JPH0364519B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel peptide obtained from the salivary glands of rats, particularly the parotid gland, which has the activity of promoting tooth fluid transport, and a method for producing the same. When a rabbit hypothalamic extract is administered to parotidectomized rats, fluid transport from the pulp of the tooth to the dentine odontogenic cell tubules is no longer observed; It has already been shown that body fluid transport is observed when administered together. From this, it is thought that hypothalamic factors act directly on the parotid gland, and that promotion of fluid transport from the dental pulp of the tooth to the dentinal odontogenic cell tubules depends on the parotid gland. Steinman et al. conducted research on caries-inducing substances and the development of dental caries in rats, and isolated a substance that promotes dental fluid transport from pig parotid gland. Yes, and isoelectric point PH7.5
revealed that it is a protein with almost no ultraviolet absorption [Endocrinology,
83, 807 (1986); Endocrinology, 106 , 1994
(1980)]. Furthermore, Steinman et al. reported that administration of this active substance to rats improves the supply of nutrients to the teeth, promotes tooth development and strengthening of tooth structure, and suppresses the incidence of dental caries. The present inventor also investigated the salivary glands of rats, especially the parotid gland.
As in the case of pigs, we expected that there would be a substance that promotes tooth fluid transport (hereinafter referred to as a "dental fluid transport promoting substance"), so we introduced this substance into the parotid glands of rats. As a result of conducting research to see if the substance existed, the existence of the substance was confirmed, and as a result of further research on the separation and purification of the substance,
We discovered a method to isolate an extremely pure tooth fluid transport promoting substance and elucidated its physical and chemical properties. The dental fluid transport promoting substance isolated in the present invention is a peptide having the following physicochemical properties. (1) Properties: White powder (2) Amino acid composition: [Table]
The above amino acid composition is based on the test peptide.
It was hydrolyzed with 6N hydrochloric acid at 110°C for 24 hours and analyzed using an automatic amino acid analyzer (resin: Hitachi Custom #2611, one-column method). However, tryptophan (Trp) was similarly determined by hydrolysis with 2.5N sodium hydroxide containing 200 μg of starch at 110° C. for 20 hours. (3) N-terminal amino acid sequence: Gly-Val-Ile-Ala
-Trp- The N-terminal amino acid sequence was determined according to the method of Gray et al. [see Biochem. J., 89 , 379 (1963)]. That is, one amino acid is removed from the N-terminus by Edman degradation, and the newly generated terminal amino acid is determined by the Dansyl method. This operation was repeated in order to determine the N-terminal amino acid. (4) C-terminal amino acid sequence: -Aag-Lys-Asx-
The Ser-Thr-Ala-Gly C-terminal amino acid sequence was determined by decomposing the test peptide with carboxypeptidase Y.
The C-terminal amino acid sequence was determined by analysis using the carboxypeptidedase method [see J.Biochem. 77 , 69 (1975)], in which amino acids are stripped one by one from the terminal and the structure of each amino acid is sequentially determined. (5) Molecular weight: Approximately 2200 The molecular weight of this peptide was measured using Sephadex (registered trademark) G-25 using 0.2N sodium citrate buffer containing 8% ethanol, pH 3.2 as the eluent.
(manufactured by Pharmacia Fine Chemicals) Measured by gel filtration. A graph plotting the relationship between the amount of eluate and molecular weight at that time is shown in FIG. (6) Ultraviolet absorption spectrum: Maximum absorption wavelength (λ _nax ) = 278 nm The above maximum absorption wavelength is for 0.80 mg of sample dissolved in 1 ml of physiological saline, optical path length of 1 cm,
This is a value measured using a Shimadzu 210 model spectrophotometer. (7) Solubility: Soluble in water and physiological saline. Insoluble in acetone. (8) Color reaction: Ninhydrin reaction positive Bieurett reaction positive Sakaguchi reaction positive (9) Partition coefficient (Kaυ value) This is the partition coefficient between the gel layer and liquid layer in gel filtration, and is calculated by the following formula. Kaυ=Ve−Vo/Vt−Vo Vt=Total volume of gel bed Ve=Eluent volume Vo=Amount of solvent outside gel particles Sephadex (registered trademark) G- as gel filtration material
25 (manufactured by Pharmacia Fine Chemicals) and a 0.05M phosphate buffer with a pH of 7.2 as the eluent.
The value is approximately 0.44. As mentioned above, the tooth fluid transport promoting substance extracted from the parotid gland of pigs by Steinman et al. has a molecular weight of 8100 as measured by disk electrophoresis on SDS-polyacrylamide gel, and an amino acid composition of 46% glycine and 28% proline. and the isoelectric point is PH
Although the substance does not have a maximum absorption wavelength (λ _nax ) in the ultraviolet absorption spectrum of 7.5, the physical and chemical properties of the peptide of the present invention indicate that at least the molecular weight, amino acid composition, and ultraviolet absorption characteristics are those extracted from pig parotid gland. This substance is clearly different from the tooth fluid transport promoting substance described above, and is considered to be a new substance that has not been described in conventional literature. The salivary glands of mammals, especially the parotid gland, contain salivary gland hormones, which are proteins and have effects such as promoting hard tissue growth, activating mesenchymal tissue, lowering serum calcium, and increasing white blood cells. It has various excellent physiological activities and is widely used as a medicine. However, the present inventor has confirmed that this salivary gland hormone does not have any effect on body fluid transport to the rat dentinal dental tubules. Therefore, it is clear that the tooth fluid transport promoting substance present in the rat parotid gland is a substance different from the salivary gland hormone in its action. That is, the tooth fluid transport promoting activity of the peptide of the present invention and salivary gland hormone was measured by the following method: Acriflavine hydrochloride was intraperitoneally administered as a fluorescent substance to 5-week-old rats at a rate of 5 mg per 100 g of body weight. Immediately thereafter, the peptide of the present invention or salivary gland hormone was intravenously administered in a liquid volume of 0.1 ml.
He was decapitated minutes later. Within 1 minute after decapitation, the mandible was extracted and frozen. Using a frozen microtome, cutting of the molar teeth perpendicular to the occlusal plane was adjusted, and the transfer of fluorescent substances to the dentin odontogenic cell tubules was observed under a fluorescence microscope. As a result, a case where the transfer was sufficiently performed is judged as positive (+), and a case where the transfer was insufficient or not carried out at all is judged as negative (-). Table 1 below shows the results of a comparison of the effects of the dental fluid transport promoting peptide of the present invention extracted from rat parotid glands and salivary gland hormones in promoting fluid transport to the teeth. [Table] The dental body fluid transport promoting substance of the present invention is produced using rat salivary glands, such as parotid glands, as a raw material, by the following steps: (a) acidifying the aqueous extract of rat salivary glands to a pH of 4.5 to 5.5; (b) passing the resulting supernatant through a molecular sieve until the molecular weight is approximately
After removing more than 30,000 components, a step of passing through a molecular sieve again to collect a fraction with a molecular weight of 1,000 to 3,500; and (c) subjecting the fraction to cation exchange, two-dimensional paper chromatography, and high-pressure electrophoresis. It can be produced through a step of collecting a fraction with a molecular weight of about 2,200. Water extraction of rat salivary gland bodies can be carried out by methods known per se. For example, a new gland body taken from a rat's salivary gland is finely chopped, about 10 times the volume of chilled acetone is added, the gland body is defatted by stirring for 1 hour while cooling, and then air-dried. and vacuum drying to obtain an acetone dry powder. Add approximately 10 times the volume of water to this dry acetone powder, and adjust the pH to around neutrality (PH6.5~
7.5), preferably after adjusting the pH to 7.0, perform extraction while stirring. Stirring is generally advantageously carried out under cooling, preferably at 0 to 5°C, with the addition of an appropriate preservative (eg toluene), for several hours, usually 2 to 3 hours. This stirred suspension is subjected to centrifugation treatment (for example, at 10,000 rpm for 20 minutes) to separate the aqueous extract. On the other hand, the residue can be discarded as is, or if necessary, the same extraction operation as described above may be repeated as many times as desired (usually one or two more times). The pH of the aqueous extract from which the residue has been removed is adjusted to 4.5 to 5.5, preferably 5.0, with an inorganic acid such as hydrochloric acid, resulting in precipitation, which is then cooled (preferably in a refrigerator; kept at about 0 to 0.5°C). It is desirable to allow the mixture to stand for several hours to one day to further complete the precipitation. Centrifuge the precipitate (e.g. 10,000 rpm for 15 minutes)
Then, separate and collect the supernatant. The collected supernatant liquid is concentrated to an appropriate amount, for example, about 1/10th of its volume, by means such as vacuum concentration, if necessary, and then passed through a flat membrane such as an ultrafiltration membrane or ultrafilter, or a hollow fiber. Subject to molecular sieve operation using a membrane. When using an ultrafiltration membrane or an ultrafiltration fiber, the fractionation operation is performed with a molecular weight cut-off of 5000~
It can be carried out by ultrafiltration using ultrafiltration membranes or ultrafiltration fibers of 50,000, preferably about 30,000. Examples of ultrafiltration membranes include:
Diaflo® PM-30 (retention limit: 30000,
Amicon) etc. can be used, and
Examples of ultraviolet fibers include Hollow
Fiber HIP30 (retention limit: 30000, manufactured by Amicon)
etc. can be used. When using an ultrafiltration membrane for separation, the supernatant obtained above is loaded with a Diaflo PM-30, for example.
Place in a 202 type stirring cell (manufactured by Amicon),
It can be fractionated by filtering under pressure with nitrogen gas of Kg/cm ³ . In addition, when using an ultrafiltration fiber, the supernatant obtained above can be circulated at high speed through a hollow fiber loaded in a DC4 type ultrafiltration device (manufactured by Amicon). Fractions similar to those obtained using membranes can be obtained. In this way, it is possible to obtain an active ingredient-containing liquid from which components having a molecular weight cut-off of about 30,000 or more have been removed. The active ingredient-containing fraction with a molecular weight cut-off of approximately 30,000 or less obtained in this way is appropriately concentrated under reduced pressure and treated with Sephadex.
After desalting using a (registered trademark) G-10 column, the mixture is concentrated again under reduced pressure. This concentrate is then mixed with water, saline buffer,
Organic solvents and mixtures thereof as eluents
Fractionation is performed using Sephadex (registered trademark) LH-20 column chromatography, and active fractions with a molecular weight cutoff of 1000 to 3500 are collected. The active ingredient-containing fraction thus obtained is adsorbed onto a cation exchange resin, and then the active portion is separated and eluted. Examples of cation exchange resins that can be used here include styrene-divinylbenzene-based strongly acidic cation exchange resins, such as Hitachi Custom Ion Exchange Resin 2611, Bio Rad
Examples include Aminex (registered trademark) A-9.
Adsorption of the above-mentioned active ingredient-containing fraction using a cation exchange resin can be carried out, for example, by cation-exchanging the active ingredient-containing fraction dissolved in a weakly acidic buffer, preferably a citrate buffer, a glycine buffer, or an acetate buffer. After adsorbing the target substance by contacting with the exchange resin, use the above buffer solution in which the ion concentration is increased by adding inorganic salts such as sodium chloride or potassium chloride, or a strong alkaline solution such as sodium hydroxide or sodium hydroxide. It can be purified by elution. The active eluate fraction obtained in this way is
Further purification can be achieved by desalting with a Sephadex (registered trademark) G-10 column and fractionation using two-dimensional paper chromatography/high-pressure electrophoresis. Two-dimensional paper chromatography/high-pressure electrophoresis uses paper as a support,
This is a method of separating peptides by first performing chromatography followed by high-pressure electrophoresis and developing a sample two-dimensionally on a sheet of paper, and is called the so-called fingerprint method. This method makes it possible to separate peptides with substantially the same molecular weight but different amino acid sequences from each other. Paper chromatography as the first dimension can be carried out according to a conventional method by adding the above active eluate to paper and developing it with a developing solvent for about 20 hours. For expansion, either the ascending method or the descending method can be used. Examples of solvents that can be used include water, alcohols such as methanol and ethanol, phenol, pyridine, ethyl acetate, acetone, diethyl ether, chloroform, and n-hexane, either singly or in combination. It is also possible to use a saturated aqueous solution prepared by mixing pyridine acetate with a solvent such as butanol that is immiscible with water. This development allows different substances to be separated from each other. Next, high-voltage electrophoresis for the second dimension is carried out in the usual manner by applying an electric field to the one-dimensionally developed paper in a direction perpendicular to the first-dimensional development. I can do it. As the buffer solution used for high-pressure electrophoresis, for example, a volatile buffer solution such as pyridine-acetic acid-water system, pH 4 to 7, etc. can be used, and since these buffer solutions can be removed by drying, they can be removed from the paper. Very convenient when extracting active substances. The extract obtained by this high-pressure electrophoresis treatment has a molecular weight cut-off of
500 ultrafiltration membrane, e.g. YC05 (manufactured by Amicon)
Gel filtration using Sephadex (registered trademark) G-10, etc., or tubes with a molecular weight cutoff of 1000, such as Spectrumpore (registered trademark) 7 (Spectrum
A fraction with a molecular weight of approximately 2200 is collected, desalted appropriately, and then freeze-dried to obtain the desired peptide with the activity of promoting tooth fluid transport. can be obtained. The tooth fluid transport promoting active peptide of the present invention thus obtained is useful for the prevention and treatment of dental caries. In other words, dental caries is caused by the formation of dental caries in dental plaque caused by the action of microorganisms.
Acid corrosion progresses due to retention of lactic acid, which is a metabolite of microorganisms, and caries progresses not only to the enamel layer but also to the dentin. There are a wide variety of factors involved in the development of dental caries, but even when the dentin is eventually caried, the ivory has the ability to protect itself by forming secondary dentin. At this time, the supply of nutritional sources is essential for the functional growth of dentin, and it is thought that substances that enhance tooth body fluid transport act to promote the formation of secondary dentin. To use the tooth fluid promoting active peptide according to the present invention as such an anti-caries agent or therapeutic agent, it can be added to a pharmaceutical preparation solution (distilled water for injection, physiological saline, phosphate buffer, glycine buffer, veronal buffer, etc.). An injection solution can be prepared by adding and dissolving the tooth fluid-promoting peptide according to the present invention.
Furthermore, in order to improve moldability, adjuvants such as sodium chloride, glycine, lactose, mannitrate, sorbitol, sucrose, hydrolyzed starch, and dextran can be added to this injection solution to form a freeze-dried preparation. Furthermore, the dental fluid transport-promoting peptide according to the present invention can be added to a commonly used toothpaste and used as an anti-caries agent and a therapeutic agent. Furthermore, this peptide can be expected to promote not only teeth but also hard tissues of the living body in general, especially ossification. Next, the present invention will be further explained by examples. Example 1 100 g of rat parotid gland was minced, cooled acetone (1) was added thereto, and the mixture was stirred for 1 hour under cooling and filtered. Excess acetone was removed by air drying and vacuum drying to obtain acetone dry powder. This acetone dry powder was added to water from Step 1, extracted with stirring for 2 hours, and then centrifuged at 10,000 rpm for 15 minutes to separate into precipitate and supernatant. 300 ml of water was added to the precipitate, extraction and centrifugation were repeated, and the resulting supernatant was combined with the previous one, and 1N hydrochloric acid was added to adjust the pH to 5.0. The precipitate was removed by centrifugation at 10,000 rpm for 15 minutes, and the supernatant was diluted with 1N sodium hydroxide to pH 7.0.
And so. Concentrate this solution under reduced pressure to 100ml,
Ultrafiltration membrane PM-30 with a molecular weight cutoff of 30,000 was used using a 202 type stirred cell filtration device (manufactured by Amicon).
(manufactured by Amicon), and the obtained filtrate was again concentrated under reduced pressure. This concentrated solution was equilibrated with 0.05M acetate buffer, pH 5.8 to Sephadex (registered trademark) G.
-10 column (1.5 x 90 cm) for desalting and elution. This eluate was concentrated under reduced pressure again and Sephadex was equilibrated with an ethanol-acetic acid-water (75:10:95) solution.
(Registered Trademark) LH-20 column (3.8 x 100 cm), and active fractions with a molecular weight cutoff of 1000 to 3500 were collected. The active fraction was then equilibrated with 0.2N sodium citrate buffer, PH3.25 using Hitachi Custom #2611.
After loading onto a column (0.9 x 55 cm), impurities were removed by sequentially washing with 0.2N sodium citrate buffer, PH4.25 and 1.2N sodium citrate buffer, PH5.28. Finally, the column was eluted with 0.2N sodium hydroxide and the active eluate fractions were collected. Next, this active elution fraction was desalted in the same manner as above.
After concentrating under reduced pressure, it was added to Toyo Paper No. 50 (42 x 45 cm) and n-butanol-pyridine-
20 using acetic acid-water (15:10:3:12) solution as a solvent.
Time unfolded. Next, pyridine-acetic acid-water (10:
After electrophoresis was performed for 90 minutes at 150 mA using the 0.4:90) solution as an electrode solution, the active portion on the paper was extracted. This active extract was desalted using an ultrafiltration membrane YC05 (manufactured by Amicon) with a molecular weight cutoff of 500 and then freeze-dried to obtain 0.45 mg of a peptide having the desired activity of promoting tooth fluid transport. This peptide showed a single band in polyacrylamide gel disc electrophoresis, the molecular weight measured by gel filtration using Sephadex (registered trademark) G-25 was approximately 2200, and the N-terminal amino acid sequence determined by Edman degradation was Gly- Val-Ile-Ala-Trp-, and the C-terminal amino acid sequence determined by carboxypeptidase Y is -Arg-Lys-Asx-Ser-Thr-
It was Ala-Gly. In addition, using 6N hydrochloric acid at 110℃
The amino acid analysis values obtained from the results of 24-hour hydrolysis were as shown below. Amino acid composition (number of residues per molecule): Asx(2), Thr(1), Ser(1), Glx(3), Pro(1), Gly
(3), Ala(2), Val(1), Ile(1), Leu(1), Lys(1), His
(1), Arg(1), and Trp(1) The purified preparations showed activity in promoting body fluid transport to the teeth at 30 ng/Kgi.υ. in rats.

[Brief explanation of drawings]

Figure (1) shows the molecular weight of the peptide of the present invention measured using a Sephadex (registered trademark) G-25 column (0.9 x 156 cm), and the vertical axis is
The amount of eluate of this substance when using a Sephadex G-25 column (0.9 x 156 cm), and the horizontal axis represents the molecular weight.

Claims (1)

[Claims] 1. The following amino acid composition: Asx(2), Thr(1), Ser(1), Glx(3), Pro(1), Gly
(3), Ala(2), Val(1), Ile(1), Leu(1), Lys(1), His
(1), Arg(1), Trp(1) where the number in brackets represents the number of amino acid residues per molecule, and the N-terminal amino acid sequence is Gly-Val-Ile-
Ala-Trp- and the C-terminal amino acid sequence is -
Arg−Lys−Asx−Ser−Thr−Ala−Gly,
A peptide having a molecular weight of approximately 2200 as measured by a gel filtration method, and a maximum absorption wavelength (λ _nax ) of ultraviolet absorption spectrum of 278 nm, which has an activity of promoting tooth fluid transport. 2 (a) Aqueous extract of rat salivary glands at pH4.5~
Step 5.5 of acidifying and removing the resulting precipitate; (b) passing the resulting supernatant through a molecular sieve until the molecular weight is approximately
After removing more than 30,000 components, a step of passing through a molecular sieve again to collect a fraction with a molecular weight of 1,000 to 3,500; and (c) subjecting the fraction to cation exchange, two-dimensional paper chromatography, and high-pressure electrophoresis. A method for producing a peptide having an activity of promoting tooth fluid transport, comprising the step of collecting a fraction having a molecular weight of about 2200.