JPH0730114B2 - Inhibin - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to INHIBIN. Inhibin is a sex hormone that can be used as a contraceptive or in livestock production.

[Conventional technology]

Inhibin is Mc Cullagh (Science, 76 , 19-20, (193
2)) was found as a water-soluble extract of testis that suppresses the appearance of castrated cells found in the pituitary gland of castrated animals. As ovarian inhibin, De Jong et al. (Nature, 263 ,
71-72, (1976), found in bovine follicular fluid, and then confirmed that a large amount of inhibin activity is present in follicular fluid of rats, pigs, monkeys, sheep, horses, humans, etc. .

At present, inhibin is a water-soluble substance that exists in both sexes, and is considered to be a substance that specifically suppresses the synthesis and secretion of FSH (follicle-stimulating hormone) from the pituitary gland. Inhibin is also involved in phenomena such as follicle development, maturation, and ovulation, and plays an important role in reproductive physiology in the hypothalamic pituitary gland system.

Of the ovarian inhibins, pigs were described by William et al. (Partial Purification of Porcine Follcular Fluid
Gonadostatin, Intraovarian Control Mechanisms, Advan
ces Experimental Medicine and Biology, vol.147, Plen
Um Press, New York, 99-116, 1982) found that the fraction with a molecular weight of 26,000 in the eluate of gel filtration chromatography with "Sephacryl S-300" of porcine follicular fluid had inhibin activity. However, in this document, inhibin has not been purified, and therefore it is not clarified whether inhibin activity is expressed by a single substance or whether there are multiple substances having inhibin activity. In fact, according to our research, at least three kinds of substances having inhibin activity have been found in porcine follicular fluid, even if it has a molecular weight of around 30,000.

[Problems to be Solved by the Present Invention]

As mentioned above, since inhibin has not been isolated as a single substance in the past, its application is not possible despite its great potential to be used as a contraceptive due to its unique physiological activity. It was Therefore, an object of the present invention is to obtain inhibin as a single substance.

[Means for solving problems]

The present inventors succeeded in separating and purifying inhibin as a single substance from porcine follicular fluid and clarifying its physical and chemical properties.

The inhibin named 32K inhibin-II of the present invention has the following properties.

(A) Molecular weight: 32 kd (SDS-gel electrophoresis method and high performance liquid chromatography method) (b) Reduction produces polypeptides with molecular weights of 20 kd and 13 kd (SDS-gel electrophoresis method), which loses activity.

(C) Adsorbed on a concanavalin A sepharose column and eluted with α-methyl mannoside.

(D) Isoelectric point: around p16 (e) Inactivated by protease treatment, but not by neuraminidase treatment and endoglycosidase H treatment.

(F) N-terminal amino acid sequence: Polypeptide having a molecular weight of 20 × 10 ³ : Ser Thr Polypeptide having a molecular weight of 13 × 10 ³ : Gly Leu (g) Exists in porcine follicular fluid and suppresses elevation of follicle-stimulating hormone .

The inhibin of the present invention can be obtained, for example, by the following method.

Collect pig ovaries and collect follicular fluid by suction or the like. Inhibin is specifically adsorbed on "Matrix Gel Red A" (Amicon) (Molecular and Cell).
ular Endocrinology, 21 , 109-117, (1981)), first,
Maffinity chromatography on this gel is performed.

Then, according to the research conducted by the present inventors, chromatography by "phenyl sepharose", "Sephacryl S-200", "DEAE Sephacryl CL6B" and finally purification by reverse phase high performance liquid chromatography were the most suitable for the purification of this substance. Are suitable. 8M urea is the most suitable eluent in these chromatography.

The activity of inhibin can be measured as follows.

(1) Rat pituitary anterior pituitary cell culture method The rat anterior pituitary is dispersed with trypsin and biocase and placed in a plastic plate so that the cell concentration is 50,000 / 0.1 ml / well. Add 0.1 ml of inhibin sample to each well and keep at 37 ° C in air containing 10% carbon dioxide gas for 72 hours. The medium is DMEM, 5% human umbilical cord serum, 2.5% FC
Use S, 5% horse serum added. The activity is expressed by the degree of inhibition of FSH release measured by radioimmunoassay for the FSH content released in the medium. 32K inhibin
II has an activity of ED ₅₀ = 1.2 ng / ml-medium.

(2) In vivo test method Male rats aged 35 days are castrated, and blood FSH rising from 6 to 24 hours after castration is measured by radioimmunoassay. The inhibin sample is intraperitoneally administered at the same time as castration, and the degree of suppression of the increase in blood FSH level after 9 hours is measured. This method requires about 5,000 times the amount of sample as compared with the method (1).

[Operation and effect of the present invention]

The ovarian inhibin of the present invention is used as a contraceptive agent for humans and can be applied to livestock production such as aligning the pregnancy period of livestock.

〔Example〕

(1) Separation and purification of inhibin Egg cells were collected from the ovary of pigs by suction, 10,000 rpm, 1
The supernatant was collected by centrifugation for an hour. To this supernatant was added 2 volumes of 50 mM Tris-hydrochloric acid buffer (pH 7.5) and buffered with the same buffer as "matrix gel red A".
Place on a column (8.0 x 25 cm), then add 0.15 M to the above buffer
After thoroughly washing with a solution containing KCl, elution was carried out with a solution containing 1.0 M urea and 1.2 M KCl added to the above buffer solution.

To the obtained eluate, add 1/4 volume of 2M KCl, put it on a "Phenyl Sepharose" column (2.5 x 30 cm), and wash thoroughly with 50 mM Tris-HCl buffer (pH 7.5) containing 1 M KCl. After that, elution was carried out with a solution containing 8 mM urea in 50 mM Tris-HCl buffer (pH 7.5).

The eluate was subjected to gel filtration chromatography using a "Sephacryl S-200" column. Tris-hydrochloric acid buffer containing 8 M urea was used as a development buffer. As a result, inhibin activity was recognized at positions of molecular weights of about 80,000 and about 30,000. Fractions with a molecular weight of 30,000 were collected, and "DEAE-Sepharose
Ion exchange chromatography was performed using a "CL6B" column (1.5 x 30 cm). The inhibin activity was separated and eluted at three sites, and the second peak had the strongest activity (Fig. 1).

Fractions of the second peak were collected and subjected to reverse phase high performance liquid chromatography as described below. Inhibin-II was eluted as a single peak at the position where the concentration of acetonitrile was 34%.

Column: "TSK-120T", 4.0 x 150 mm, elution: (A) 10%
Trifluoroacetic acid (TFA): acetonitrile: water = 1: 10:
90, (B) 10% TFA: acetonitrile: water = 1: 60: 40 linear gradient.

The changes in the amount of protein, inhibin activity and the like in the above purification steps are as shown in Table 1.

The obtained standard was re-reversed phase high performance liquid chromatography, S
It was single by DS-gel electrophoresis, and its inhibin activity was about 5,000 times higher than that of follicular fluid. About 0.7 mg of purified preparation was obtained per egg cell.

(2) Physical properties of 32K inhibin-II (a) The molecular weight by reverse phase high performance liquid gel filtration chromatography (column TSK-3000SW) is 32 kd.

(B) The molecular weight by SDS-gel electrophoresis is 32 kd.

(C) It is easily inactivated by a reducing agent and an oxidizing agent, and two polypeptides having a molecular weight of about 20 kd and about 13 kd are produced by SDS-gel electrophoresis.

(D) The N-terminal amino acid of the above polypeptide was Ser The Ala Pro with 20 kd and Gly Leu Gln Cys with 13 kd.

(E) 32K inhibin-II is a glycoprotein because it is adsorbed on a concanavalin A sepharose column and eluted with α-methylmannoside.

(F) 32K inhibin-II is inactivated by protease treatment (trypsin 60%, pepsin 85%,
It is inactivated by 70% by chymotrypsin, 95% by pronase, and 98% by thermolysin. ).

(G) 32K inhibin-II was treated with inoraminidase,
It is not inactivated by treatment with endoglycosidase H.

(H) Isoelectric point: around p16

[Brief description of drawings]

FIG. 1 shows the elution pattern of 32K inhibin-II by ion exchange chromatography.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Kenji Samukawa 1520-24 Kano Ko, Kiyotake-cho, Miyazaki-gun, Miyazaki Prefecture (72) Inventor Toshiyuki Matsuo 6653A-104 Kihara, Kiyotake-cho, Miyazaki-gun, Miyazaki Prefecture (72) Masao Igarashi Gunma Gunma 357-4, 4-chome, Hiyoshi-cho, Maebashi, Japan

Claims (1)

[Claims] 1. Inhibin having the following properties. (A) Molecular weight: 32 kd (SDS-gel electrophoresis method and high performance liquid chromatography method) (b) Reduction produces polypeptides with molecular weights of 20 kd and 13 kd (SDS-gel electrophoresis method), which loses activity. (C) Adsorbed on a concanavalin A sepharose column and eluted with α-methyl mannoside. (D) Isoelectric point: around p16 (e) Inactivated by protease treatment, but not by neuraminidase treatment and endoglycosidase H treatment. (F) N-terminal amino acid sequence: Polypeptide having a molecular weight of 20 × 10 ³ : Ser Thr Polypeptide having a molecular weight of 13 × 10 ³ : Gly Leu (g) Exists in porcine follicular fluid and suppresses elevation of follicle-stimulating hormone .