. That is, the sulfate group is not substantially transferred to the 4-sulfated N-acetylgalactosamine except for the hydroxyl group at the 6-position, and the sulfate group is not substantially transferred to the hexuronic acid residue.

The sulfate group donor is preferably 3'-phosphoadenosine 5'-phosphosulfate (hereinafter also referred to as "PAPS").

Preferably, the activity of transferring the non-reducing end of the glycosaminoglycan to N-acetylgalactosamine 4-sulfate residue and the activity of internal N-acetylgalactosamine 4-sulfate are reduced.
The transfer activity to sulfate residues is described in J. Biol. Chem., 261, 447.
As determined by the measurement method described in 0-4475 (1986), it is preferable that the transfer activity to an internal residue is larger.

(2) Substrate specificity: Sulfate groups are transferred to chondroitin sulfate A derived from whale cartilage, chondroitin sulfate C derived from shark cartilage, and dermatan sulfate derived from pig skin. Chondroitin sulfate E from squid cartilage, keratan sulfate from bovine cornea, heparan sulfate from bovine kidney, CD
Sulfate groups are not substantially transferred to SNS-heparin. C
DSNS-heparin (completely desulfated, N-resul
Fated heparin means heparin in which glucosamine residues are N-sulfated after complete desulfation.

As is apparent from the above-mentioned action, even in the case of chondroitin sulfate C, the hydroxyl group at the 4-position of some N-acetylgalactosamine residues is sulfated,
If the hydroxyl group at the 6-position of the residue is not sulfated, the sulfate group is transferred by the preparation of the present invention.
Further, although a sulfate group is not substantially transferred to chondroitin sulfate E derived from squid cartilage, even in the case of chondroitin sulfate E, the hydroxyl group at the 4-position of some N-acetylgalactosamine residues is sulfated. If the hydroxyl group at the 6-position of the residue is not sulfated, the sulfate group is transferred by the preparation of the present invention.

(3) Activation: 20 mM Mn²⁺, M
g²⁺, Ca²⁺, Sr²⁺, Ba²⁺Or Co ²⁺In the presence of
Increased activity (usually 2 to 4 times compared to absence)
I do. 0.1 M NaCl, 0.1 M KCl or
Activity in the presence of 0.15 mg / ml protamine (
It always increases (2 to 3 times compared to the absence).

(4) Inhibition: Transfer of a sulfate group to chondroitin sulfate A is inhibited by chondroitin sulfate E. The transfer of a sulfate group to chondroitin sulfate A is inhibited by dermatan sulfate. Chondroitin sulfate A inhibits the transfer of sulfate groups to dermatan sulfate.

Here, "inhibited" usually means
When the same amount of glycosaminoglycan as the substrate and glycosaminoglycan as the inhibitor are present, it means that the transfer of sulfate groups to the substrate is 35% or less as compared with the absence of the inhibitor. I do.

The preparations according to the invention preferably further have the following physicochemical properties: (5) Molecular weight: about 63 kD, about 54 kD (SDS-polyacrylamide gel electrophoresis; under non-reducing conditions) (6) Optimum pH: around 6.2

The preparation of the present invention preferably further has the following physicochemical properties. (7) Optimal Ca ²⁺ concentration: about 20 mM (when chondroitin sulfate A is the substrate). About 100 mM (when dermatan sulfate is the substrate). (8) Km value: about 5x10 ^-7 M (3'-phosphoadenosine 5'-phosphosulfate) about 1.3 x 10 ^-7 M (dermatan sulfate derived from porcine skin) from about 1.1 × 10 ^-6 M (from whale cartilage Chondroitin sulfate A)

The above physicochemical properties can be measured by the methods described in the examples below.

The preparation of the present invention shows a substantially uniform band on SDS-polyacrylamide gel electrophoresis (PAGE). Here, "substantially uniform" means that a band is confirmed to be sufficiently purified to establish a sulfated property, and indicates a single band. It does not mean. Typically, the conditions for SDS-PAGE include the conditions described in Examples below.

The preparation of the present invention is preferably derived from squid cartilage.

The preparation of the present invention preferably has a specific activity of 100,000 U / mg or more (more preferably, 150,000 U / mg or more). The specific activity can be calculated by measuring the activity of the preparation and the amount of the protein (using the standard reaction mixture used for evaluating the characteristics of the preparation of the present invention) in accordance with the method described in the Examples below.

The preparation of the present invention can be produced by the method for producing the preparation of the present invention described below.

The method for producing the preparation of the present invention provided by the present invention comprises at least the following steps. (Step 1) A step of obtaining an extract from an organism containing a sulfotransferase contained in the preparation of the present invention. (Step 2) A step of bringing the extract into contact with protamine to form a precipitate. (Step 3) A step of removing the precipitate generated in Step 2.

In step 1, the organism containing a sulfotransferase contained in the preparation of the present invention is not particularly limited as long as it contains the above sulfotransferase. Separated living tissue, cells separated from living tissue, cells modified by genetic engineering, and the like can be used. Such an organism can be selected by screening the organism using the properties of the preparation of the present invention revealed by the present invention as an index. Preferably, the organism is squid cartilage. As a method for obtaining the extract from the organism, a known extraction method can be used, and usually, a method in which the organism is crushed in a buffer and the supernatant is obtained by centrifugation is used. The crushing method is appropriately selected depending on the properties of the organism.

In the step 2, usually, the extract is brought into contact with the protamine by adding protamine to the extract. This produces a precipitate. The amount of protamine is selected based on the amount of proteoglycan contained in the extract, which varies depending on the type of organism and the like. Selection of this amount can be performed by a method as described in Example 1 below.

The removal of the precipitate in step 3 can be carried out by a commonly used solid-liquid separation means such as centrifugation.

The above-mentioned production method comprises an affinity chromatography step using heparin as a ligand and adenosine 3 ', 5'-diphosphate (3', 5 '
It is preferable that the method further includes an affinity chromatography step using -ADP) as a ligand.

Affinity chromatography is a method in which a ligand is bound to a carrier, a protein having an affinity for the ligand is specifically adsorbed to the ligand, impurities are removed by washing, followed by elimination and purification. is there.

Binding of heparin and 3 ', 5'-ADP to a carrier can be carried out by a known method, and those bound to a carrier can be obtained as a commercial product (for example, Heparin Sepharose CL). -6B (Amersham Pharmaci
a Biotechnology), 3 ', 5'-ADP-agarose (Sigma-Ald
rich Japan)).

The conditions for adsorption, washing and desorption can be selected by methods known to those skilled in the art. For example, in the case of an enzyme derived from squid cartilage, the conditions described in Example 1 below can be used.

The order and number of the affinity chromatography steps using heparin as a ligand and the affinity chromatography steps using 3 ′, 5′-ADP as a ligand are not particularly limited, and are selected so as to obtain a desired purification degree. You.

The above production method may further include a known purification step such as gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography and the like.

In addition, it should be noted that the conditions in the above-mentioned various known methods mentioned above are based on the fact that the properties of the preparation of the present invention have been clarified by the present invention.
Those skilled in the art can easily select.

The present invention further provides a method for producing chondroitin sulfate E using the preparation of the present invention. This method comprises reacting the preparation of the present invention with chondroitin sulfate A,
Isolating the resulting chondroitin sulfate E.

The conditions for causing the preparation of the present invention to act on chondroitin sulfate A are not particularly limited as long as the above-mentioned effects are sufficiently exerted, but the activity of the preparation of the present invention as described above increases. Conditions are preferred.

The chondroitin sulfate E can be isolated according to a known method.

It is known that chondroitin sulfate E has physiological activities such as immunological activity and anti-infective property.

Further, the preparation of the present invention may be able to create a novel bioactive sugar chain as a drug material.

[0046]

The present invention will be described below in more detail with reference to examples. However, these should not limit the technical scope of the present invention.

Hereinafter, an enzyme having an activity of transferring a sulfate group to the hydroxyl group at position 6 of GalNAc (4-SO ₄ ) is referred to as “GalNAc (4-SO ₄ )”.
4S-6ST ". First, the measurement method used in the examples will be described.

(1) Measurement of sulfotransferase activity Sulfotransferase activity (GalNAc4S-6ST activity)
Has been reported (J. Biol. Chem., 246, 7357-7365 (1971);
J. Biol. Chem., 268, 21968-21974 (1993)). That is, the standard reaction mixture is 2.
5 μmol Tris-HCl, pH 8.0, 1 μmol CaCl ₂ , 1 μmol reduced glutathione, 25 nmol (as glucuronic acid) chondroitin sulfate A (derived from whale cartilage, 4-SO ₄ : 6-SO ₄ = 74.2: 2
2.9, Seikagaku Corporation), 50 pmol [ ³⁵ S] PAPS (about 5.0 ×
10 ⁵ cpm) and enzyme in a final volume of 50 μl.
The reaction mixture was incubated at 25 ° C. for 20 minutes, and the reaction was stopped by immersing the test tube in boiling water for 1 minute. After terminating the reaction, ³⁵ S-labeled glycosaminoglycan was subjected to ethanol precipitation and a Fast Desalting Column (Amersham) according to the method described previously (J. Biol. Chem., 268, 21968-21974 (1993)). Pharmaci
a Biotechnology) and the radioactivity was measured.

In the characterization of the preparation of the present invention, it was found that the above reaction mixture developed for the crude enzyme was not optimal, so that 2.5 μmol imidazole-HCl, pH 6.8 was added to 2.5 μmol Tris-HCl What was added to the reaction mixture instead of pH 8.0 was used as the standard reaction mixture.

One unit (U) of the enzyme is 1 pmol per minute.
Is defined as the amount required to catalyze the transfer of sulfate groups.

(2) Measurement of uronic acid The uronic acid was measured by the method described in Anal. Biochem., 4, 330-334 (1962).

(3) Measurement of protein Protein was measured by the Bradford method using bovine serum albumin as a standard (Anal. Biochem., 7).
2, 248-254 (1976)). Bio-based protein assay reagents
Rad's was used.

[0053]

Example 1 Purification of GalNAc4S-6ST (Preparation of Preparation of the Present Invention) All operations were performed at 4 ° C.

(1) Preparation of Crude Extract The head of a squid (Ommastrephes sloani pacificus) was dissected.
Remove cartilage and remove soft tissue by wiping with cotton cloth
And cut into slices with a razor. 3x 480 g slices
Amount of protease inhibitor (5 μM Nα-p-tosyl-
L-lysine chloromethyl ketone, 3 μM N-tosyl-L-phenyl
Lulanine chloromethyl ketone, 30 μM phenylmethyl
Containing sulfonyl fluoride and 3 μM pepstatin A)
Ice-cold extraction buffer (0.5 M NaCl, 10 mM Tris-HCl,
pH 7.2, 20 mM MgCl_Two, 2 mM CaCl _Two, 10 mM 2-mercap
Triethanol, 0.5% Triton X-100, and 20% glycerol
And a Polytron homogenizer (Kinematica)
5 times for 30 seconds at speed 7 setting
And homogenized. Homogenate for 24 hours
After gently stirring, centrifuge at 100,000 xg for 60 minutes.
Was. The clear supernatant fraction was used as a crude extract.

(2) Protamine precipitation The crude extract contained a large amount of proteoglycan. Thus, an attempt was made to remove proteoglycans with protamine sulfate. Various amounts of protamine sulfate were added to the crude extract obtained in the above (1). Generate the precipitate at 10,000 xg, 10
The uronic acid content (black circles) and sulfotransferase activity (open circles) of the supernatant fraction were measured (FIG. 1). As a result, it was found that proteoglycan can be efficiently removed from the crude extract as an insoluble complex with protamine sulfate. Protamine sulphate was added with stirring to the crude extract at a final concentration of 0.66 mg per μmol uronic acid. The generated precipitate was removed by centrifugation at 100,000 × g for 30 minutes to obtain a supernatant (clear solution).

(3) Dimatrex Gel Red (Dye
matrex Gel Red) A chromatography The clarified solution obtained in the above (2) was mixed with a buffer A containing 0.5 M NaCl (10 mM Tris-HCl, pH 7.2, 20 mM MgCl ₂ , 2 mM
CaCl ₂ , 10 mM 2-mercaptoethanol, 0.1% Triton X-
Dimatrex gel (Millipore Corp.) Red A column (2.2 × 2) equilibrated with 100 and 20% glycerol)
4.4 cm). The column was eluted stepwise with 920 ml of buffer A containing 0.5 M NaCl, 920 ml of buffer A containing 1 M NaCl and buffer A containing 2 M NaCl.
Most of the sulfotransferase activity is in 1 M NaCl
Eluted in fractions. The eluate was collected in each 15 ml fraction. The elution profile is shown in FIG. Fractions containing GalNAc4S-6ST activity, indicated by horizontal bars in FIG.
dialyzed against Buffer A containing l.

(4) First Heparin Sepharose CL-6
B Chromatography The dialysate obtained in the above (3) was prepared by equilibrating a buffer solution A containing 0.05 M NaCl with heparin sepharose CL-6B (Amers
ham Pharmacia Biotechnology) column (2.2 x 26.5 cm)
Applied. The column was washed with 1000 ml of buffer A containing 0.05 M NaCl. The adsorbed substance is 0.05 M
Elution was with a linear gradient (1 l) of 1 M NaCl. The eluate was collected in 12 ml fractions each. The elution profile is shown in FIG. The fractions containing the sulfotransferase activity indicated by the horizontal bar in FIG. 3 were collected and dialyzed against buffer A containing 0.05 M NaCl.

(5) First 3 ′, 5′-ADP-agarose chromatography The dialysate obtained in the above (4) was converted into a buffer B containing 0.05 M NaCl (10 mM Tris-HCl, pH 7.2, 3 ', 5'-ADP-agarose (Sigma-Aldrich Japa) equilibrated with 10 mM 2-mercaptoethanol, 0.1% Triton X-100, 5% glycerol
n) Apply to column (1.2 × 8.5 cm). Set column to 0.
Washed with buffer B containing 05 M NaCl. Sulfotransferase activity was eluted with a linear gradient (150 ml) from 0.05 M to 5 M NaCl in buffer B. After elution with a gradient, elution was subsequently carried out with 150 ml of buffer B containing 5 M NaCl. The elution profile is shown in FIG. Eluate each
Collected in 6 ml fractions. Fractions containing sulfotransferase activity (indicated by horizontal bars in FIG. 4) were collected, and 0.05 MN
It was dialyzed against buffer B containing aCl.

(6) Second 3 ′, 5′-ADP-agarose chromatography The dialysate obtained in the above (5) was equilibrated with buffer B containing 0.05 M NaCl to obtain 3 ′, 5′-agarose. ADP-agarose column (1.2
× 8.5 cm). Elution conditions are the first 3 ', 5'-
Same as ADP-agarose chromatography. Fractions containing sulfotransferase activity were collected and 0.05 M
Dialysis was performed against buffer A containing NaCl.

(7) Second heparin sepharose CL-6
B Chromatography The dialysate obtained in (6) above was applied to a heparin sepharose CL-6B column (0.9 × 1.4 cm) equilibrated with buffer A containing 0.05 M NaCl. Column is 0.05 M Na
Washed with 10 ml of buffer A containing Cl. The adsorbed sulfotransferase was added to 10 ml of buffer A containing 1.0 M NaCl.
Eluted. Fractions containing sulfotransferase activity were collected and dialyzed against buffer A containing 0.05 M NaCl. This step was performed for concentration of the enzyme solution. The purified enzyme was stored at -20 ° C.

The results of the purification of sulfotransferase from squid cartilage are summarized in Table 1. In addition, since the apparent enzyme activity was significantly increased by the protamine treatment, the purification degree of each step was expressed as a relative value of each step with respect to the value of the protamine fraction. In addition, since the protein concentration of the second heparin sepharose CL-6B fraction was too low for direct measurement, it was reported previously (J. Biol. Chem., 268,
21968-21974 (1993)).

[0062]

[Table 1] Table 1 Purification of GalNAc4S-6ST ──────────────────────────────────── Total step volume Activity Total protein Specific activity Purity (ml) (10 ^-3 × U) (mg) (10 ^-5 × U / mg) (fold) ────────────────── ────────────────── Crude extract 2580 1.1 10090--Protamine precipitation 2560 28.2 7830 0.0000036 1.0 TIMEMATREX KALELET A 1545 15.8 560 0.000028 7.9 First heparin sepharose CL- 6B 212 9.2 55.1 0.0017 46 1st 3 ', 5'-ADP-acylose 180 1.4 0.32 0.045 1240 2nd 3', 5'-ADP-acylose 125 0.40 ND ND ND 2nd heparin sepharose CL-6B 6.3 0.25 0.0015 1.6 45 800 ──────────────────────────────────── No ND measurement

Since the slices of squid cartilage were too hard to homogenize with a glass homogenizer, homogenization with a polytron homogenizer was effective for efficient extraction of sulfotransferase. The addition of 0.5 M sodium chloride to the extraction buffer increased the activity yield by a factor of 1.35. After centrifugation of the homogenate at 100,000 × g, a clear supernatant solution (crude extract) was obtained, but the crude extract contained a large amount of proteoglycan.
Proteoglycans could be removed by centrifugation as insoluble protamine-proteoglycan complexes. After protamine precipitation, most of the uronic acid-containing substances present in the crude extract were removed, and the sulfotransferase activity was increased (Table 1, FIG. 1). Purified GalNAc4S-6
ST was prepared from squid cartilage-derived CS as shown in Example 2 below.
Since the activity was strongly inhibited by -E, the increase in activity after protamine precipitation was considered to be mainly due to the removal of proteoglycans, and not due to the stimulating effect of protamine.

Since GalNAc4S-6ST was not eluted with 0.5 M NaCl but was eluted with 1 M NaCl in Dimatrex Gel Red A agarose chromatography (FIG. 2), the supernatant fraction after protamine precipitation was subjected to dialysis. Without this, it could be applied to Dimatrex Gel Red A agarose. As seen with many of the glycosaminoglycan sulfotransferases, GalNAc4S-6ST was also adsorbed to heparin Sepharose CL-6B and from this column about 0.75
It was eluted with M NaCl (FIG. 3). Heparin Sepharose CL
The concentration of NaCl required for elution from -6B was higher than the concentration at which chondroitin 6-sulfotransferase (C6ST) and chondroitin 4-sulfotransferase (C4ST) were eluted from the same column. FIG. 4 shows the elution pattern of sulfotransferase from 3 ′, 5′-ADP-agarose. The concentration required for elution was about 5M. This concentration is determined by using heparan sulfate N-deacetylase / N-sulfotransferase (J. Biol. Chem., 270, 4172-4179 (199).
5)), glucosaminyl 3-O-sulfotransferase (J.
Biol. Chem., 271, 7645-7653 (1996)) and the concentration required for elution of chondroitin 4-sulfotransferase. Good purification of GalNAc4S-6ST was obtained by re-chromatography on 3 ', 5'-ADP-agarose. The second 3 ', 5'-ADP-agarose fraction was concentrated using a small heparin sepharose CL-6B column to give a purified GalNAc4S-6ST fraction (preparation of the present invention).

The fraction from each purification step was analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). That is, polyacrylamide gel electrophoresis in SDS was performed using a 10% polyacrylamide gel under non-reducing conditions according to a previous report (Nature, 227, 680-685 (1970)). Protein bands were detected by silver staining. 2-mercaptoethanol was removed from the SDS-PAGE sample buffer to avoid spurious results in silver staining. FIG. 5 shows the results. 63 kDa and 54 k in the second heparin sepharose fraction
Two overlapping broad protein bands of Da were predominantly stained (FIG. 5, lane 6).

As described above, GalNAc4S-6ST was purified to a specific activity of about 45,000-fold relative to the supernatant fraction after protamine precipitation, and to apparently uniform purification.

[0067]

Example 2 Characterization of Purified GalNAc4S-6ST (Preparation of the Present Invention) (1) Optimum pH 0.05 M imidazole-HCl contained in the standard reaction mixture was
The activity was measured according to the method for measuring sulfotransferase activity described above, except that the buffer was replaced with 0.05 M buffer at various pHs. The results are shown in FIG. The optimum pH was about 6.2.

(2) Influence of Various Compounds The activity was measured according to the above-described method for measuring sulfotransferase activity except that various compounds were added to the standard reaction mixture.

The reduced form of glutathione was GalNAc4S-6S at 20 mM.
Although T activity was stimulated 1.3-fold, other sulfhydryl compounds such as dithiothreitol and 2-mercaptoethanol had minimal effects on GalNAc4S-6ST at the same concentration.

The activity was increased 3- to 4-fold by various divalent ions (20 mM) such as Mn ²⁺ , Mg ²⁺ , Ca ²⁺ , Sr ²⁺ , Ba ²⁺ and Co ²⁺ . An approximately 2-fold increase in activity was observed in the presence of 0.1 M NaCl, 0.1 M KCl, or 0.15 mg / ml protamine.

(3) Measurement of Km Sulfotransferase activity was measured using [
³⁵ S] PAPS, dermatan sulfate from pig skin or chondroitin sulfate A (4-SO
₄ : 6-SO ₄ = 74.2: 22.9), except that the concentration was changed, and the activity was measured according to the method for measuring sulfotransferase activity described above.

The apparent Km for PAPS is 5.0 × 10 ⁻⁷ M
(FIG. 6B). The Km for dermatan sulfate and the Km for chondroitin sulfate A are 1.3 × 10 ^−7, respectively.
M and 1.1 × 10 ⁻⁶ M (FIG. 7).

(4) Receptor Substrate Specificity Purified GalNAc4S-6ST was incubated with various glycosaminoglycans in the presence of various amounts of CaCl ₂ . The glycosaminoglycan (receptor), whale cartilage-derived chondroitin sulfate _{A (4-SO 4: 6} -SO 4 = 74.2: 22.
9), shark cartilage-derived chondroitin sulfate _{C (4-SO 4: 6} -SO
₄ = 26.9: 55.1), chondroitin sulfate C from human half moon
(4-SO ₄ : 6-SO ₄ = 13.4: 81.0, a gift from Dr. Habuchi, Aichi Medical University), dermatan sulfate from pig skin, chondroitin from squid skin (Biochim. Biophys. Acta, 616, 2)
08-217 (1980)), bovine cornea-derived keratan sulfate, bovine kidney-derived heparan sulfate, CDSNS-heparin, and squid cartilage-derived chondroitin sulfate E (J.B.
Prepared by the method described in iol. Chem., 252, 4570-4576 (1977) (eluted with 1.5 M NaCl from DEAE-Sephadex A-50). Glycans are manufactured by Seikagaku Corporation. That is, instead of chondroitin sulfate A, 25 nmol (as galactosamine for chondroitin sulfate and dermatan sulfate, and as glucosamine for heparan sulfate, CDSNS-heparin and keratan sulfate) was used, and various amounts of CaCl ₂ were used. The activity was measured according to the above-described method for measuring sulfotransferase activity except that was added.

FIG. 8 shows the results. As shown in FIG. 8, the purified GalNAc4S-6ST has a sulfate group as chondroitin sulfate A.
(Closed circles), chondroitin sulfate C (open circles and closed triangles) and dermatan sulfate (open triangles). Low activity was observed against squid skin chondroitin (open squares). Squid cartilage chondroitin sulfate E, keratan sulfate, heparan sulfate and CDSNS-heparin did not become receptors. The optimal concentration of CaCl ₂ was 20 mM for chondroitin sulfate A and chondroitin sulfate C, but was 100 mM for dermatan sulfate.

When comparing two chondroitin sulfates,
The activity was higher for those derived from shark cartilage (open circles) than those derived from human half moon (solid triangles). GlcA
The ratio of β1-3GalNAc (4-SO ₄ ) units / GlcAβ1-3GalNAc (6-SO ₄ ) units is 0.49 for chondroitin sulfate C derived from shark cartilage,
The amount of chondroitin sulfate C from human half moon was 0.17.
The lower the ratio of _{GalNAc (4-SO 4) /} GalNAc (6-SO 4), it was less active. Incorporation into chondroitin sulfate A was significantly inhibited by chondroitin sulfate E. In the presence of an equal amount of chondroitin sulfate E, incorporation into chondroitin sulfate A was reduced to 35% of the control (open diamonds in FIG. 8).

Next, the antagonism of the receptor substrate was examined. The activity was measured according to the sulfotransferase activity assay described above, except that 25 nmol chondroitin sulfate A was replaced with various amounts of chondroitin sulfate A (CSA) and dermatan sulfate (DS) shown below the graph in FIG. . After digesting ³⁵ S-labeled glycosaminoglycan with chondroitinase ACII or chondroitinase ABC (both manufactured by Seikagaku Corporation), 3 times the volume of ethanol containing 1.3% potassium acetate is added, and the mixture is centrifuged. did. ³⁵ S radioactivity which became soluble in ethanol after chondroitinase digestion was measured. FIG. 9A shows the result of calculating the incorporation of ³⁵ SO ₄ into CSA from the radioactivity contained in the ethanol-soluble fraction after chondroitinase ACII digestion. B
And C, subtracting the incorporation of ³⁵ SO ₄ into DS by subtracting the radioactivity contained in the ethanol-soluble fraction after chondroitinase ACII digestion from the radioactivity contained in the ethanol-soluble fraction after chondroitinase ABC digestion. The result calculated by the following is shown. As a result, the sulfation of chondroitin sulfate A by purified GalNAc4S-6ST was inhibited by dermatan sulfate, and the reverse inhibition was similarly observed. This means
This suggests that the sulfation of both chondroitin sulfate A and dermatan sulfate is catalyzed by the same enzyme, ie, at least the same catalytic site.

(5) Examination of Sulfation Position In order to determine the position of the sulfate group transferred to chondroitin sulfate A (CS-A) and dermatan sulfate (DS), ³⁵ S-labeled glycosami produced by sulfotransferase reaction was determined. Noglycan is digested with chondroitinase ACII or chondroitinase ABC (and further digested with chondro-6-sulfatase (manufactured by Seikagaku Corporation) if necessary), and the digestion product is superdex 30 gel (Amersham Pharmacia)
Biotechnology) Chromatography and Partisil (P
artisil) -10 SAX (Whatman) HPLC.

The sulfotransferase reaction was performed using 25 nmol (as galaxamine) CS-A, except that CaCl ₂ was added to a final concentration of 100 mM when DS was used as the receptor.
Alternatively, the measurement was performed using DS according to the above-described method for measuring sulfotransferase activity.

Digestion with chondroitinase ACII or chondroitinase ABC was performed using ³⁵ S-labeled glycosaminoglycan, 1.25 μmol Tris-acetate buffer, pH 7.5, 2.5 μg bovine serum albumin, and 50 mU chondroitinase ACII or chondroitinase ABC. The reaction was performed at 37 ° C. for 4 hours with a reaction mixture containing itinase ABC in a final volume of 25 μl. After completion of the reaction with chondroitinase ACII or chondroitinase ABC, the reaction mixture was immersed in a boiling water bath for 1 minute. If for further digestion with chondro-6-sulfatase, chondro-6-sulfatase and (75 mU) was added to the reaction mixture, incubation at 37 ° C., for degradation of [Delta] Di-diS _E 30
Continued for minutes. Further, it continued for 5 hours for the degradation (extended degradation) of GalNAc. The sample for HPLC analysis was digested with 10 nmol ΔDi-diS _E (and 30 nmol GalNAc (4,6-bisSO ₄ ) in the case of extended decomposition) added to ³⁵ S-labeled glycosaminoglycan. .

Superdex 30 gel chromatography was performed as follows. Superdex 30 16/16 column was equilibrated with 0.2 M NH ₄ HCO _3. The flow rate was 1 ml / min. The eluate was collected in fractions of 1 ml each. Partisil-10
SAX HPLC is, Partisil 10-SA equilibrated with 10 mM KH ₂ PO ₄
This was performed using an X column (4.6 mm × 25 cm). Column
10 mM KH ₂ PO ₄ for 10 min and then developed with a linear gradient of 10~450 mM KH ₂ PO _4. The flow rate was 1 ml / min and the column temperature was 40 ° C. The eluate was collected in 0.5 ml fractions each.

The abbreviations of the disaccharide used here are as follows. ΔDi-0S: 2-acetamido-2-deoxy-3-O-
(β-D-gluco-4-enpyranosyluronic acid) -D-galactose, ΔDi-6S: 2-acetamido-2-deoxy-3-O- (β-D
-Gluco-4-enpyranosyluronic acid) -6-O-sulfo-D-galactose, ΔDi-4S: 2-acetamido-2-deoxy-3-O
-(β-D-gluco-4-enpyranosyluronic acid) -4-O-sulfo
-D- galactose, ΔDi-diS _D: 2- acetamido-2-deoxy -3-O- (2-O- sulfo-beta-D-gluco-4 Enpi Rano sill uronic acid) -6-O-sulfo - D- galactose, ΔDi-diS _E: 2-
Acetamide-2-deoxy-3-O- (β-D-gluco-4-enpyranosyluronic acid) -4,6-bis-O-sulfo-D-galactose.

FIG. 10 shows the results of analysis of chondroitinase ACII or chondroitinase ABC digest by Superdex 30 gel chromatography.

The digestion product obtained from ³⁵ S-labeled CS-A by digestion with chondroitinase ACII or chondroitinase ACII + chondro-6-sulfatase was analyzed by HPLC.
The results of the separation are shown in FIG. 11 and show that the digested product obtained from ³⁵ S-labeled DS was digested with chondroitinase ABC or chondroitinase ABC + chondro-6-sulfatase.
FIG. 12 shows the results of the PLC separation. FIG. 13 shows the results of HPLC separation of digestion products obtained from ³⁵ S-labeled glycosaminoglycans by prolonging digestion by chondro-6-sulfatase.

[0084] If ^{the 35} S-labeled CS-A was digested with chondroitinase ACII, major radioactivity was detected at the position of ΔDi-diS _E (C in B and 11 in FIG. 10). While ³⁵
S-labeled DS was hardly degraded by chondroitinase ACII digestion (FIG. 10D). ³⁵ when digested with S-labeled DS with chondroitinase ABC, the major radioactive, similar to that observed with chondroitinase ACII digested CS-A, was detected at the position of [Delta] Di-diS _E (FIG. 10 E and FIG. 12C). To sulfuric acid group [Delta] Di-diS _E Check to make sure that a ³⁵ S radioactivity, chondroitinase ACII or chondroitinase decomposition product obtained by ABC digested further digested with chondro-6-sulfatase, The sample was subjected to SAX HPLC (B and D in FIG. 11 and B and D in FIG. 12). Chondro-6
- After degradation in sulfatase, the [Delta] Di-diS _E was externally added
UV absorption (B in FIG. 11 and B in FIG. 12) and radioactivity (D in FIG. 11 and D in FIG. 12) disappeared, and UV absorption and radioactivity shifted to the positions of ΔDi-4S and inorganic sulfate, respectively. These results indicate that ³⁵ SO ₄ is not the internal GlcAβ1-3Ga of CS-A.
lNAc _(4-SO 4) GalNAc included in the unit _(4-SO 4) Internal IdoAα1-3GalNAc _(4-SO 4) residues or DS GalNAc included in the unit (4-S
This clearly shows that the residue was transferred to the 6th position of the O ₄ ) residue. ³⁵ S sign
If CS-A was digested with chondroitinase ACII, and ^{3 5}
When S-labeled DS was digested with chondroitinase ABC, a small amount of radioactivity was detected at the position of GalNAc (4,6-bisSO ₄ ) (C in FIG. 11 and C in FIG. 12). GalNAc (4,6-bis
The radioactivity detected at the position of (SO ₄ ) was not lost after chondro-6-sulfatase digestion (D in FIG. 11 and D in FIG. 12). GalNAc (4,6-bisSO _4), when the [Delta] Di-diS _E is completely decomposed, it has been found that is only partially degraded in chondro-6-sulfatase. However, when the incubation with chondro-6-sulfatase is extended, more than 95% of GalNAc (4,6-bisSO ₄ )
It was decomposed into (4-SO ₄ ) (FIG. 13B). In this condition, G
The radioactivity observed at the position of alNAc (4,6-bisSO ₄ ) disappeared (C and D in FIG. 13). These results indicate that purified GalN
This shows that Ac4S-6S was able to transfer a sulfate group to position 6 of both the internal and non-reducing terminal GalNAc (4-SO ₄ ) residues of chondroitin sulfate and DS.

Squid skin chondroitin was purified from GalNAc4S-6.
Weakly sulfated by ST. Squid skin chondroitin
The sulfate group transferred to squid chondroitin is mainly composed of GlcAβ1-3GalNAc units and the content of 4-sulfated disaccharide units is less than 0.2% of the total repeating disaccharide units (data not shown). It was predicted to be located at position 6 of the residue. However, a sulfotransferase reaction was performed using 25 nmol (as galactosamine) squid skin chondroitin according to the above-described method for measuring sulfotransferase activity, and the obtained ³⁵ S-labeled chondroitin was digested with chondroitin ACII and then subjected to SAX-HPLC. as the eluent, ³⁵ S radioactivity detected in GalNAc (4,6-bisSO ₄₎ and [Delta] Di-diS _E, the [Delta] Di-6S were not detected radioactivity (Figure 1
4). These results indicate that the activity of GalNAc4S-6ST
Indicates that (4-SO ₄ ) is absolutely required.

[0086]

According to the present invention, sulfation such as an action of specifically transferring a sulfate group from a sulfate group donor to a hydroxyl group at the 6-position of N-acetylgalactosamine 4-sulfate residue of glycosaminoglycan. Provided is a sulfotransferase preparation whose properties have been established and which have been purified to such an extent that they have a sufficiently high specific activity. The present sulfotransferase preparation can be used for producing a polysaccharide having a desired sulfation mode.

[Brief description of the drawings]

FIG. 1 shows the amount of protamine sulfate added to a crude extract derived from squid cartilage, and the sulfotransferase activity (open circles) and glucuronic acid in the supernatant fraction obtained by centrifuging the crude extract containing protamine sulfate. The relationship with the amount (black circle) is shown.

FIG. 2 shows the elution profile of Dimatrex Gel Red A column chromatography. Filled circles indicate sulfotransferase activity, open circles indicate protein concentration,
The arrow indicates the starting position of elution with the buffer containing the indicated concentration of NaCl.

FIG. 3 shows the elution profile of the first heparin sepharose CL-6B column chromatography. Solid circles indicate sulfotransferase activity, open circles indicate protein concentration, and broken lines indicate NaCl concentration.

FIG. 4 shows the elution profile of the first 3 ′, 5′-ADP-agarose column chromatography. Filled circles indicate sulfotransferase activity, open circles indicate protein concentration,
The dashed line indicates the concentration of NaCl.

FIG. 5: Results of analysis of each fraction in the purification step by SDS-polyacrylamide gel electrophoresis (electrophoresis photograph)
Is shown. Lane 1, crude extract. Lane 2, supernatant fraction after protamine precipitation. Lane 3, dimatrex gel red A fraction. Lane 4, first heparin sepharose CL-4
B fraction. Lane 5, first 3 ', 5'-ADP-agarose fraction.
Lane 6, second heparin sepharose CL-4B fraction. The molecular weight standards were myosin (205 kDa), β-galactosidase (116 kDa), phosphorylase b (97.4 kDa), bovine serum albumin (66 kDa), egg albumin (45 kDa) and carbonic anhydrase (29 kDa). Was.

FIG. 6 shows the effect of pH on sulfotransferase activity (A) and the results of measuring Km (B) on PAPS. In A, the black square is sodium acetate and the white square is ME
S-NaOH, closed circles indicate imidazole-HCl, open circles indicate Tris-HCl buffer. In B, the value on the vertical axis of the reciprocal plot indicates 1 / (pmol / min / μg protein).

FIG. 7 shows the effect of chondroitin sulfate A and dermatan sulfate concentrations on sulfotransferase activity.

FIG. 8 [ ³⁵ S] PAPS by sulfotransferase
₃ shows the incorporation of ³⁵ SO ₄ into various glycosaminoglycans from. Receptors include chondroitin sulfate A (solid circles), chondroitin sulfate C from shark cartilage (open circles), chondroitin sulfate C from human meniscus (closed triangles), dermatan sulfate (open triangles), and chondroitin from squid skin (open squares). It was used. Open diamonds indicate incorporation into chondroitin sulfate A in the presence of 25 nmol (as galactosamine) chondroitin sulfate E.

FIG. 9 shows inhibition of chondroitin sulfate A (CSA) sulfation by dermatan sulfate (DS) and inhibition of DS sulfation by CSA. The values below the graph indicate CSA and D in the reaction mixture.
Indicates the amount of S. A shows incorporation of ³⁵ SO ₄ into CSA.
B and C show incorporation of ³⁵ SO ₄ into DS.

FIG. 10: Superdex 30 column of chondroitinase ACII or chondroitinase ABC digest of ³⁵ S-labeled product derived from chondroitin sulfate A or dermatan sulfate by incubation with [ ³⁵ S] PAPS and purified GlcNAc4S-6ST. 2 shows a chromatographic elution profile. A: ³⁵ S-labeled chondroitin sulfate A before chondroitinase digestion, B: ³⁵ S-labeled chondroitin sulfate A chondroitinase ACII digestion product,
C: chondroitinase digestion of ³⁵ S-labeled dermatan sulfate, D: chondroitinase ACII digestion product of ³⁵ S-labeled dermatan sulfate, E: chondroitinase
3 shows the results of analysis of ABC digestion products. Arrows indicate Vo, blue dextran, ΔDi-diS _E (1) and GalNAc (4,6-bisS
O ₄ ) Indicates the position of (2).

FIG. 11 shows the results of HPLC separation of a digestion product obtained from ³⁵ S-labeled chondroitin sulfate A by digestion with chondroitinase ACII or chondroitinase ACII + chondro-6-sulfatase. A and C after chondroitinase ACII digestion, B and D after chondroitinase ACII digestion.
+ After chondro-6-sulfatase digestion. A and B
Indicates absorbance at 232 nm, and C and D indicate ³⁵ S radioactivity. Arrows indicate 1: ΔDi-0S, 2: GalNAc (6-SO ₄ ), 3: GalNAc (4-S
O ₄ ), 4: ΔDi-6S, 5: ΔDi-4S, 6: GalNAc (4,6-bisSO ₄ ),
^{_{7: SO 4 2-, 8:}} ΔDi-diS D and 9: shows the elution position of ΔDi-diS _E.

FIG. 12 shows the H of digestion products obtained from ³⁵ S-labeled dermatan sulfate by digestion with chondroitinase ABC or chondroitinase ABC + chondro-6-sulfatase.
The result of PLC separation is shown. A and C are after chondroitinase ABC digestion, and B and D are after chondroitinase ABC + chondro-6-sulfatase digestion. A and B are 232
The absorbance at nm, C and D indicate ³⁵ S radioactivity. The elution position of the standard substance indicated by the arrow is the same as in FIG.

FIG. 13 shows the results of HPLC separation of digestion products obtained from ³⁵ S-labeled glycosaminoglycans by prolonged digestion with chondro-6-sulfatase. A and B show the elution profiles of standard ΔDi-diSE and GalNAc (4,6-bisSO ₄ ) before and after digestion with chondro-6-sulfatase for 5 hours (chondro-6-sulfatase extended digestion).
Monitored at 0 nm absorbance. C is chondro-6-sulfatase extended digest of chondroitinase ACII digest of ³⁵ S-labeled chondroitin sulfate A, and D is chondro-6-sulfatase extended digest of ³⁵ S-labeled chondroitin sulfate ABC of dermatan sulfate. The results are shown. The elution position of the standard substance indicated by the arrow is the same as in FIG.

FIG. 14. Digestion with chondroitinase ACII resulted in ³⁵
Fig. 4 shows the results of HPLC separation of digestion products obtained from S-labeled squid skin chondroitin. The elution position of the standard substance indicated by the arrow is the same as in FIG.

Claims (8)

[Claims] 1. An SDS-Polyac having the following physicochemical properties:
Substantially homogeneous band in lilamide gel electrophoresis
, A sulfotransferase preparation. (1) Action: From sulfate group donor, glycosaminoglycan
At position 6 of the N-acetylgalactosamine 4-sulfate residue of
A sulfate group is specifically transferred to a droxyl group. (2) Substrate specificity: chondroitin sulfate from whale cartilage
Acid A, chondroitin sulfate C from shark cartilage, pig skin
The sulfate group is transferred to dermatan sulfate. Squid cartilage
Conventional chondroitin sulfate E, keratan sulfate from bovine cornea
Acid, heparan sulfate from bovine kidney, CDSNS-Hepari
The sulfate group is not substantially transferred to the compound. (3) Activation: 20 mM Mn²⁺, Mg²⁺, Ca²⁺, S
r²⁺, Ba²⁺Or Co ²⁺Increases in the presence of.
0.1 M NaCl, 0.1 M KCl or 0.15
Activity increases in the presence of mg / ml protamine. (4) Inhibition: Chondroitin sulfate E causes chondroitin
Transfer of a sulfate group to itin sulfate A is inhibited. Dermata
Transfer of sulfate groups to chondroitin sulfate A
Transfer is inhibited. By Chondroitin Sulfate A, Dell
Transfer of sulfate groups to matane sulfate is inhibited. 2. The sulfotransferase preparation according to claim 1, which further has the following physicochemical properties. (5) Molecular weight: about 63 kD, about 54 kD (SDS-polyacrylamide gel electrophoresis; under non-reducing conditions) (6) Optimum pH: around 6.2 3. The sulfotransferase preparation according to claim 1, which further has the following physicochemical properties. (7) Optimal Ca ²⁺ concentration: about 20 mM (when chondroitin sulfate A is the substrate). About 100 mM (when dermatan sulfate is the substrate). (8) Km value: about 5 × 10 ⁻⁷ M (3′-phosphoadenosine 5′-phosphosulfate) about 1.3 × 10 ⁻⁷ M (dermatan sulfate from pig skin) about 1.1 × 10 ⁻⁶ M (chondroitin sulfate A derived from whale cartilage) 4. The sulfotransferase preparation according to claim 1, which is derived from squid cartilage. 5. The method according to claim 1, comprising at least the following steps:
5. The method for producing the sulfotransferase preparation according to any one of items 4 to 4. (Step 1) A step of obtaining an extract from an organism containing a sulfotransferase contained in the sulfotransferase preparation according to any one of claims 1 to 4. (Step 2) A step of bringing the extract into contact with protamine to form a precipitate. (Step 3) A step of removing the precipitate generated in Step 2. 6. The method according to claim 5, wherein the organism is squid cartilage. 7. The method according to claim 5, further comprising an affinity chromatography step using heparin as a ligand and an affinity chromatography step using adenosine 3 ′, 5′-diphosphate as a ligand, performed after the step 3. The production method described in 1. 8. A method for producing chondroitin sulfate E, comprising allowing the sulfotransferase preparation according to any one of claims 1 to 4 to act on chondroitin sulfate A, and isolating the produced chondroitin sulfate E. Method.