JPH0625299A - Protein capable of transporting vitamin e - Google Patents

Abstract

PURPOSE:To obtain a new protein, having a specific amino acid sequence, a specified isoelectric point and a specific molecular weight, capable of efficiently carrying out the intracellular transportation of vitamin E capable of preventing phospholipids from undergoing peroxidizing reaction leading to unsaturated fatty acids and useful as medicines, etc. CONSTITUTION:The objective protein, capable of transporting vitamin E and having partial structures of formulas I and II, 5.1 pI isoelectric point and about 30500 molecular weight is obtained by homogenizing a fresh liver of an SD rat (male) in a buffer solution, centrifuging the homogenate, collecting a supernatant, adding 1NHCl thereto, regulating the pH to 5.0, removing a precipitate, then collecting a fraction saturated with 30-60% ammonium sulfate according to an ammonium sulfate salting out method, dissolving the resultant fraction in a buffer solution, carrying out the gel filtration, collecting an active fraction having about 30000 molecular weight, passing the obtained fraction through an anion exchange column, eluting the prepared fraction with a concentration gradient at 0-0.3 M common salt concentration in the buffer solution, subsequently successively passing the fraction through a hydroxyapatite column and a gel filtration column and performing the purification.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a vitamin E-specific transport protein.

[0002]

2. Description of the Related Art Vitamin E is widely distributed in biological membranes in vivo and is considered to play a role of preventing the peroxidation reaction of phospholipids to unsaturated fatty acids and its expansion by its antioxidant action. . In addition, microcirculatory activating action, effects on lipid metabolism, etc. are known, and in recent years, many studies have been conducted with emphasis on the relationship between aging and adult diseases and vitamin E. Regarding the intracellular transport of vitamin E, there are some reports that a vitamin E binding protein exists in the cytoplasm of rat liver (Rajaram, OV et al, B.
iochem. Biophys. Res. Commu
n. , 52, 459-465, 1973. , Catig
nani, G .; L. , Biochem. Biophy
s. Res. Commun. , 67, 66-72, 19
75. , Catignani, G .; L. et al, B
iochem. Biophys. Acta. , 497,
349-357, 1977. Behrens. W.
A. et al, Nutr. Rep. Int. , 25,
107-112, 1982. However, there are still many unclear points, and nothing is known about its structure.

[0003]

[Problems to be Solved by the Invention] Vitamin E in vivo
The purpose is to provide a specific transport protein.

[0004]

[Means for Solving the Problems] The present inventors
As a result of earnest studies on the physiological mechanism of the, the complete purification method of the vitamin E-specific transport protein from the soluble fraction of rat liver was established. Furthermore, when the partial structure was analyzed, it was found that it was a novel specific transport protein, and the present invention was completed. That is, the present invention has an isoelectric point pI5.1 having a partial structure of SEQ ID NOS: 1 and 2, a vitamin E-specific transport protein I having a molecular weight of about 30500, and an isoelectric point pI5.I having a partial structure of SEQ ID NO: 3. It relates to a vitamin E-specific transport protein II having a molecular weight of 0 and a molecular weight of about 30,500. This protein is a useful substance that efficiently transports vitamin E in cells. Hereinafter, the purification method and structure confirmation of the proteins I and II of the present invention will be described in detail with reference to Examples.

[0005]

【Example】

Reference Example 1 Vitamin E transport activity measurement method Blj R. et al. Et al. (Bloj R. et al,
J. Biol. Chem. , 252, 1613-161
9, 1977. ). That is, liposomes and mitochondria are used, and the liposomes are prepared as a composition of egg yolk phosphatidylcholine, dicetylphosphate, butylhydroxytriene, and α- ¹⁴ C tocopherol (Mowri. H. et. Al. Eur.
J. Biochem. , 117, 537-542, 19
81. ). The mitochondria used are those prepared from rat liver according to a conventional method. In the presence of liposomes and mitochondria, a sample was added to confirm the transport protein in each purification step and the vitamin E specificity of the transport protein using the amount of α- ^14C tocopherol transported from the liposome to mitochondria as an index.

Example 1 Purification Method of Vitamin E Transport Protein Twenty fresh livers of SD rats (male) were mixed with 2-3 times the amount of 0.25 M sucrose, 1 mM EDTA, 10
Add mM Tris-HCl buffer (pH 7.4), homogenize, centrifuge at 10,000 × G for 20 minutes, and collect the supernatant. 1N HCl is added dropwise to this to adjust the pH to 5.0, and then the same centrifugation is performed to obtain a supernatant. Next, a 30% to 60% ammonium sulfate saturated fraction is obtained by the ammonium sulfate salting-out method, and the column fraction is subjected to the following column operations for purification. First Sephade
x G75 (superfine) column (size, 4
5 × 1000 mm) and 10 mM KH ₂ PO ₄ ·
5 mM 2-mercaptoethanol, 10%
Using Glycerol (pH 6.8) (buffer solution A) as a solvent, gel filtration fractionation of the above ammonium sulfate salting out is carried out to fractionate an active fraction having a molecular weight of about 30,000.

Then, DE fully buffered with the same buffer A was used.
Using an AE-Sepharose CL-6B column (size, 10 × 250 mm), the Sephadex
After adsorbing the active fraction collected on the G75 column, concentration gradient elution is carried out at a salt concentration of 0 to 0.3 M in the same buffer (150 ml each). 3 ml aliquots are collected and active fraction number 3
Collect 7-45. Next, a Hydroxyapatite column (size, 4 × 9) sufficiently buffered with buffer A
0 mm) and after adsorbing the active substance obtained by the above operation, 70 ml of 10 mM and 200 mM KH ₂ PO ₄
Gradient elution is performed with (other composition is the same as buffer A). Fractionation is carried out in 3 ml portions and the active fraction number 19-26 is collected. This active fraction was added to 25 mM Histidine / H
Cl, 5 mM 2-mercaptoethanol,
Polybuffer 94excha well buffered with 10% Glycerol (pH 6.2) (buffer B)
After being adsorbed on an nger column (size, 10 × 250 mm), it is eluted with polybuffer 74 (pH 4.0) to perform pH gradient fractionation from pH 6.2 to 4.0. As a result, as shown in FIG. 1, two activity peaks I and II were observed at the isoelectric points 5.1 and 5.0, and each was separated.

Finally, blue Sepharose C
Purify and unify activity peaks I and II using an L-6B column. Activity peak I is 5 mM KH ₂ PO ₄ , 5 m
M 2-mercaptoethanol, 10% Gl
Blu well buffered with ycerol (pH 6.8)
eSepharose CL-6B column (size, 6
X 150 mm), and then the salt concentration in the same buffer was 0
Gradient elution is performed at -1 M to obtain a single active fraction. Activity peak II is 1 mM KH ₂ PO ₄ , 5 mM 2
-Mercaptoethanol, 10% Glyce
blue well buffered with roll (pH 7.4)
Sepharose column (size, 6 x 150 mm)
And collect as a non-adsorbed fraction, and then under the same conditions as for activity peak I, blue Sepharose CL
-6B column is applied and the same operation is performed to obtain a single active fraction.

The yield and specific activity of each purification step are as shown in Table 1.

[Table 1]

Confirmation of the unity of the purified sample is made by O.K. Farre
SDS-using 15% slab gel according to the method of 11.
Polyacrylamide gel electrophoresis was performed. (O.
Farrell P. H. J. Biol. Che
m. , 250, 4007-4021, 1975). Figure 2
Activity peaks I and II, that is, transport proteins I and II
Both showed a single band located at a molecular weight of about 30,500.

Example 2 Analysis of Amino Acid Analysis Value and Amino Acid Sequence As shown in Table 2, the amino acid composition values of transport proteins I and II were analyzed by an amino acid analyzer, and there is not much difference between them. The amino acid sequence was analyzed using a sequencer. Since no information was obtained for both proteins as they were, it was suggested that the N-terminus may be blocked. Therefore, both proteins were hydrolyzed with lysyl endopeptidase, and the resulting fragments were purified by high performance liquid chromatography and analyzed by the same sequencer. As a result, the sequences of SEQ ID NOS: 1 and 2 were confirmed from the fragment of transport protein I, and the sequence of SEQ ID NO: 3 was confirmed from the fragment of transport protein II. When these amino acid sequences were compared with a sequence database, no protein having a similar sequence was found and it is a novel transport protein.

Reference Example 2 Substrate Specificity According to the method described in Reference Example 1, the transport activities of α-tocopherol for structural analogs and cholesterol were compared by competitive inhibition experiments (Table 3). As a result, it was shown that not only α-tocopherol but also β, γ, δ-tocopherol exhibited an inhibitory effect to a small extent, and the purified protein also recognized these tocopherols. However, tocopherol quinone, tocopherol acetate, cholesterol, etc. had no inhibitory effect, suggesting that this protein specifically recognizes the structure of tocopherol. The two isoforms did not differ in terms of substrate specificity.

[Brief description of drawings]

FIG. 1 is an elution diagram of the isoelectric focusing method.

FIG. 2 is an SDS-polyacrylamide electrophoresis diagram. B: Transport protein I, C: Transport protein II, A, D: Molecular weight marker

[Sequence list]

SEQ ID NO: 1 Sequence length: 21 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Lys Ala lle Phe Asp Leu Glu Gly Trp Gln lle Ser His Ala Phe 5 10 15 Gln lle Thr Xaa Ser Val 20 SEQ ID NO: 2 Sequence length: 12 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence lle Ser Asn Ser Thr Ser Pro Thr (Gln) Asn (Ser) 5 Phe Val Ala SEQ ID NO: 3 Sequence length: 9 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

[Table 2]

[Table 3]

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] June 22, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be amended] Detailed explanation of the invention

[Correction method] Change

[Correction content]

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a vitamin E-specific transport protein.

[0002]

2. Description of the Related Art Vitamin E is widely distributed in biological membranes in vivo and is considered to play a role of preventing the peroxidation reaction of phospholipids to unsaturated fatty acids and its expansion by its antioxidant action. . In addition, microcirculatory activating action, effects on lipid metabolism, etc. are known, and in recent years, many studies have been conducted with emphasis on the relationship between aging and adult diseases and vitamin E. Regarding the intracellular transport of vitamin E, there are some reports that a vitamin E-binding protein exists in the cytoplasm of rat liver (Rajaram, OV et al, B.
iochem. Biophys. Res. Commu
n. , 52, 459-465, 1973. , Catig
nani, G .; L. , Biochem. Biophy
s. Res. Commun. , 67, 66-72, 19
75. , Catignani, G .; L. et al, B
iochem. Biophys. Acta. , 497,
349-357, 1977. Behrens. W.
A. et al, Nutr. Rep. Int. , 25,
107-112, 1982. However, there are still many unclear points, and nothing is known about its structure.

[0003]

[Problems to be Solved by the Invention] Vitamin E in vivo
The purpose is to provide a specific transport protein.

[0004]

[Means for Solving the Problems] The present inventors
As a result of earnest studies on the physiological mechanism of the, the complete purification method of the vitamin E-specific transport protein from the soluble fraction of rat liver was established. Furthermore, when the partial structure was analyzed, it was found that it was a novel specific transport protein, and the present invention was completed. That is, the present invention has an isoelectric point pI5.1 having a partial structure of SEQ ID NOS: 1 and 2, a vitamin E-specific transport protein I having a molecular weight of about 30500, and an isoelectric point pI5.I having a partial structure of SEQ ID NO: 3. It relates to a vitamin E-specific transport protein II having a molecular weight of 0 and a molecular weight of about 30,500. This protein is a useful substance that efficiently transports vitamin E in cells. Hereinafter, the purification method and structure confirmation of the proteins I and II of the present invention will be described in detail with reference to Examples.

[0005]

EXAMPLES Reference Example 1 Vitamin E Transport Activity Measurement Method Blj R. et al. Et al. (Bloj R. et al,
J. Biol. Chem. , 252, 1613-161
9, 1977. ). That is, liposomes and mitochondria are used, and the liposomes are prepared as a composition of egg yolk phosphatidylcholine, dicetylphosphate, butylhydroxytriene, and α- ¹⁴ C tocopherol (Mowri. H. et. Al. Eur.
J. Biochem. , 117, 537-542, 19
81. ). The mitochondria used are those prepared from rat liver according to a conventional method. In the presence of liposomes and mitochondria, a sample was added to confirm the transport protein in each purification step and the vitamin E specificity of the transport protein using the amount of α- ^14C tocopherol transported from the liposome to mitochondria as an index.

Example 1 Purification Method of Vitamin E Transport Protein Twenty fresh livers of SD rats (male) were mixed with 2-3 times the amount of 0.25 M sucrose, 1 mM EDTA, 10
Add mM Tris-HCl buffer (pH 7.4), homogenize, centrifuge at 10,000 × G for 20 minutes, and collect the supernatant. 1N HCl is added dropwise to this to adjust the pH to 5.0, and then the same centrifugation is performed to obtain a supernatant. Next, a 30% to 60% ammonium sulfate saturated fraction is obtained by the ammonium sulfate salting-out method, and the column fraction is subjected to the following column operations for purification. First Sephade
x G75 (superfine) column (size, 4
5 × 1000 mm) and 10 mM KH ₂ PO ₄ ·
5 mM 2-mercaptoethanol, 10%
Using Glycerol (pH 6.8) (buffer solution A) as a solvent, gel filtration fractionation of the above ammonium sulfate salting out is carried out to fractionate an active fraction having a molecular weight of about 30,000.

Then, DE fully buffered with the same buffer A was used.
Using an AE-Sepharose CL-6B column (size, 10 × 250 mm), the Sephadex
After adsorbing the active fraction collected on the G75 column, concentration gradient elution is carried out at a salt concentration of 0 to 0.3 M in the same buffer (150 ml each). 3 ml aliquots are collected and active fraction number 3
Collect 7-45. Next, a Hydroxyapatite column (size, 4 × 9) sufficiently buffered with buffer A
0 mm) and after adsorbing the active substance obtained by the above operation, 70 ml of 10 mM and 200 mM KH ₂ PO ₄
Gradient elution is performed with (other composition is the same as buffer A). Fractionation is carried out in 3 ml portions and the active fraction number 19-26 is collected. This active fraction was added to 25 mM Histidine / H
Cl, 5 mM 2-mercaptoethanol,
Polybuffer 94excha well buffered with 10% Glycerol (pH 6.2) (buffer B)
After being adsorbed on an nger column (size, 10 × 250 mm), it is eluted with polybuffer 74 (pH 4.0) to perform pH gradient fractionation from pH 6.2 to 4.0. As a result, as shown in FIG. 1, two activity peaks I and II were observed at the isoelectric points 5.1 and 5.0, and each was separated.

Finally, blue Sepharose C
Purify and unify activity peaks I and II using an L-6B column. Activity peak I is 5 mM KH ₂ PO ₄ , 5 m
M 2-mercaptoethanol, 10% Gl
Blu well buffered with ycerol (pH 6.8)
eSepharose CL-6B column (size, 6
X 150 mm), and then the salt concentration in the same buffer was 0
Gradient elution is performed at -1 M to obtain a single active fraction. Activity peak II is 1 mM KH ₂ PO ₄ , 5 mM 2
-Mercaptoethanol, 10% Glyce
blue well buffered with roll (pH 7.4)
Sepharose column (size, 6 x 150 mm)
And collect as a non-adsorbed fraction, and then under the same conditions as for activity peak I, blue Sepharose CL
-6B column is applied and the same operation is performed to obtain a single active fraction.

The yield and specific activity of each purification step are as shown in Table 1.

[Table 1]

Confirmation of the unity of the purified sample is made by O.K. Farre
SDS-using 15% slab gel according to the method of 11.
Polyacrylamide gel electrophoresis was performed. (O.
Farrell P. H. J. Biol. Che
m. , 250, 4007-4021, 1975). Figure 2
Activity peaks I and II, that is, transport proteins I and II
Both showed a single band located at a molecular weight of about 30,500.

Example 2 Analysis of Amino Acid Analysis Value and Amino Acid Sequence As shown in Table 2, the amino acid composition values of transport proteins I and II were analyzed by an amino acid analyzer, and there is not much difference between them. The amino acid sequence was analyzed using a sequencer. Since no information was obtained for both proteins as they were, it was suggested that the N-terminus may be blocked. Therefore, both proteins were hydrolyzed with lysyl endopeptidase, and the resulting fragments were purified by high performance liquid chromatography and analyzed by the same sequencer. As a result, the sequences of SEQ ID NOS: 1 and 2 were confirmed from the fragment of transport protein I, and the sequence of SEQ ID NO: 3 was confirmed from the fragment of transport protein II. When these amino acid sequences were compared with a sequence database, no protein having a similar sequence was found and it is a novel transport protein.

Reference Example 2 Substrate Specificity According to the method described in Reference Example 1, the transport activities of α-tocopherol for structural analogs and cholesterol were compared by competitive inhibition experiments (Table 3). As a result, it was shown that not only α-tocopherol but also β, γ, δ-tocopherol exhibited an inhibitory effect to a small extent, and the purified protein also recognized these tocopherols. However, tocopherol quinone, tocopherol acetate, cholesterol, etc. had no inhibitory effect, suggesting that this protein specifically recognizes the structure of tocopherol. The two isoforms did not differ in terms of substrate specificity.

[Sequence Listing] SEQ ID NO: 1 Sequence length: 21 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Lys Ala lle Phe Asp Leu Glu Gly Trp Gln lle Ser His Ala Phe Gln 5 10 15 lle Thr Xaa Ser Val 20 Sequence number: 2 Sequence length: 12 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence lle Ser Asn Ser Thr Ser Pro Thr (Gln) Asn (Ser) Phe Val Ala 5 10 SEQ ID NO: 3 Sequence length: 9 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

[Procedure Amendment 2]

[Document name to be amended] Statement

[Name of item to be corrected] Brief description of the drawing

[Correction method] Change

[Correction content]

[Brief description of drawings]

FIG. 1 is an elution diagram of the isoelectric focusing method.

FIG. 2 is an SDS-polyacrylamide electrophoresis diagram.

[Explanation of symbols] A, D molecular weight marker B transport protein I C transport protein II

Claims (2)

[Claims] 1. Having a partial structure of SEQ ID NOS: 1 and 2,
Vitamin E transport protein I having the following physical properties: isoelectric point: pI 5.1 molecular weight: about 30500 2. A vitamin E transport protein II having the partial structure of SEQ ID NO: 3 and having the following physical properties: isoelectric point: pI 5.0 molecular weight: about 30500.