The method of obtaining receptors lactogenic hormones


(57) Abstract:

The invention relates to biology, to methods of allocation of hormone receptors. From the milk of animals is isolated and washed fat globules. Treat them with the detergent Triton X-100. The resulting solubilized membranes of the fat globules of the milk clean. Using affinity chromatography allocate receptors. As the activated sorbent use prolactin immobilized on epoxyoctadecane TSK-gel. Sorbent get in the incubation buffer at pH of 7.4, and 9.0. The invention allows to obtain receptors lactogenic hormones in pure form and to increase the efficiency of their production. 4 Il. ^

The invention relates to the field of biology, the study of materials by separation into components using chromatography, in particular to a method of allocating hormone receptors using conventional liquid and affinity chromatography, high pressure component of dairy products.

Known methods for isolating receptors lactogenic hormones from animal organs, based on the extraction of protein fractions, in particular, from the amniotic fluid and from the pituitary glands of animals. However, these methods require a large number of the/P> Closest to the claimed solution is the method of determining the concentration of receptors lactogenic hormones (author's certificate N 1751665 from 30.07.92, authors Larina, M. M. and Fedoseeva Century A.).

The essence of the prototype method is as follows: the membranes of the fat globules containing receptors lactogenic hormones, isolated from the milk of cows and subjected receptor analysis by incubation of these membranes labeled with I125prolactin and native prolactin in the incubation buffer.

Then the membrane is separated from the buffer by centrifugation and determine the number associated with the receptor labeled prolactin radioactivity of the samples.

The dissociation constant and concentration of receptors lactogenic hormones is determined by the schedule of Scatchard by regression analysis (using the program "Delta", developed at MSU for two model binding).

The disadvantage of the prototype is that the selected receptors lactogenic hormones associated with the membranes of the fat globules of milk, which does not ensure the purity of the obtained receptors of hormones. This circumstance, in turn, allows to receptors only quantitative analysespider includes the use of receptor analysis time-consuming, costly, and associated with radioactive drugs.

The purpose of the claimed solution is to increase the efficiency of the method.

This goal is achieved by the fact that in the known method for immobilization of prolactin used activated sorbent - epoxyoctadecane TSK-gel, and the incubation carried out for 2 hours at a temperature of 25oC and the pH of the buffer from 7.4 to 9.0.

The claimed method allows to obtain receptors lactogenic hormones in pure form and their use in scientific and biomedical research physiology and biochemistry of the lactogenic functions of the body.

The essence of the claimed method consists in the following: from the milk of different animal species secrete fat globules and carefully washed. The membranes of the fat globules solubilizing using the detergent Triton X-100. From the received solubilisate using affinity chromatography allocate lactogenic receptors in pure form. The receptor solution is concentrated, then the examination of physico-chemical and biological properties of the receptors for this species and the species using high performance liquid chromatography high pressure (URHVD) (Fig. 1).A.

Fresh milk is filtered through a cotton filter. Filtered secrete milk fat globules by centrifugation for 40 min at 10oC at 3000 rpm Selected fat globule washed with warm water, dispense in portions and placed in storage in the refrigerator at -40oC.

2. Solubilization of the membranes of the fat globules of milk (MIGM).

The solubilization MGGM perform using the detergent Triton X-100: frozen fat globule add the required amount of 1% Triton X-100 in 0.04 M phosphate buffer with pH 7.4.

Emulsion a mixture of fat globules incubated in a solution of detergent with constant stirring for 3 hours at 30oC.

After incubation solubilized containing residues membranes and released fat, centrifuged 1 hour at 0oC at 3000 rpm Then a thick layer of fat is removed from the centrifuge cups and solubilized filtered through conventional filters to remove the remaining pieces of fat, then through the microfilters for cleaning of large casein micelles and the remnants of the membranes of the fat globules.

3. Release solubilisate from Triton X-100.

It is known that the detergent Triton X-100 causes kompleksoobrazovateleyj to use a very simple cell, filled with sorbent TSK HW-40 (Fig. 2). The cell is pre-washed with buffer (0.04 M phosphate buffer with pH 7.4, containing 0.1% albumin). The buffer is completely removed from the cells by centrifugation for 10 min at 1500 rpm

After removal of the buffer in cells contribute portions of solubilisate and again centrifuged under the same conditions. Each cell is able to be completely cleaned of 1% Triton X-100 30 ml solubilisate. Simultaneously applying the 4 cells in the centrifuge K-70 allows you to clean from the washing liquid 120 ml solubilisate.

The adsorption of Triton X-100 showed that when the ratio of solubilisate containing 1% Triton X-100, and gel TSK HW-40 1:3 by volume passing Triton X-100 in the cell is negligible and is 1.4% or less of the original quantity.

Purified solubilized of MIGM placed in storage in the freezer at -20oC.

4. Purification of receptors lactogenic hormones.

Purification of receptors lactogenic hormones isolated from MIGM, from various components of membrane protein and nonprotein nature performed using affinity chromatography. For its implementation the authors suggest the use of a reactive sorbent, containing as active the ptx2">

Graft copolymerization of the gel is carried out in the redox environment in the presence of salts suryamaninagar - Ce(NH4)2(NO3)6that initiate graft copolymerization.

5. Immobilization of prolactin on the activated sorbent - epoxyoctadecane TSK-gel.

It is known that during attachment of ligands to an activated sorbent epoxy groups react with the amine groups of the ligands, forming a covalent bond. The degree of annexation is determined by the primary amino groups of the ligand and depends on the pH, composition of buffer, temperature and time of incubation.

Additionally, the accession of a protein depends to some extent and properties of this protein.

In the research the authors found the optimal values of these parameters: pH, composition of buffer, temperature and time of incubation for the adsorption of prolactin activated sorbent.

In the proposed method, the authors suggest the use of bovine prolactin (produced by "Hormone", Moscow) with an activity of 20 IU. As the incubation buffer serves 0.5 M Na2CO3. Incubation of the sorbent with prolactin carried out at peremeshivanie 2 hours of incubation. These experiments are shown in Fig. 3.

Under these conditions associated with gel 51% contained in the incubation buffer prolactin.

When increasing the incubation time to 4 hours at 25oC the amount of bound prolactin increases to 58%.

Because some number of activated groups in the sorbent remain free and after joining ligands, these groups are usually blocked by an excess of primary amine - Econoline, glycine, glucosamine and other Authors propose to use for this purpose a 10% solution of Tris-OH in 0.5 M Na2CO3pH 9.00. Incubation Tris-OH buffer sorbent is carried out at 25oC for 3 hours with constant stirring. Then the sorbent is washed.

The washed sorbent with immobilized prolactin is used for experiments or placed in storage in the refrigerator at 4oC in 0.5 M NaCl solution.

Binding of LH receptors to immobilized on epoxyoctadecane TSK-gel prolactin sorbent incubated with solubilization of MIGM for 18 hours at 25oC in 0.04 M phosphate buffer with pH 7.4. Then the sorbent is transferred into glass filters SCHOTT and thoroughly washed with water, buffer, and finally again with water.

4CH3COO), pH 4.2, containing 0.1% Triton X-100.

The sorbent immobilized on its surface a complex of prolactin-receptor incubated in a solution of ammonium acetate for 1 hour at 25oC. Then the mixture of sorbent solution is transferred into a special cell (Fig. 2) with a TSK HW-40 gel and centrifuged. During this operation, the solution with the desorbed with complex receptors LH is released from Triton X-100.

Purified using affinity chromatography LH receptors have in a solution of very low concentration, which is beyond the sensitivity of the UV detector. The authors propose to concentrate solutions, containing the LH receptor, using emissioni filters CX-10 (Millipore). One filter CX-10 is able to filter 20 ml of fluid for 6-7 hours. The authors performed the concentration of a solution of receptor 20 times.

In Fig. 4 shows the chromatogram of the purified and concentrated LH receptors from MIGM. The chromatogram clearly shows three peaks. The first peak is held in the free volume gelfilte column (TSK 4000 SW) and has a molecular weight of at least 2000 BC. Obviously, this peak represents the receptor cluster, solubilization with MIGM.

The second peak on chrome

The third peak on the chromatogram represents the decay products of the LH receptor with a molecular weight of less than 10 KD.

Thus, control the quality of the received receptors.


1. Description of the invention to A. S. N 1751665 authors: M. Larina, M. and Fedoseeva Century A. "a Method for determining the concentration of the lactogenic receptors".

2. Biol Chem. Hoppe - Seyler, vol. 374, pp. 271-279, 1993. Johrke, B. Kruger, T. Viengutz, The Celycolylation as Source of the Variability in controls Patterns of Jnolividl al Human Amniotic Fluids.

The method of obtaining receptors lactogenic hormones, including the selection of the fat globules of milk by centrifugation, characterized in that the fat globules of milk treated with the detergent Triton X-100, obtained solubilized membranes of the fat globules of milk cleanse, receptors allocate using affinity chromatography on activated sorbent, which is used as prolactin, immobilized on epoxyoctadecane TSK-gel obtained in the incubation buffer at pH from 7.4 to 9.0.


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