JPS60166622A - Serum calcium lowering substance px - Google Patents

Abstract

NEW MATERIAL:The serum calcium lowering substance PX [molecular weight, 20,000-30,000 (gel-filtration analysis with Sephadex); isoelectric point, 3-5pH; having sensitivity to trypsin; physiological activity, at least having the activity to lower the serum calcium level and keeping the activity even by heating at 80 deg.C for 5min]. USE:It has activities to lower the serum calcium level and decrease the peripheral leucocyte, and useful as a remedy for bulbospinal paralysis, muscular dystrophy, etc. PREPARATION:The acetone-dried powder of swine pancreas is suspended in a buffer solution, and extracted by stirring under cooling. The extract liquid is centrifuged, and the supernatant liquid is added with an organic solvent and centrifuged. The recovered supernatant liquid is added with an organic solvent, and the precipitate is recovered by centrifugal separation. The precipitate is dissolved in a buffer solution, fractionated by gel-filtration, again fractionated with ion exchange material, eluted by increasing the salt concentration of the buffer solution, and the obtained active fraction is dialyzed with water to obtain the objective serum calcium lowering substance PX.

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical Field] The present invention relates to a novel protein which is a substance extracted from pig pancreas and has at least blood ln calcium lowering activity.

[Prior art]

Regarding a serum calcium-lowering substance extracted from pig pancreas, there have already been research results by Takaoka1, Sahara2, -Se3 et al. It is reported to be effective against muscular dystrophy 5.8 and muscular dystrophy 7.

In addition, Sahara et al. purified this substance to almost a single peak,
It was found to be a protein with a molecular weight of 655,000 and an isoelectric point pl+4.84.

In addition, -se established a new extraction method, with a molecular weight of 10,
000 or less serum calcium lowering substance was obtained.

The present inventors adopted organic solvent fractionation and salting-out fractionation when extracting a serum calcium-lowering substance from pig pancreas, and succeeded in obtaining a new serum calcium-lowering substance with a molecular weight of 120,000 to 30,000. The present invention was completed by naming this substance PX, a serum calcium lowering substance.

[Disclosure of the invention]

The present invention is a protein that can be separated and purified by gel filtration, ion exchange chromatography, adsorption chromatography, etc. after organic solvent fractionation and salting-out fractionation of an aqueous extract of dried powder of porcine pancreas A7. Molecular weight measured by gel filtration analysis using Dex is 20,000-30,0
00, the isopoint is pl+3 to 5, is sensitive to trypsin, is stable to heat (remains active after heating at 80° C. for 25 minutes), and has at least a serum calcium-lowering effect.

(Extraction and fractionation method) To extract and fractionate the serum calcium-lowering substance PX from porcine pancreas, first perform water extraction using acetone dry powder of porcine pancreas as a starting material, centrifugation, concentration, organic solvent fractionation, and salting-out fractionation. This is carried out by appropriately combining chromatography, gel filtration, ion exchange chromatography, adsorption chromatography, etc.

Specifically, it is extracted and fractionated, for example, by the following method.

Acetone dry powder of porcine pancreas is suspended in a buffer solution and extracted with cooling stirring. As the buffer, for example, about 0.1M phosphate buffer, Tris buffer, etc. (pl+6 to 8) can be used.

Centrifuge the extract to separate it into supernatant and precipitate. 30 for supernatant
% concentration, an organic solvent (for example, a ketone such as acetone, an alcohol such as methanol or ethanol) is added, stirred, allowed to stand, and the supernatant is collected by centrifugation. Furthermore, the above-mentioned organic solvent is added to the supernatant to give a concentration of 60%, and the precipitate is collected by centrifugation.

The precipitation is generally carried out in a 0.01-0.05M phosphate buffer (
After dissolving in a mild (i solution) such as pl+6 to 8), dialyze against the same buffer.

Ammonium sulfate is dissolved in the dialysis fluid to 80% saturation, left to stand, and the precipitate is collected by centrifugation.

The precipitate is generally prepared in a 0.01-0.05M phosphate buffer (
pl! After dissolving in a buffer such as 6 to 8), it is fractionated by gel filtration.

As the IH body used for gel filtration, for example, molecule i
Suitable gel filtration carriers for molecular sieving of compounds with t 5,000 to] 00,000 include, for example, Sephadex, agarose, biogel, Sephacryl, and the like.

These carriers are equilibrated in advance with a buffer solution of about p116 to 8 before use.

The active fraction obtained by the above gel filtration was Am-1c
Concentrate on Diaflow 5ystenl and fractionate with an ion exchanger.

Ion exchangers used include anion exchange cellulose, Sephadex (e.g. DEAE, QAE, EC
TEOLA, etc.).

These tH forms are equilibrated in advance with a low ionic strength buffer solution of about pl+6 to 8 before use.

Elute by increasing the salt concentration of the buffer to obtain an active fraction.

The active fraction was dialyzed against low ionic strength buffer and Aml-co
After concentration using the Diaflow System, fractionation is performed using a desorbent.

At this time, examples of the desorbent used include hydroxyapatite, calcium phosphate gel cellulose, and the like.

These carriers are equilibrated with a low ionic strength phosphate buffer solution of pl+6 to 8 before use.

Elute by increasing the salt concentration of the phosphate buffer to obtain an active fraction.

After dialysis against water, lyophilization is performed.

The serum calcium-lowering substance PX is thus obtained.

(Characteristics of the substance of the present invention) (Gel filtration analysis using 11 molecular weight Sephadex G-100 was performed under the following conditions, and the molecular weight of the serum calcium lowering substance px was 20,000 to 30,000.

Conditions: Column size: 16X750 Dragon Solvent diphosphate buffer Development flow rate: 4ml/hr.

The molecular weight was determined by comparison with a standard protein of known molecular weight (Figure 1).

In addition, in FIG. 1, the Kav value means the following: Kav value - (Ve Vo) / (t/c - Vo) ■e
: Sample elution volume Vo: Excluded volume ■C; Total volume of head (2) Isoelectric point Preparative electrofocus
When the isoelectric point was determined using ng (LK118100), the isoelectric point of the serum calcium-lowering substance PX was ρ113~
It was 5.

(3) Enzyme-sensitive trypsin was added to the blood ln calcium-lowering substance PX at a weight ratio of ]:]OO, and ammonium acetate buffer (p
The sensitivity was examined by allowing the cells to react for 13 hours at 37° C. (l+8.0).

The results showed sensitivity to trypsin (Table 1)
.

The presence or absence of sensitivity was determined by examining the surface IN calcium lowering activity described below.

(4) Temperature-stable serum calcium-lowering substance PX was dissolved in phosphate buffer (pH 7,
0), incubated at 80°C for 5 minutes, and examined for activity.

As a result, although the substance was found to have a lower molecular weight, its activity remained (Table 2).

(5) Serum calcium lowering activity White domestic rabbits weighing 2-3 kg are used as experimental animals.

First, blood is collected from the auricular artery and serum is collected.

After blood collection, 1 mg of the serum calcium-lowering substance PX is dissolved in 1 ml of physiological saline, and 1 ml per body weight (kg) is administered through the auricular vein. Note that only physiological saline was administered as a control.

4.5 hours after administration, blood is again collected from the auricular artery and serum is collected.

The amount of calcium in serum before administration of this substance and 4.5 hours after administration is determined, and the percentage of the after-administration value relative to the pre-administration value is calculated.

As a method for quantifying serum calcium, atomic absorption spectrometry, OCPC colorimetry, chloroanilate colorimetry, etc. are generally used.

As a result, the serum calcium lowering substance PX was 73.3±3.5% while the control showed 94.7±2.1%.

(6) Peripheral leukocyte depletion effect White domestic rabbits weighing 2-3 kg are used as experimental animals.

First, blood is collected from the auricular artery and mixed with an anticoagulant. EDTA, citric acid, heparin, etc. are generally used as anticoagulants.

After blood collection, 1 mg of the serum calcium-lowering substance PX is dissolved in 1 ml of physiological saline, and 1 ml per body weight (kg) is administered through the auricular vein. In addition, only physiological saline was administered as a control.

1.5 hours after administration, blood is collected from the auricular artery and mixed with an anticoagulant.

Measure the white blood cell count before and 1.5 hours after administration of this substance, and calculate the percentage of the after-administration value to the pre-administration value.

To measure the number of white blood cells, an automatic blood cell counter, a counting board, etc. are generally used.

As a result, the serum calcium-lowering substance PX was 39.1±6.4% while the control showed 102.9±10.7%.
%showed that.

Since the serum calcium-lowering substance px of the present invention has serum calcium-lowering effects and peripheral leukocyte-reducing effects, it is useful, for example, as a therapeutic agent for myasthenia gravis, muscular dystrophy disease, and the like.

The serum calcium-lowering substance PX is administered parenterally (especially intramuscularly) in the form of a pharmaceutical formulation with a suitable and conventional pharmaceutically acceptable carrier.

Pharmaceutical formulations may take the form of injections and the like.

When administered intramuscularly, the serum calcium-lowering substance PX is usually administered at a dose of 0.1 to 10 mg once a day, but the dose may vary depending on age, body weight, and/or the severity of the condition being treated and response to treatment. sell.

Example 1 750 ml of 0.1 M +-lishydroxymethylaminomethane-hydrochloric acid buffer (1118,0) containing 2% sodium chloride was added to 50 g of porcine pancreas acetone dry powder,
Extract for 1 hour while cooling and stirring.

The extract is centrifuged for 45 minutes at 10,000 g or more in a refrigerated centrifuge to collect 11*. Cold acetone is added to the supernatant to give a concentration of 30%, and the mixture is allowed to stand for 1 hour. Centrifuge this for 45 minutes at 10,000 g or more in a refrigerated centrifuge, and then ? Collect j.

Further, cold acetone was added to make the concentration 60%, and the mixture was allowed to stand for 1 hour. This was heated to 3,000 g or more in a refrigerated centrifuge.
Centrifuge for 0 minutes and collect the precipitate.

Precipitate in 0.05M phosphoric acid mild ih solution (pH 7.0) 20
0 ml and dialyzed against the same buffer overnight.

Ammonium sulfate is added to the dialyzed fluid to make it 80% saturated, and then left to stand for 1 hour. 3,000 yen in a refrigerated centrifuge
Centrifuge for 30 minutes at higher than g and collect the precipitate.

Add the precipitate to 0.05 M phosphate buffer (pH 7, 0) 20
The active fraction was obtained by dissolving the solution in 0 ml and applying it to a Sephadex G-100 column equilibrated with the same buffer.

The active fraction was transferred to Am1con Diaflow System.
m (YM-5) and applied to DEAE-Sephadex A-50. 0.05M+ Lis-HCl buffer (
pl+7.5) is eluted by increasing the salt concentration to obtain an active fraction.

The active fraction was transferred to Am1con Iliaflow Syst.
Concentrate with em (YM-5) and apply to hydroxyapatite. The active fraction is obtained by elution by increasing the salt concentration of 0.01M phosphate buffer (pH 7.0).

References +11 Yoshito Takaoka: Hormones and Clinical Studies 3.957-988 (1955) (2
) Tadashi Sahara et al.: Abstracts of the 89th Annual Meeting of the Pharmaceutical Society of Japan: 584. (196
9) (3) - Light leakage: Nagasaki Medical Journal 52 (3), 173-188 (1
977) (4) Yoshito Takaoka et al.: Nagasaki Medical Journal 10.51-57 (1966) (5)
Yoshito Takaoka et al.: Nagasaki Medical Journal 13.28-35 (1,969) (6
) Yoshito Takaoka et al.: Japan Clinical 3L 345-349 (1973) (7) Yoshito Takaoka et al.: 11 Clinical Practice 23, 824-834 (1974) Table 1 Enzyme sensitivity of serum calcium-lowering substances PX Table 2 Serum calcium-lowering substances px Thermal stability* The value before administration is taken as 100%.

2

[Brief explanation of drawings]

FIG. 1 is a diagram showing the molecular weight measurement of the serum calcium-lowering substance PX of the present invention using Sephadex G-100. 1-Thousand Tochrome C 2-Equine Myoglobin 3-Serum Calcium Lowering Substance PX 4-Chymotrypsinogen 5-Egg Yolk Albumin 6-Bovine Serum Albumin

Claims (1)

[Claims] PX is a protein that can be extracted from the pancreas of the liver and has the following characteristics: - A molecular weight of 20.00 as measured by gel filtration analysis using Sephadex.
0 to 30,000, ■ Isoelectric point is p113 to 5, ■ Sensitive to lipsin, ■ Has at least serum calcium lowering activity as physiological activity, ■ Heating at 80°C for 5 minutes also inhibits the above (■) Activity does not decrease.