JP2631294B2 - Epidermal cell differentiation inhibitor - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an epidermal cell differentiation inhibitory factor.

2. Description of the Related Art Staphylococcus aureus has a strong pathogenicity, once called pathogenic staphylococcus, and even though various antibiotics are being developed, it is a clinically important pathogen as a causative agent of various purulent diseases and the like. It is one of. Most of the skin infections caused by the Staphylococcus aureus are purulent diseases, and the purulent skin lesions are characteristic of the skin such as desquamation, erosion, blisters, as well as the so-called four major signs of inflammation. Various microscopic symptoms are shown in a complex manner, and the diversity is understood to be due to the diversity of the virulence factors produced by S. aureus. As for the above Staphylococcus aureus, it is known that the same strain produces more than 30 extracellular products, and that one extracellular product has various biological activities [Wadstro.
m, T., Ann. NY Acad. Sci., 236 , 343-361 (1974); Rogols
ky, M., Microbiol. Rev., 43 , 320-360 (1979)] make it difficult to elucidate the role of extracellular products in staphylococcal infection. Moreover, most of the extracellular products have no clear relationship with the above-mentioned skin lesions, and, of course, have not been isolated or purified.

On the other hand, skin infections caused by Staphylococcus aureus are thought to undergo a pathogenic process involving attachment, invasion and proliferation of staphylococci to the skin, and the first invaded tissue is the epidermis. The epidermis is composed of several layers of cells in a series of differentiation processes.Maintaining tissue differentiation by maintaining this differentiation ability regulates regeneration, but Staphylococcus aureus infected to some damaged epidermis, It is thought that the pathogenic factor is produced at that location, and as a result, the disorder of the tissue structure of the epidermis and the decline of the physiological function appear.

Problems to be Solved by the Invention It is an object of the present invention to provide the above-mentioned active factor as a new substance having an activity of inhibiting the differentiation of epidermal cells in vitro.

The present inventors have been examining the influence of the extracellular product produced by the above-mentioned Staphylococcus aureus on the differentiation of epidermal cells. In the process, the method of Ispa et al. [Cell, 19 , 245] -254 (1980)], using mouse epidermal cells cultured in vitro to inhibit the in vitro differentiation of the skin cells into extracellular products of Staphylococcus aureus isolated from skin lesions. After confirming the presence of the factor, subsequent studies have succeeded in isolating the active factor, and have now completed the present invention.

Means for Solving the Problems According to the present invention, it has an N-terminal 48 amino acid sequence represented by the following formula (1) and has a molecular weight of about 3 determined by SDS-PAGE.
At 2000 daltons, the isoelectric point is about 8.2, stable in the range of pH 5-10, decreased activity in the more acidic region, shows pH stability completely deactivated at pH 3, and 60 ° C, 30 minutes Epidermal cell differentiation inhibitor (EDIN), which exhibits thermal stability that is completely inactivated by heat treatment
nhibitor) is provided.

Formula (1): Ala-Asp-Val-Lys-Asn-Phe-Thr-Asp-Leu-4sp-Glu-Ala-Thr-Lys-Trp-Gly-Asn-Lys-Leu-Ile-Lys-Gln-Ala -Lys-Try-Ser-Ser-Asp-Asp-Lys-Ile-Ala-Leu-Tyr-Glu-Tyr-Thr-Lys-Asp-Ser-Lys-Ile-Asn-Gly-Asp-Leu-Arg -The physiological activity of the epidermal cell differentiation inhibitory factor (EDIN) of the present invention, that is, the activity of inhibiting epidermal cell differentiation, can be determined by the method described in detail in Examples below.

The EDIN of the present invention comprises Staphylococcus aur
eus) and is specified in that it has the above amino acid sequence and molecular weight. Heretofore, there has been only a small amount of exfoliative toxin (ET: Exfoliative Toxin) as the activity that has been clarified as an extracellular product derived from Staphylococcus aureus, but the reported molecular weight of ET is About 24
000 daltons (Kondo, I., Sakurai, S. and Sarai,
Y., Infect. Immun., 8 , 156-164 (1973)], EDIN of the present invention.
Is clearly distinguished from the ET in its molecular weight, amino acid composition and amino acid sequence.
Has no activity, i.e., no activity to induce epidermis detachment by intradermal administration to neonatal mice.

The EDIN of the present invention is also specified by having the following various physicochemical properties.

1) Unity: Pentax PE, which was obtained by dialyzing 6 μg of EDIN of the present invention against 10 mM phosphate buffer (pH 6.8) and preliminarily equilibrating with the same buffer
As a result of linear-gradient elution to a C101 (manufactured by Asahi Optical Co., Ltd.) column up to 0.5 M phosphate buffer (pH 6.8), the activity is obtained as a single peak.

Further, the EDIN of the present invention shows the same migration profile by SDS-PAGE in the presence or absence of the reducing agent β-mercaptoethanol, and migrates as a single band by Coomassie brilliant blue (CBB) staining. Thus, it is identified as a single protein.

2) Isoelectric point by chromatofocusing: As a result of chromatofocusing performed using a PBE94 (Pharmacia Fine Chemicals) column at a pH in the range of 6.0 to 9.0, this substance was eluted around pH 8.2, and basic protein was eluted. (PI = about 8.2).

3) pH stability and thermal stability: The EDIN of the present invention is stable in the pH range of 5-10. The activity is reduced in the more acidic region, and completely inactivated at pH 3. This factor is completely inactivated by heat treatment at 60 ° C. for 30 minutes.

 The details of these are as shown in the examples below.

The EDIN of the present invention is useful in elucidating the physiological functions of epidermal cells, the mechanism of bacterial infection, etc., and has various skin infectious diseases, since it has an epidermal cell differentiation inhibitory activity specific to it as described above. Useful for treatment.

 Hereinafter, the method for producing the EDIN of the present invention will be described in detail.

The EDIN of the present invention can be used, for example, for staphylococcal burn-like syndrome (St.
aphylococcal Scalded Skin Syndrome (SSSS) can be obtained by culturing staphylococci isolated from the skin of a patient. Representative examples of the above-mentioned microorganisms of origin include Staphylococcus aureus E-1 (Staphylococcus aureus E-1).
-1) A strain can be exemplified. The strain was isolated and identified from the skin of a 1-year-old boy diagnosed with SSSS at the Hiroshima Prefectural Hospital Internal Medicine, and was distributed by Dr. Kuwahara of the Hospital Internal Medicine. The strain is unacceptable for deposit at the Japan Institute of Fine Arts and is stored in a state that can be distributed by the present inventors.

The culture of the above-mentioned source microorganism can be performed according to various methods in the same manner as the usual culture of this kind of microorganism. For example, the strain is Trypticase-soybroth.
Broth) Slope medium (Becton Dickinson and Co., MD, USA)
Can be subcultured.

The growth is, for example, TY medium (10 g of yeast extract /
After culturing at 37 ° C. for 24 hours in a tryptic case 17 g /, NaCl ₅ g /, K ₂ HPO ₄ 2.5 g /) in the presence of 10% CO ₂ ,
It can be carried out by shaking culture at 24 ° C for 24 hours.

The culture solution obtained by the above culture can be subjected to operations such as centrifugation and membrane filtration according to a conventional method to obtain a desired culture supernatant containing EDIN.

The production of a crude sample containing EDIN from the culture supernatant thus obtained can be carried out by appropriately combining conventionally known various operations. Examples of the above operations include treatment with a protein precipitant such as ammonium sulfate precipitation, centrifugation, dialysis, ultrafiltration, and concentration.

The purification of the crude product can also be performed by various known methods, for example, gel permeation, liquid chromatography, electrophoresis, affinity chromatography, chromatofocusing, reversed-phase high-performance liquid chromatography, or a combination thereof. The above various chromatography operations are described in more detail, for example, by Sephadex G-75.
Gel per column chromatography using (Pharmacia Fine Chemicals), PBE94 (manufactured by Dojo)
Cation exchange column chromatography using TSK gel SP-Toyopearl 650M (manufactured by Tosoh), TSK gel butyl toyopearl 650
Hydrophobic binding column chromatography using M (manufactured by Dojo), high-speed ion exchange chromatography using TSK gel SP-5PW (manufactured by Dojo), Pentax (PENTAX)
High-performance hydroxyapatite chromatography using PEC101 (manufactured by Asahi Optical Co., Ltd.) or the like, high-speed gel permeation column chromatography using TSK gel G3000SW (manufactured by Tosoh) or the like can be used. SDS-PAGE was performed according to the method of Laemmli [Laemuli, UK, Na
, 227 , 680-685 (1970)].

Thus, the EDIN of the present invention can be isolated and purified.

The EDIN can be effectively used for the various uses described above by utilizing its unique physiological activity.

Examples Hereinafter, examples will be given to explain the present invention in more detail. The physiological activity of EDIN was measured and evaluated by the following method.

[Measurement of epidermal cell differentiation inhibitory activity (1) Eagle MEM containing no preparation Ca ²⁺ medium for epidermal cell culture (Eagle's Minimum Essen
tial Medium (Joklik-modified)) to penicillin G75U /
ml and streptomycin (Grand Island Biological
Co., NY, USA), 50 μg / ml, and Chelex-100 Resin (Chelex-100 Resin, Bio Rad Lab. Richmond, CA, US).
The fetal calf serum (FCS) treated in A) was added to 10%, and the Ca concentration was adjusted to 0.05 mM with a 0.3 M CaCl ₂ solution to obtain a low-concentration Ca medium (LCM).

A high concentration Ca medium (HCM) having a Ca concentration adjusted to 1.0 mM was obtained in the same manner as described above. The Ca concentration in each of the above media was measured using an atomic absorption spectrometer (Hitachi, Model 207).

(2) Culture of epidermal cells The back skin was collected from ICR-based CD-1 neonatal mice,
% Trypsin (Osaka Univ. Micro Laboratory) at 4 ° C for 24 hours, then extract only the epidermis under a stereoscopic microscope (Olympus KKXTr type), and further treat it with 0.25% trypsin at 37 ° C for 1 hour. Cells were isolated. Dye exclusion test using trypan blue (Schrek, R., Arch.Pathol., 37 , 319-323).
(1944)], and 1 × 10 ⁶ cells were placed in a 35 mm plastic Petri dish (Becton Dickinson and Co., USA).
Cell / Well, Cell Well
ls, 24-well flat bottom, Corning, NY, USA) 3 × 10 ⁵
Cells were inoculated so as to give cells / well, and the cells were
The cells were cultured for 5 days in the presence of 5% CO _{2 and} used for the experiment.

(3) Observation of morphological differentiation of epidermal cells Epidermal cells cultured in LCM were transferred to HCM, and their morphological changes were observed under an inverted microscope (Nikon MTD, manufactured by Nippon Kogaku) over time for 48 hours.

(4) Quantification of differentiation of epidermal cells The quantification of differentiation of epidermal cells was performed by the method of Peel et al. [Peehl, D .;
M.and Ham, RG, Growth and differentiation of human
keratinocytes without a feeder or conditioned med
In Vitro, 16 , 516-525 (1980)].

That is, epidermal cells cultured in LCM were transferred to HCM and cultured for 48 hours, and then treated with 0.25% trypsin + 0.02% EDTA to obtain a cell suspension. A part of the cell suspension was collected and microscopically analyzed using a hemocytometer. After counting the number of cells under Nikon OPTIPHOT, Nippon Kogaku, the cells were treated with 2% SDS-20 mM dithiothreitol at 90 ° C. for 10 minutes to form an insoluble envelope (Cornified Envelop).
e) was extracted. After the obtained insoluble envelope number was calculated using a hemocytometer, the ratio of the insoluble envelope in all cells was calculated, and this was defined as% cornified cells (% Cornified Cells).

(5) Detection and Quantification of Epidermal Cell Differentiation Inhibitory Activity When transferring epidermal cells cultured in LCM using a 35 mm plastic petri dish to HCM, a test sample was added, and the methods described in (3) and (4) above were performed. The differentiation inhibitory activity was detected and quantified.

As a simple assay, when epidermal cells cultured using cell wells are transferred to HCM, phosphate buffered saline (PB
Add the test sample twice-diluted by S) and add (3)
The differentiation inhibitory activity was detected by the method described above.

Example 1 (1) Culture of bacteria and preparation of culture supernatant Trypticase-soy broth
h) Staphylococcus aureus E-1 subcultured on a slant medium (Becton Dickinson and Co., MD, USA)
One platinum loop of (Staphylococcus aureus E-1) was added to a TY medium (yeast extract 10 g /, trypticase 17 g /, Na
Cl5g /, K ₂ was inoculated into HPO ₄ 2.5g /) 30ml, after 37 ° C. for 24 hours under static culture, transferred to the same medium 3, 37 ° C. in ₂ the presence 10% CO
The cells were cultured under shaking for 24 hours. The culture solution was centrifuged at 5000 × g for 20 minutes, and the obtained supernatant was filtered with a filter having a pore size of 0.22 μm (Millipore Co., Mass, USA).
To give a culture supernatant preparation.

(2) Preparation of crude preparation by salting out and concentration of ammonium sulfate Solid ammonium sulfate was added to the culture supernatant preparation obtained in the above (1) so as to be 60% saturated, and the mixture was allowed to stand at 4 ° C. for 24 hours. g
For 30 minutes to obtain a precipitate. This was dialyzed against a 50 mM phosphate buffer (pH 7.0) to obtain a crude sample.

Next, the crude sample was ultrafiltered using a Pellicon Lab Cassette System (Millipore Co., USA) to give a molecular weight of 100
More than 00 fractions were collected and concentrated about 50-fold to prepare a concentrated culture solution (CCF).

As a result of examining the epidermal cell differentiation inhibitory activity of the above crude preparation, the epidermal cells cultured in LCM show a beautiful paving stone-like arrangement peculiar to epithelial cells, and the individual cells are small and relatively uniform. Clear cell gaps were observed. When the cells were transferred to HCM to induce differentiation, the cells underwent significant morphological changes. That is, the cells gradually expanded and desmosomes were formed between the cells. The cells eventually undergo vertical stratification, and cells with corneal membranes (keratinocytes; Corni
fied Cell) began to increase. 48 hours after induction of differentiation,
Individual cells significantly increased in size, and a large number of keratinocytes were observed.

When an appropriate amount (2.5-20 μg protein / ml) of a crude preparation obtained from the ammonium sulfate salting-out fraction is added to induce differentiation by transferring to HCM, the morphological differentiation induced by Ca is completely suppressed. Was. That is, although each cell was slightly larger than the control, a clear cell gap was observed. The appearance of keratinocytes was hardly observed.

When the cells were further cultured in HCM from which the crude preparation had been removed, each cell differentiated in the same manner as in the case where the crude preparation was not added. When the cells cultured for 48 hours in the HCM with or without the crude preparation were transferred to the non-added LCM, the non-added cells no longer
It was not possible to return to the original pavement-like arrangement and the growth potential was not restored. However, the added cells began to undergo morphological changes several minutes after being transferred to the LCM, and returned to the original cobblestone-like arrangement two hours later, and a division image of the cells was observed 24 hours later.

From the above, it was shown that the differentiation inhibition by the crude preparation was reversible.

Next, in order to quantify the differentiation inhibitory activity, the percentage of differentiated cells was determined in accordance with (4) of the above [Measurement of epidermal cell differentiation field inhibitory activity].
When the (% cornified cells) were calculated, the percentage of differentiated cells in the crude sample-added cells was remarkably lower than that in the non-added cells, which was almost the same as that of the epidermal cells cultured in LCM.

(3-1) Purification by chromatography To the culture supernatant sample obtained in the above (1), solid ammonium sulfate was added so as to be 60% saturated, and the mixture was allowed to stand at 4 ° C for 24 hours.
For 30 minutes to obtain a precipitate. Each of the following chromatographic operations was carried out on a crude sample obtained by dialyzing this against 10 mM Tris-HCl buffer (pH 7.5).

In addition, the protein quantification in each operation was performed using a bovine serum albumin (BSA) as a standard and a dye fixation method [dye fixation me
thod, Bio Rab Lab., Bradford, MM, Anal. Biochem., 72 , 2
48-254 (1976)].

(I) Sephadex G-75 (Pharmacia Fine Chemicals Co., Ltd., particle diameter 40-120 μm, 650 × 26 mm, flow rate 15
The column was subjected to gel filtration using 10 mM Tris-HCl buffer (pH 7.5).

Each fraction obtained by the gel filtration was measured using the simple assay method according to (5) of [Measurement of Epidermal Cell Differentiation Inhibiting Activity], and the activity was determined by comparing the elution position of the standard protein with the molecular weight of 12500- Elution was found to be in the range of 43,000 daltons.

For this reason, in the subsequent purification operation, a concentrate (CCF) obtained by concentrating the fraction having a molecular weight of 10,000 or more due to the above-mentioned ultrafiltration was used as a starting material.

(Ii) CCF dialyzed beforehand against 10 mM phosphate buffer (pH 5.0) is added to a TSK gel SP-Toyopearl 650M (Tosoh, flow rate 2 ml / min) column equilibrated with the same buffer. Then, when the non-adsorbed fraction was washed away with the same buffer in an amount several times the column volume, the activity was adsorbed on the column, and no activity was observed in the fraction passing therethrough. Next, a linear gradient elution of NaCl was performed to obtain 120-210 mM NaCl.
Elution of activity was observed in the vicinity, and this was collected.

(Iii) The above active fraction was added to 10 mM Tris-HCl buffer + 0.1 M N
TS dialyzed against aCl (pH 7.0) and equilibrated with the same buffer
K-gel butyl Toyopearl 650M (Tosoh, flow rate 2 ml
/ Min) applied to the column. The non-adsorbed fraction is washed away with several times the column volume of the same buffer, and then 10 mM Tris-HCl buffer + 1 M NaCl (pH 7.0) to 10 mM phosphate buffer + 30%
Elution with linear gradient up to ethylene glycol (pH 7.0) was carried out, and the active fraction adsorbed on the column was reduced to 20.
It was eluted and collected around -30% ethylene glycol.

(Iv) The above active fraction was treated with 10 mM phosphate buffer + 0.1 M Na ₂ SO ₄
(PH 6.8), and then ultra-concentrated using Mollcut (Millipore Co.) and equilibrated with the same buffer.
Using a column (manufactured by Tosoh Corporation), high performance liquid chromatography gel permeation (flow rate: 1 ml / min) was performed to obtain activity as a single peak.

(3-2) Purification by chromatography (i) A concentrate (CCF) of a fraction having a molecular weight of 10,000 or more due to the above-mentioned ultrafiltration was used as a starting material, and was previously used as a 50 mM phosphate buffer (pH 5.0). ), Added to a TSK gel SP-5PW (Tosoh, flow rate 1 ml / min) column equilibrated with the same buffer, and the non-adsorbed fraction was diluted with several times the column volume of the same buffer. Upon washing out, the activity was adsorbed on the column, and no activity was observed in the flow-through fraction. Next, a linear gradient elution was performed from 50 mM phosphate buffer (pH 5.0) to 50 mM phosphate buffer + 0.5 M NaCl (pH 5.0), and the active fraction eluted around 170-230 mM NaCl. Collected.

(Ii) The active fraction was dialyzed against 25 mM phosphate buffer (pH 6.8), and PENTAX PEC101 (Asahi Optical Co., Ltd., flow rate 1 ml /
Min) added to the column. The non-adsorbed fraction was washed away with several times the column volume of the same buffer, followed by linear gradient elution to 0.5 M phosphate buffer (pH 6.8).
Active fractions eluted around 0.25M were collected.

(Iii) The above active fraction was added to 10 mM phosphate buffer + 0.1 M Na ₂ SO
₄ After dialysis against (pH 6.8), mol cut (Millipore C
o.), ultra-high concentration was performed, and high performance liquid chromatography gel permeation (flow rate 1 ml / min) was performed using a TSKG3000SW (manufactured by Tosoh) column equilibrated with the same buffer. As a result, the activity was obtained as a single peak.

FIG. 1 shows the results of the high performance gel permeation chromatography. In the figure, the horizontal axis is the fraction N
o., the vertical axis is the absorbance at 280 nm, Indicates an active fraction.

Table 1 below shows a summary of each of the above purification steps.

The activity unit (U) in Table 1 is represented by the reciprocal of the amount of protein required for suppressing morphological differentiation and suppressing keratinous membrane formation within 48 hours.

(4) SDS-PAGE electrophoresis 4. Sodium dodecyl sulfate (SDS) -polyacrylamide gel (PAGE) was prepared according to the method of Laemmli [Laemuli, UK, Nat.
ure, 227 , 680-685 (1970) with some modifications.

As the polyacrylamide gel, 3% was used for concentration and 10% was used for separation. Staining was performed using Coomassie Brilliant Bull (CBB).

As a result of the SDS-PAGE performed on the active fraction (EDIN of the present invention) obtained in the final step of the purification step,
N migrated slightly above the carbonic anhydrase between the molecular weight markers carbonic anhydrase (molecular weight 31k) and ovalbumin (molecular weight 45k), confirming its molecular weight to be approximately 32,000 daltons.

In addition, the above EDIN in the presence and absence of reducing agent β-mercaptoethanol, as a result of repeating the SDS-PAGE,
Both electrophoresis profiles were the same and migrated as a single band with CBB staining. From this, the EDIN of the present invention was identified as a single protein having no subunit structure.

(5) Unicity of EDIN 6 μg of the final sample obtained in the above purification step (3-2) (iii) was dialyzed against 10 mM phosphate buffer (pH 6.8), and the same buffer was used in advance. Pentax equilibrated with liquid
As a result of performing a linear gradient elution up to 0.5 M phosphate buffer (pH 6.8) using a PEC101 column, EDIN activity was eluted as a single peak.

FIG. 2 shows the results of the high-speed hydroxyapatite chromatography in the same manner as in FIG.

(6) Chromatofocusing Using the PBE94 (Pharmacia Fine Chemicals) column equilibrated with 25 mM ethanolamine-acetic acid (pH 9.5) for the partially purified sample obtained in the above purification step (3-1) (i), Polybuffer 96-acetic acid (pH 6.0)
As a result of performing chromatofocusing in the range of pH 9.0-6.0, the above activity was eluted around pH 8.2.

(7) Epidermal cell differentiation inhibitory activity of EDIN The EDIN obtained in the final step (3-2) (iii) of the purification step was used to inhibit epidermal cell morphological differentiation and Cornif
The minimum amount required for suppressing the appearance of ied Cells was determined according to the above-mentioned activity measurement method.

As a result, the minimum amount was about 0.018 μg protein / ml.

The activity of the above EDIN was reversible as in the case of the crude sample.

(8) pH stability The partially purified sample obtained in the purification step (3-2) (i) was converted to a 50 mM buffer solution of pH 2.0-11.0 [citrate-phosphate buffer: pH2-6, phosphoric acid Salt buffer: pH 7.0, glycine-NaO
H: pH8-11], after dialysis at 4 ° C for 24 hours, at 4 ° C in PBS,
The residual activity was examined using the dialyzed material for 2 hours.

As a result, the EDIN activity was stable in the pH range of 5-10. A decrease in activity was observed in the more acidic region, and the activity was completely inactivated at pH 3.

(9) Thermal stability The activity of the final purified sample is determined using DRY THERMOUNIT TAH-1 (Ocean), which has been heat-treated (40-100 ° C, 30 minutes) and quenched in an ice bath. Was performed.

As a result, the activity of EDIN was completely inactivated by the heat treatment at 60 ° C. for 30 minutes.

(10) Amino acid composition of EDIN About 2 μg of the purified EDIN solution was placed in a hard glass sample tube (manufactured by Nidec Rika Glass Co., Ltd., 6 × 50 mm), placed in a hydrolysis vial (manufactured by Pierce), dried under vacuum, 6N−
Hydrochloric acid (containing 1% phenol) (200 μl) was placed in the reaction vial, sealed under reduced pressure, and hydrolyzed at 130 ° C. for 4 hours.

After the reaction, 0.02N-hydrochloric acid (400 µ) was added to the sample tube, transferred to a sample tube for amino acid analysis, and 250 µ of the sample was automatically injected into a Hitachi high-speed amino acid analyzer (manufactured by Hitachi, Ltd.) to analyze the amino acid composition. In addition, as a detection method, an OPA (orthophthalaldehyde) method was used. This method does not detect proline and cystine.

Each amino acid separated and identified is quantified by a calibration curve prepared using three standard concentrations of standard amino acids (50 picomoles, 250 picomoles, and 1250 picomoles each), and contains four phenylalanines so that the molecular weight is about 32,000. The composition ratio was calculated.

 The results obtained are shown in Table 2 below.

(11) N-terminal amino acid sequence of EDIN Using a purified EDIN solution equivalent to about 500 picomoles, a gas phase protein sequencer (manufactured by Applied Biosystems, Inc., type 470A) was used to obtain up to 48 amino acid residues from the N-terminal of EDIN. The sequence was determined. PTH obtained in each reaction cycle
-The amino acid solution was dried by vacuum centrifugation, dissolved in 33% acetonitrile aqueous solution, and separated and identified by reversed-phase high performance liquid chromatography for PTH-amino acid analysis (manufactured by Beckman).

The amino acid sequence determined as described above was as follows.

Ala-Asp-Val-Lys-Asn-Phe-Thr-Asp-Leu-Asp-Glu-Ala-Thr-Lys-Trp-Gly-Asn-Lys-Leu-Ile-Lys-Gln-Ala-Lys-Tyr- Ser-Ser-Asp-Asp-Lys-Ile-Ala-Leu-Tyr-Glu-Tyr-Thr-Lys-Asp-Ser-Ser-Lys-Ile-Asn-Gly-Asp-Leu-Arg-

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing the results of high-performance gel permeation chromatography. FIG. 2 is a view showing the results of high-speed hydroxyapatite chromatography.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical indication (C12N 1/20 C12R 1: 445) (C12P 21/02 C12R 1: 445)

Claims (1)

(57) [Claims] (1) It has an amino acid sequence of 48 N-terminals represented by the following formula (1) and has a molecular weight of about 32000 determined by SDS-PAGE.
Dalton, isoelectric point of about 8.2, stable in the pH range of 5-10,
Epidermal cell differentiation characterized by a decrease in activity in a more acidic region, a pH stability that completely inactivates at pH 3, and a thermostability that completely inactivates at 60 ° C for 30 minutes. Inhibitor. Formula (1): Ala-Asp-Val-Lys-Asn-Phe-Thr-Asp-Leu-Asp-Glu-Ala-Thr-Lys-Trp-Gly-Asn-Lys-Leu-Ile-Lys-Gln-Ala -Lys-Thr-Ser-Ser-Asp-Asp-Lys-Ile-Ala-Leu-Tyr-Glu-Tyr-Thr-Lys-Asp-Ser-Ser-Lys-Ile-Asn-Gly-Asp-Leu-Arg