JP2967945B2 - Gonadotropic hormones in fish - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a gonadotropin of fish obtained by extracting from the pituitary gland of a perch fish.

[0002]

2. Description of the Related Art Gonadotropin (Gonadotropin, hereinafter abbreviated as GTH) is a kind of pituitary hormone, and is mainly GTH of mammals, namely, luteinizing hormone (LH), and Follicle stimulating hormone (Folliclc hormone, F
(Abbreviated as SH), the activity and structure of which have been studied. For example, for human LH, B. Schome et al., J. Clin. Endocrinol.Metab., 618 (1973), and for sheep LH, H. Papkoff et al., J. An. Chem.
c., 93 1531 (1971), for human FSH, B. Schome et al., J. Clin. Endocrinol.Metab., 39 199 (1974)
In addition, about horse FSH, P. Rathnam et al.
hem., 253 5355 (1978).

On the other hand, GTH in fish, especially teleost fish, is described in carp [E. Buruzawa-Gerard et al .; Gen. Comp. Endocri
nol., 38 421 (1979)], salmon [Edward M. Donalson et al .;
Gen. Comp. Endocrinol., 18 469 (1972), flounder (T. Bun
NG. Et al .; Gen. Comp. Endocrinol., 34 408 (1978)]. However, regarding the structure, mammalian GTH has a subunit structure consisting of an α-chain and a β-chain, and the respective structures have been clarified.
Very few examples have determined the structure of both the chain and the β chain.
Carp [YS Chang et al .; Int. J. Peptide Protein Res., 325]
56 (1988)], eel [CS Lin et al .; Eur. J. Bio-chem., 1
86 105 (1989)] and white salmon (JP-A-63-304997). In fish, the presence of several types of differentiated GTH such as LH and FSH in mammals is only reported in chum salmon (JP-A-60-67500).

[0004]

Therefore, an object of the present invention is to provide a teleost fish (Tclcostici) that can be obtained from fish other than these and that is important as a marine resource such as tuna. ) To provide gonadotropins that are widely applicable to fish belonging to
Determining the structure of the gonadotropin may lead to the development of research on the gonadotropin at the molecular level in fish or the path to mass production by synthesis of the hormone.

[0005]

Means for Solving the Problems As a result of diligent research, the present inventors have extracted GTH from the pituitary gland of perciformes, and
GTH was found to be widely applicable to fish belonging to the teleost of the teleost, and furthermore, the GTH was identified as a polypeptide, and the physicochemical properties such as amino acid sequence were analyzed. The present invention has been completed.

That is, the present invention provides: Gonadotropic hormone of fish obtained by extracting from the pituitary gland of perciformes. 2. 2. The gonadotropin I of fish according to 1 above, wherein the gonadotropin of the fish is a polypeptide having the following physicochemical properties. (I) Amino acid composition (%); Cys 11.2 Asp 8.7 Glu 8.7 Ser 7.7 Gly 7.1 His 3.6 Arg 3.6 Thr 8.3 Ala 3.6 Pro 6.6 Tyr 5.6 Val 6.6 Met 2.0 Ile 6.2 Leu 3.1 Phe 3.3 Lys 5.1 (ii) Molecular weight; 30,000-40,000 (iii) Solubility; easily soluble in water ( 2. iV) sugar content; around 20% 2. The gonadotropin II of fish according to the above 1, wherein the gonadotropin of fish is a polypeptide having the following physicochemical properties. (I) Amino acid composition (%); Cys 10.5 Asp 9.1 Glu 7.7 Ser 6.7 Gly 3.3 His 3.8 Arg 3.8 Thr 8.6 Ala 3.8 Pro 8.1 Tyr 5.3 Val 6.7 Met 2.9 Ile 4.8 Leu 3.8 Phe 5.3 Lys 5.7 (ii) molecular weight; 30,000-40,000 (iii) solubility; readily soluble in water ( iV) Sugar content; around 16% 4. A fish gonadotropin I-α or II-α polypeptide having the amino acid sequence of SEQ ID NO: 1. 5. A fish gonadotropin I- β polypeptide having the amino acid sequence of SEQ ID NO: 2. 6. A fish gonadotropin II-β polypeptide having the amino acid sequence of SEQ ID NO: 3. 7. 2 above having the polypeptide according to 4 or 5 above.
The gonadotropin I described. 8. (3) having the polypeptide according to (4) or (6) above;
Gonadotropin II as described. It is about.

Hereinafter, the present invention will be described in detail. The teleostei of teleost fish can be classified into three groups: herring fish group, middle fish group and sea bass fish group, the most evolved group of sea bass fish group, the most primitive group of herring fish group, middle fish group Are considered to be a group with characteristics intermediate to those of the herring fish school and the sea bass fish school. Among them, most of the marine fish belong to the school of sea bass. When aiming at the cultivation of this school of perch fish, if GTH is administered to the fish to increase the reproductive line, it is useful for egg collection (promotion of egg maturation, promotion of spawning), etc. It is optimal to use GTH extracted from the pituitary gland.

The GTH of the present invention can be obtained from the pituitary gland of the Mackerel family Thunnus obcsus belonging to the order Perch by alcohol extraction. That is, an ethanol solution (for example, 35
%), Ammonium acetate (for example, 10%), phenylmethylsulfonyl fluoride (for example, 1.5 mM), ethylenediaminetetraacetic acid (for example, 5 mM), and the mixture is moderately (eg, for 15 seconds) with a homogenizer under ice-cooling. 5 times), and extract in ice-cooling while stirring for a certain time (for example, 2 hours). Refrigerated centrifugation (eg, 15,000 rpm
The precipitate fraction was removed at 4 ° C. for 30 minutes, the same applies hereinafter), ethanol (for example, 80% final concentration) was added to the supernatant, and the mixture was allowed to stand under ice-cooling overnight, followed by refrigerated centrifugation. Apply.
Remove the supernatant and add ammonium bicarbonate buffer (for example, 100m
M, pH 9.0, hereinafter the same), dialyzed sufficiently against the same buffer, and then subjected to DEAE Sephacel column chromatography. For equilibration and washing, an ammonium bicarbonate buffer is used. Elution is performed by increasing the ionic strength of the buffer. The powder obtained by collecting and freeze-drying the active fractions is dissolved in an ammonium bicarbonate buffer (for example, 10 mM, pH 9.0) and subjected to reversed-phase high performance liquid chromatography. Equilibration is performed with the same buffer containing acetonitrile (for example, 15%), and elution is performed with a linear gradient of acetonitrile (for example, 15 to 45%). The active fractions are collected and lyophilized to obtain the GTH of the present invention as a white powder.

The GTH of the present invention obtained by these operations
Are two types of polypeptides, GTH I and GTH II,
Each peptide can be hydrolyzed with hydrochloric acid at 110 ° C. for 24 hours, added with phenyl isocyanate (PITC), and then subjected to high performance liquid chromatography manufactured by JASCO Corporation for amino acid composition analysis. The physicochemical properties of these GTH I and GTH II are shown in (i) to
It is shown in (vi). (I) Amino acid composition; as shown in Table 1 below.

Table 1 Amino acid composition (%) {amino acid GTH I GTH II} ─── Cys 11.2 10.5 Asp 8.7 9.1 Glu 8.7 7.7 Ser 7.7 6.7 Gly 7.1 3.3 His 3.6 3.8 Arg 3.6 3.8 Thr 8.3 8.6 Ala 3.6 3.8 Pro 6.6 8.1 Tyr 5.6 5.3 Val 6.6 6.7 Met 2.0 2.9 Ile 6.2 4.8 Leu 3.1 3.8 Phe 3.3 5.3 Lys 5.1 5.7 ─────────────────── (ii) Amino acid sequence of GTH I; SEQ ID NO: 1 and SEQ ID NO: 2
Has the sequence of the polypeptide shown in FIG.

The amino acid sequence of GTH II has the polypeptide sequence shown in SEQ ID NO: 1 and SEQ ID NO: 3. (Iii) molecular weight; both GTH I and GTH II molecules are 30,000-4
(Iv) Solubility; both GTH I and GTH II molecules are readily soluble in water. (V) Sugar content; GTH I is around 20%, GTH II is around 16%. (Vi) Color and shape of substance; GTH I and Both molecules of GTH II are white powders.
By applying to reversed-phase high-performance liquid chromatography, two peptides, GTH I-α and GTH I-β, were obtained from GTH I, and two types of GTH I-α, GTH II-β were obtained from GTH II. The peptide is obtained.

The amino acid sequence analysis of the peptides constituting GTH obtained by this operation can be carried out by using a 477A type protein sequencer manufactured by Applied Biosystems on the peptide fragments obtained by treatment such as enzyme digestion. This GTH
I-α and GTH II-α both have the amino acid sequence shown in SEQ ID NO: 1, GTH I-β has the amino acid sequence shown in SEQ ID NO: 2, and GTH II-β has the amino acid sequence shown in SEQ ID NO: 3, respectively.

The GTH of the present invention can stimulate the gonads of fish belonging to teleosts and teleosts, and can promote the efficiency of seed production in marine fish culture.
Hereinafter, examples of the present invention will be described.

[0014]

【Example】

[0015]

Example 1 (Extraction and Purification of Bigeye Tuna GTH) 500 g of a solution consisting of 35% ethanol, 10% ammonium acetate, 1.5 mM PMSF and 5 mM EDTA were prepared by adding 20 g of the pituitary gland of Bigeye Tuna.
The mixture was ground in a Teflon homogenizer (100 rpm) and extracted with stirring under ice cooling for 2 hours. Refrigerated centrifugation (10,000rpm, 4 ℃, 30
min.) to remove the precipitate, and add a final concentration of 80% to the supernatant.
Then, ethanol was added thereto, and the mixture was allowed to stand under ice-cooling overnight. Again, refrigerated centrifugation was performed in the same manner as above, and the supernatant was removed to obtain a precipitated fraction. The precipitate fraction was dissolved in 40 ml of 100 mM ammonium bicarbonate buffer, pH 9.0, dialyzed against the same buffer, and equilibrated with the same buffer.
(1.5 × 30 cm). After washing well with the same buffer, elution was performed with 200 mM ammonium bicarbonate buffer, pH 9.0, 300 mM
The reaction was performed sequentially with M bicarbonate buffer and pH 9.0, and the solution was collected per 5 ml. 300 mM ammonium bicarbonate buffer shown in FIG. 1,
The fractions eluted at pH 9.0 are collected, lyophilized and
20 mg of powder were obtained. To 1 mg of this powder was added and dissolved 1 ml of a solution containing 10 mM ammonium bicarbonate buffer, pH 9.0, and 15% acetonitrile, and the mixture was dissolved in Asahip.
The sample was subjected to high performance liquid chromatography equipped with an ak C4P-50 column (4.6 x 250 mm). Elution was performed with a linear gradient of 15 to 45% acetonitrile containing the same buffer for 40 minutes (flow rate: 0.8 ml / min). The active fractions shown in FIG. 2 were collected and freeze-dried. This purification by high performance liquid chromatography was repeated 20 times to obtain about 4 mg and about 2 mg of a white powder, namely, two kinds of polypeptides, bluefin tuna GTH I and GTH II.

[0016]

Example 2 (Structural analysis of GTH I-α, GTH II-β, GTH II-α and GTH II-β peptides) GTH I and GTH II obtained from bluefin tuna were reduced with dithiothreitol, respectively. After pyridylethylation with vinylpyridine, the mixture was subjected to high performance liquid chromatography equipped with a TSK-gel ODS120T column equilibrated with 20% acetonitrile containing 0.1% trifluoroacetic acid (hereinafter referred to as TFA). Elution: 20-60 with 0.1% TFA
% Acetonitrile with a linear gradient of 40 min.
Two types of peptides, GTH I-α and GTH I-β, were obtained from TH I, and two types of peptides, GTH II-α, GTH II-β, were obtained from GTH II.

For GTH I-α, this sample was subjected to Edman degradation 40 times using a gas-phase sequencer (type 477A protein sequencer manufactured by Applied Biosystems, the same applies hereinafter), and the amino acid of the N-terminal 40 residues was analyzed. The type and the order of binding were determined. C-terminal residues were determined by carboxypeptidase treatment and amino acid analysis for amino acids released over time. Further, a sample obtained by treating a sample with lysyl endopeptidase and separating and purifying the sample using reverse-phase high performance liquid chromatography is obtained.
For each kind of peptide fragment, the type of amino acid and the order of conjugation were determined using a gas phase sequencer. From the above results, it was revealed that GTH I-α has the amino acid sequence shown in SEQ ID NO: 1.

For GTH I-β, the sample was subjected to 40 endoman digestions using a gas-phase sequencer and the N-terminal 40
The amino acid type of the residues and the order of linkage were determined. In addition, for the six peptide fragments obtained by subjecting the sample to trypsin treatment and separation and purification using reversed-phase high performance liquid chromatography, the types of amino acids and the order of binding were determined using a gas phase sequencer. From the above results, it was revealed that GTH I-β has the amino acid sequence shown in SEQ ID NO: 2.

For GTH II-α, the samples were subjected to 40 Edman degradations using a gas phase sequencer to determine the type of amino acids and the order of attachment of the N-terminal 40 residues. C-terminal residues were determined by carboxypeptidase treatment and amino acid analysis for amino acids released over time. In addition, the sample is treated with lysyl endopeptidase,
About six kinds of peptide fragments obtained by separation and purification using reversed-phase high performance liquid chromatography,
The types of amino acids and the order of binding were determined using a gas-phase sequencer. From the above results, it was revealed that GTH II-α has the same amino acid sequence as GTH I-α shown in SEQ ID NO: 1.

For GTH II-β, the sample was subjected to 40 Edman degradations using a gas phase sequencer to determine the type of amino acid and the order of attachment of the N-terminal 40 residues. In addition, for the six peptides obtained by subjecting the sample to lysyl endopeptidase treatment and separation and purification using reversed-phase high-performance liquid chromatography, the types of amino acids and the order of binding were determined using a gas-phase sequencer.
The amino acid sequence at the C-terminus was obtained by further treating the C-terminal peptide obtained by lysyl endopeptidase treatment with trypsin and separating and purifying the C-terminal peptide separated and purified using reversed-phase high-performance chromatography using a gas-phase sequencer. The type and the order of binding were determined. From the above results, it was revealed that GTH II-β has the amino acid sequence shown in SEQ ID NO: 3.

[0021]

Example 3 (Gonadal stimulating hormone activity of GTH I and GTH II) Ovaries isolated from the bluefin tuna at the spawning stage were fragmented into 20 mg size, and cultured in culture medium (140 mM sodium chloride, 5 mM calcium chloride, 0.8 mM, respectively). Magnesium sulfate,
1 mM sodium dihydrogen phosphate, 1.5 mM calcium chloride, 2 mM sodium bicarbonate, 0.088% Eagle MEM, 0.05
Organ culture was carried out in a plastic multiwell plate filled with HEPES buffer containing 7.5% glucose, 0.02% gentamicin sulfate, and 1% bovine serum albumin (pH 7.5). Then, GTH I and GT obtained from bigeye tuna
After adding HII to 8, 40, 200, and 1,000 ng / ml, respectively, the cells were cultured by shaking at 20 ° C. for 18 hours. After completion of the culture, the culture supernatant was collected and subjected to radioimmunoassay,
Testosterone and estradiol-17 in culture supernatant
The amount of β was measured. As a result, as shown in FIGS. 3 and 4 , GTH I and GTH II of the present invention promoted the production of testosterone and estradiol-17β, which are steroid hormones that directly promote gonad maturation, in a concentration-dependent manner.

[0022]

As described above, according to the present invention, the existence of at least two types of tuna gonadotropin and the amino acid sequence thereof have been elucidated for the first time by the present invention. Thus, research on the molecular level of gonadotropin in fish can be expected to develop, and mass production by synthesis of the hormone is also possible. In particular, the gonadotropin produced by the present invention can be widely applied to fish belonging to teleosts and teleosts, and is extremely useful in that it can promote high efficiency of seed production in marine fish culture. It is.

[0023]

[Sequence list] SEQ ID NO: 1 (1) Sequence length: 94 (2) Sequence type: amino acid (3) Number of chains:-(4) Topology: linear (5) Sequence type: peptide ( 6) origin (a) organism: bigeye pituitary (7) SEQ features: GTH I-.alpha. and amino acid sequence of GTH II-alpha (8) SEQ: 5 10 TyrProAsnValAspLeuSerAsnMetGly 15 20 CysGluGluCysThrLeuLysLysAsnAsn 25 30 ValPheSerArgAspArgProIleTyrGln 35 40 CysMetGlyCysCysPheSerArgAlaPhe 45 50 ProThrProLeuLysAlaMetLysThrMet 55 60 ThrIleProLysAsnIleThrSerGluAla 570 ThrCysCysValAlysHisSerTyrGlu7580ThrGluValAlaGlyIleArgValArgAsn8590HisThrAcy sequence of the sequence of the straight chain (the sequence of the amino acid sequence of the amino acid sequence of the amino acid sequence). ) Sequence type: Peptide (6) Origin (a) Organism: Pituitary gland of Big Bluefin Tuna (7) Sequence features: Amino acid sequence of GTH-I β chain (8) Sequence: 510 GlyGlnGlyCysSerTryGlyCisHisProl 20 Ile 35 40 CysGluGlyGlnCysTyrLeuGluAspPro 45 50 ValTyrIleSerHisAspGluGlnLysIle 55 60 CysAsnGlyAspTrpSerTyrGluValLys 65 70 HisIleGluGlyCysProValGlyValThr 75 80 TyrProValAlaArgAsnCysGluCysThr 85 90 AlaCysAsnThrGlyAsnThrTyrCysGly 95 100 ArgLeuProGlyTyrValProSerCysPro SerPhe SEQ ID NO: 3 (1) sequence Length: 115 (2) sequence type: the number of amino acids (3) Chain :-(4) Topology: linear (5) Sequence type: peptide (6) Origin (a) Organism name: Bachimaguro pituitary (7) SEQ Features: GTH-II beta amino acid sequence of the chain (8) SEQ: 5 10 PheGlnLeuProProCysGlnLeuIleAsn 15 20 GlnThrValSerValGluLysGluGlyCys 25 30 AlaSerCysHisProValGluThrThrIle 35 40 CysSerGlyHisCysIleThrLysAspPro 45 50 ValIleLysIleProPheSerLysValTyr 55 60 GlnHisValCysThrTyrArgAspPheTyr 65 70 TyrLysThrPheGluLeuProAspCysPro 75 80 ProGlyValAspProThrValThrTyrPro 85 90 ValAlaLeuSer ysHisCysGlyArgCys 95 100 AlaMetAspThrSerAspCysThrPheGlu 105 110 SerLeuGlnProAspPheCysMetAsnAsp 115 IleProPheTyrTyr

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a view showing an elution pattern when a precipitate fraction obtained by ethanol precipitation of a pituitary gland ethanol extract supernatant of a bluefin tuna is subjected to DEAE Sephacel column chromatography.

FIG. 2 is a view showing an elution pattern when an active fraction obtained by DEAE Sephacel column chromatography is applied to an Asahipak C4P-50 column.

FIG. 3 is a graph showing the testosterone producing effect of GTH.

FIG. 4 is a graph showing the effect of GTH on estradiol 17β production.

Continuation of the front page (72) Inventor Yasuhiko Sasamoto 3-2-9 Tsukishima, Chuo-ku, Tokyo Ocean Fisheries Inside the Ocean Research Institute Co., Ltd. (56) References JP-A-60-67500 (JP, A) JP-A-63-304997 (JP, A) (58) Fields investigated (Int. Cl. ⁶ , DB name) C07K 14/59 CA (STN) REGISTRY (STN) BIOSIS (DIALOG) WPI (DIALOG)

Claims (6)

(57) [Claims] 1. A gonadotropic hormone of a fish obtained by extracting from the pituitary gland of a perciform fish, which comprises the following (a):
Or a gonadotropin of fish which is any of (b). (a) in the I-α polypeptide of gonadotropin of fish having an amino acid sequence in which one or several amino acids may be deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1 and in the amino acid sequence of SEQ ID NO: 2 1
Or a gonadotropin I of a fish having an I-β polypeptide of a gonadotropin of a fish having an amino acid sequence in which several amino acids may be deleted, substituted or added. (B) In the amino acid sequence of SEQ ID NO: 1 Alternatively, the gonadotropic hormone II-α polypeptide of fish having an amino acid sequence in which several amino acids may be deleted, substituted or added, and the amino acid sequence of SEQ ID NO: 3
Or gonadotropin II of fish having a gonadotropin II-β polypeptide of fish having an amino acid sequence in which several amino acids may be deleted, substituted or added 2. A fish gonadotropin I-α or II-α polypeptide having an amino acid sequence in which one or several amino acids may be deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1. 3. A fish gonadotropin I-β polypeptide having an amino acid sequence in which one or several amino acids may be deleted, substituted or added in the amino acid sequence of SEQ ID NO: 2. 4. A fish gonadotropin II-β polypeptide having an amino acid sequence in which one or several amino acids may be deleted, substituted or added in the amino acid sequence of SEQ ID NO: 3. 5. Gonadotropin I of fish having the polypeptide according to claim 2 and 3 and having the following physicochemical properties. (I) Amino acid composition (%); Cys 11.2 Asp 8.7 Glu 8.7 Ser 7.7 Gly 7.1 His 3.6 Arg 3.6 Thr 8.3 Ala 3.6 Pro 6.6 Tyr 5.6 Val 6.6 Met 2.0 Ile 6.2 Leu 3.1 Phe 3.3 Lys 5.1 (ii) Molecular weight: 30,000- 40,000 (iii) Solubility; easily soluble in water (iv) Sugar content; around 20% 6. Gonadotropin II of fish having the polypeptide of claim 2 and 4, and having the following physicochemical properties. (I) Amino acid composition (%); Cys 10.5 Asp 9.1 Glu 7.7 Ser 6.7 Gly 3.3 His 3.8 Arg 3.8 Thr 8.6 Ala 3.8 Pro 8.1 Tyr 5.3 Val 6.7 Met 2.9 Ile 4.8 Leu 3.8 Phe 5.3 Lys 5.7 (ii) Molecular weight: 30,000- 40,000 (iii) Solubility; easily soluble in water (iv) Sugar content; around 16%