JPS61263999A - Novel peptide - Google Patents

Abstract

NEW MATERIAL:The osteocalcin (salt) of formula (Thr is tyrosine; Leu is leucine; Asp is aspartic acid; Ser is serine; Gly is glycine, Ala is alanine; Pro is proline; Gla is gamma-carboxyglutamic acid; Lys is lysine; Arg is arginine; Val is valine; Cys is cysteine; Asn is asparagine; His is histidine; Ile is isoleucine; Phe is phenylalanine). USE:Labeling reagent for the determination of osteocalcine in blood which can be used as an index of osteometabolism. PREPARATION:For example, both epiphyses of a fumur of a beagle are cut off, marrow is removed from the bone, and the bone is pulverized and suspended in an aqueous solution of EDTA to effect the extraction. The extracted liquid is subjected to gelfiltration, the amino acid analysis is carried out for each fraction and a fraction containing Gla is freeze-dried to obtain the peptide of formula.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Application of the Invention The present invention relates to an osteocalcin peptide useful as a labeling reagent that is an important indicator for understanding the state of bone metabolism.

Prior Art Osteocalcin is a protein that contains a large amount of mineralized bone and contains Gla (r-carboxyglutamic acid), a vitamin-dependent Ca-binding amino acid.
Also known as protein (BGP), to date, human,
Cows, swordfish, cats, cows, chickens, and rats are known (Pr1ce, P.A.).

etal Proc, Natl, Acad, Sci,
U, S,, 73, 33711-3373, (/ri7
B) H Shimomura, H, etal, J
.

Biochem...? Otsu, '10! ;-'II/,
(/de, r4')]. Radioimmunoassays utilizing labels and antibodies derived from natural osteocalcin have also been described.
A., etal, J.

Cl1n, Invest, At g7gNl#3(/
Rig.

)].

Problems to be Solved by the Invention The present inventors isolated osteocalcin from cortical bone of dogs.
They purified it, determined its structure, and confirmed that it was a new substance.

Means for Solving the Problems The present invention has been made based on the above knowledge, and is based on the above findings.
Leu-Gla-Pro-Lys-Arg-Gla-7
"I -C,y,s -G Io-1°"-""-1゛°-"°"-Cys-Asp-Glu-Leu-Ala
-Asp-His-11e-Gly-Phe-OH[:
I) (In the formula, T is L-tyrosine, Leu is L-leucine, Asp is L-aspartic acid, Ser is L-serine, Gly is glycine, Ala is L-alanine, Pro is L-proline, and Gla is r-carboxyglutamic acid,
Lys is L-lysine, Arg is L-arginine, Val
is L-valine, Cys is L-cystine, Asn is L-
asparagine, His is L-histidine, Ile is L-
isoleucine (Phe indicates L-phenylalanine) or a salt thereof.

First, in carrying out the present invention, the peptide may be extracted from dog bones, isolated, and purified. It's okay.

An example of the extraction method will be described below.

For example, after degreasing and drying dog bones, grind them and use EDT.
Demineralize and extract with a solution of No. 8, collect the supernatant by centrifugation, etc.
It is desalted and separated using a dialysis membrane, etc., and then separated and purified by gel/l/? filtration and high performance liquid chromatography.

EXAMPLES The present invention will now be described with reference to Examples, but the present invention is not limited to these Examples.

Example / Collect the femurs of the large S heads of Peagle, cut both tube ends, remove the internal bone marrow, cut into 2 to 37 small pieces, and place in acetone.
After degreasing at 0°C for 22 hours, it was dried.

Grind the dried bones to obtain 33 ft of bone powder, 0.3 M E
It was suspended in 3Qml of DTA (pHgO) and deashed and extracted at 4Z°C for 10 days. After centrifuging this <300OR
PM, 10 minutes), the supernatant was collected, desalted by dialysis against 007M ammonium bicarbonate using a dialysis membrane, and lyophilized to obtain a crude component containing osteocalcin. Set this to 0 in advance. /M ammonium bicarbonate buffered Sephadex G-100 column ('1. II x 7 g
on) and fill with 0. /M ammonium bicarbonate. / 0.2 ml fractions were collected, and the ultraviolet absorption of these fractions was measured using Omμ.
7～711.73～ri Otsu, 97～//4', //3～
After freeze-drying each of /112, approximately / ~ of the progeny was weighed,
Dissolve 03ml of 2.5N sodium hydroxide, degas,
After sealing the tube and hydrolyzing for 2 hours at 10° C., amino acid analysis revealed that fractions 75 to 2 were osteocalcin fractions containing 1-carboxyglutamic acid (Gla).

Freeze-dried product of this fraction 7S-Otsu (10 Otsu mg)
) for high performance liquid chromatography Nucleosi15
C1a column (g
A fraction showing a single ultraviolet absorption peak at 3 mμ was collected and freeze-dried to obtain canine osteocalcin (1) having the following physicochemical properties.

Amino acid analysis obtained about canine osteocalcin/ηN-)te
Amino acid analysis was carried out at l/ml <dissolved at /10°C for 2q hours, and the amino acid composition (theoretical values in parentheses) when Leu=5 was as follows.

Asp Otsu O/f61, 5erQ Ri Otsu (+), G I u
lA23(41゜Pro5.gg(61, G l
y291(s), Al a,, 20ff(21+V
al/93 (2), Cys-CysOgll (1),
I le /(1)2(1).

Leu! ; (51, Tyr2011(2), P
he105 (11°Lys/15(1), HisOri9
(+), ArglOg (l1 elemental analysis value actual value C: 5.20!r% H: 63g% N: /l
'Ω% calculated value C: 3/, 9A% H: A0% N: /l
A7! ;%C189H278066N46S11・3H
It was calculated as 20 (MW, 4'3g, 5').

Using the isoelectric point Bio-Rad method, gOμV of canine osteocalcin was applied to a 70% polyacrylamide gel at 300V,
Electrophoresis was performed for 111 hours at 30°C, precipitated with 725% trichloroacetic acid, and gel scanner (Isco mode)
When measuring the U absorption of 2 g Opm with 3 g of Otsu, P
I=3.9.

Test example/Preparation of canine osteocalcin antibody Canine osteocalcin (3~) Bovine serum albumin (B
sA) Conventional method using (10~) and gluculaldehyde
This was subjected to a cross-linking reaction and purified using a Sephadex G-100 column to obtain osteocalcin-B S of /2~
A. Obtaining the immunogen of the conjugate.

Add this /m'j/ml (physiological saline) to an equal amount of kelp IJ
- After emulsifying with Tofreund's adjuvant and immunizing rabbits or goats four times, antiserum containing each antibody was obtained by a conventional method.

Test Example 2 Blood Osteocalcin Measurement Method 100 μt of sample serum was added with antiserum containing Hangyo Osteocalcin rabbit antibody in buffer CO, 0/M phosphoric acid, 07%.
Gelatin, 0/% NaN3.23 m M E D T
A-62Na, 0. /3M NaC1,0,/
%Tween20 and adjusted the pH to \\711] to 7
100μt of 000x dilution and 200μt of buffer
Add t and leave overnight. This is green wood
-Hunter method (Biochem, J, g9
/ / II-/ 23 (/RI Otsu 3)) Method t 1-Osteocalcin 100A
,000 cpm) and left overnight at <Z°C, 100 pL of rabbit IgG solution (/20 pfl/ml) and 100 μt of goat anti-rabbit IgG antiserum were added and incubated at 37°C for 2 hours. Centrifuge the reaction at <300 °C.
Orpm 30mln), and the radioactivity of the precipitate was counted. On the other hand, instead of 100μt of the above sample serum, 100μt of this buffer solution was added (indicated by X---X in the figure).
From this, the osteocalcin content in the serum can be determined.

Test Example 3 Elcatonin, EGFL h-PTH (/-311),
h-insulin, h-calcitonin buffer 10011
Immune cross-reactivity of canine osteocalcin with domestic rabbit antibodies was investigated using samples containing 3 ng, 10 ng, 1100 n, and 1100 n of canine osteocalcin in Test Example 1, respectively. , Figure 1 (X- in the figure)
--Xi peptide of the present invention, ○□○; elcatonin, low --- mouth, EGF, ・---, h-PTH (/-3
Il), △...-△; h-insulin, mu--4H
h CT is shown. ), as shown, selectively reacts with large osteocalcin, elcatonin, EGF, h-
No cross-reactivity was shown with PTH (/-1 g-311), h-insulin, and h-calcitonin.

Effect of the invention It is a useful substance for measuring temperature.

[Brief explanation of the drawing]

FIG. 1 shows the standard curve and cross-reactivity of radioimmunoassay of the peptide of the present invention.

Claims (1)

[Claims] (1) Formula [There is an amino acid sequence] (In the formula, Tyr is L-tyrosine, Leu is L-leucine, Asp is L-aspartic acid, Ser is L-serine,
Gly is glycine, Ala is L-alanine, Pro is L
- Proline, Gla is γ-carboxyglutamic acid, L
ys is L-lysine, Arg is L-arginine, Val is L-valine, Cys is L-cysteine, Asn is L-asparagine, His is L-histidine, He is L-isoleucine, and Phe is L-phenylalanine) A peptide represented by or a salt thereof.