RU2159251C1 - Method of preparing lactogenic hormone receptors - Google Patents

Abstract

FIELD: biochemical methods. SUBSTANCE: fat globules are isolated from animal milk, washed, and treated with Triton X-100 (detergent). Resulting fat globule membrane solubilizate is purified and subjected to affinity chromatography to isolate receptors. As active sorbent, prolactine immobilized on epoxyactivated TSK gel is used. Sorbent is prepared by incubation in buffer with pH 7,4-9,0. EFFECT: increased purity of receptors and enhanced efficiency of their production. 4 dwg, 5 ex

Description

 The invention relates to the field of biology, to the study of materials by separation into components using chromatography, in particular to methods for isolating hormone receptors using conventional high-pressure liquid and affinity chromatography from components of dairy products.

 Known methods for isolating receptors of lactogenic hormones from animal organs, based on the extraction of protein fractions, in particular, from amniotic fluid and from the pituitary gland of animals. However, these methods require a large amount of source material, laborious preparative purification, a complex extraction process.

 Closest to the claimed solution is a method for determining the concentration of receptors of lactogenic hormones (copyright certificate N 1751665 from 07.30.92, authors Larina MM and Fedosimova VA).

The essence of the prototype method is as follows: fat globule membranes containing lactogenic hormone receptors are isolated from cow's milk and subjected to receptor analysis by incubating these membranes with I ¹²⁵ labeled prolactin and native prolactin in incubation buffer.

 The membranes are then separated from the buffer by centrifugation and the amount of labeled prolactin bound to the receptors is determined by the radioactivity of the samples.

 The dissociation constant and the concentration of lactogenic hormone receptors are determined according to Scatchard's graph by regression analysis (using the Delta program developed at Moscow State University for a two-center binding model).

 The disadvantage of the prototype is that the selected receptors of lactogenic hormones are associated with the membranes of the fat globules of milk, which does not ensure the purity of the obtained hormone receptors. This circumstance, in turn, allows only quantitative analysis with receptors and does not allow the study of their physicochemical and biological properties.

 In addition, the known method includes the use of receptor analysis - time-consuming, expensive and associated with radioactive drugs.

 The aim 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 the immobilization of prolactin using an activated sorbent - epoxy-activated TSK-gel, and incubation is carried out for 2 hours at a temperature of 25 ^o C and a pH of from 7.4 to 9.0 buffer.

 The claimed method allows to obtain receptors of lactogenic hormones in pure form and use them in scientific and biomedical studies of the physiology and biochemistry of the lactogenic function of the body.

 The essence of the claimed method is as follows: fat globules are isolated from milk of various animal species and washed thoroughly. The membranes of the fat globules are solubilized with the Triton X-100 detergent. Pure lactogenic receptors are isolated from the obtained solubilizate by affinity chromatography. The receptor solution is concentrated, then a study of the physicochemical and biological properties of the receptors for this species and this individual is carried out using high-performance liquid chromatography high pressure (HPLC) (Fig. 1).

 Examples of specific performance.

 Example 1. Isolation and processing of fat globules of milk.

Fresh milk is filtered through cotton filters. Fat globules are isolated from filtered milk by centrifugation for 40 minutes at 10 ^° C at 3000 rpm. The selected fat globules are washed with warm water, dosed in portions and stored in a refrigerator at -40 ^o C.

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

 The solubilization of MFGM is carried out using a Triton X-100 detergent: the required amount of 1% Triton X-100 in 0.04 M phosphate buffer with a pH of 7.4 is added to frozen fat globules.

The emulsion mixture of fat globules is incubated in a solution of detergent with constant stirring for 3 hours at 30 ^o C.

After incubation, the solubilizate containing the remains of the membranes and the released fat is centrifuged for 1 hour at 0 ^° C at 3000 rpm. Then a dense layer of fat is removed from the centrifuge cups, and the solubilizate is filtered through conventional filters to remove the remaining pieces of fat, then through microfilters to clean large casein micelles and the remains of the membranes of the fat globules.

 3. The release of the solubilizate from Triton X-100.

 It is known that the detergent Triton X-100 causes complexation of the main lactogenic hormone - prolactin.

 To clean the solubilizate from Triton X-100, the authors propose using a very simple cell filled with TSK HW-40 sorbent (Fig. 2). The cell is pre-washed with buffer (0.04 M phosphate buffer with a pH of 7.4 containing 0.1% albumin). The buffer is completely removed from the cells by centrifugation for 10 min at 1500 rpm.

 After removing the buffer, portions of the solubilizate are introduced into the cells and centrifuged again under the same conditions. Each cell is able to completely purify 1% Triton X-100 with 30 ml of solubilizate. At the same time, the use of 4 cells in a K-70 centrifuge allows 120 ml of solubilizate to be removed from the detergent.

 A study of the adsorption of Triton X-100 showed that with a ratio of solubilizate containing 1% Triton X-100 and TSK HW-40 1: 3 gel by the volume of Triton X-100 passing through the cells, it is negligible and amounts to 1.4% or less of the initial amount .

The purified solubilizer from MZHGM is stored in a freezer at -20 ^o C.

 4. Purification of receptors for lactogenic hormones.

 The purification of lactogenic hormone receptors isolated from MFHM from various components of the protein and non-protein membranes is carried out using affinity chromatography. For its implementation, the authors propose using a reactive sorbent containing epoxy rings as active groups and obtained by graft copolymerization of TSK HW-65 gel with glycidyl methacrylate.

Grafted gel copolymerization is carried out in a redox environment in the presence of cerium ammonium nitrate salts - Ce (NH ₄ ) ₂ (NO ₃ ) ₆ , which initiate grafted copolymerization.

 5. Immobilization of prolactin on an activated sorbent - epoxy-activated TSK-gel.

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

 In addition, the attachment of a particular protein depends to some extent on the properties of this protein.

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

In the inventive method, the authors propose using beef prolactin (manufactured by Hormon, Moscow) with an activity of 20 IU. As an incubation buffer, 0.5 M Na ₂ CO _{3 is} proposed. Incubation of the sorbent with prolactin is carried out with stirring at 25 ^o C. The optimal conditions for the binding of prolactin are provided in a buffer with a pH of 9.0 at 25 ^o C for 2 hours of incubation. The experimental data are shown in FIG. 3.

 Under these conditions, 51% of the prolactin contained in the incubation buffer binds to the gel.

With an increase in incubation time up to 4 hours at 25 ^o C, the amount of bound prolactin increases to 58%.

Since some activated groups in the sorbent remain free even after ligands are attached, these groups are usually blocked by an excess of the primary amine — ethocolamine, glycine, glucosamine, etc. The authors propose using a 10% solution of Tris-OH in 0.5 M Na ₂ CO for this purpose. ₃ , pH 9.00. Incubation of Tris-OH buffer with the sorbent is carried out at 25 ^o C for 3 hours with constant stirring. Then the sorbent is washed.

The washed sorbent with immobilized prolactin is used for experiments or stored in a refrigerator at 4 ^o C in a 0.5 M NaCl solution.

To bind LH receptors with prolactin immobilized on an epoxy-activated TSK gel, the sorbent is incubated with solubilizate from MFGM for 18 hours at 25 ^° C in 0.04 M phosphate buffer with a pH of 7.4. Then the sorbent is transferred to Schott glass filters and washed thoroughly with water, buffer and finally again with water.

The desorption of receptors with the PRL receptor complex is carried out using a 0.05 M solution of ammonium acetate (NH ₄ CH ₃ COO), pH 4.2, containing 0.1% Triton X-100.

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

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

 In FIG. Figure 4 shows a chromatogram of purified and concentrated LH receptors from MHFM. Three peaks are clearly visible in the chromatogram. The first peak takes place in the free volume of the gel filter column (TSK SW 4000) and has a molecular weight of at least 2000 KD. Obviously, this peak represents receptor clusters solubilized with MFGM.

 The second peak in the chromatogram has an apparent molecular weight of 56 KD and is a monomeric form of the LH receptor.

 The third peak in the chromatogram represents the decomposition products of LH receptors with a molecular weight of less than 10 KD.

 Thus, quality control of the obtained receptors is carried out.

List of references
1. Description of the invention to A.S. N 1751665 Authors: Larina M.M. and Fedosimova V.A. on "A method for determining the concentration of lactogenic receptors."

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

Claims (1)

 A method of obtaining lactogenic hormone receptors, including the selection of milk fat globules by centrifugation, characterized in that the milk fat globules are treated with Triton X-100 detergent, the obtained solubilizate of the milk fat globule membranes is purified, the receptors are isolated by affinity chromatography on an activated sorbent, using prolactin immobilized on an epoxy-activated TSK gel, obtained by incubation at a buffer pH of 7.4 to 9.0.

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