RU2599511C2 - Method of production enriched by sphyngomyelinsintasis extract with using saponin - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry, namely to production from human tissues of the protein extract is enriched with sphyngomyelinsintase 1. Method consists in sequential extraction of tissue, selected from tissue of human's renal cortex, bland or testicle with buffer containing saponin in concentration of 0.05 and 1 mg/ml.

EFFECT: method described above, allows to obtain extracts with high output of sphyngomyelinsintase 1.

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Description

FIELD OF THE INVENTION

The present invention relates to the field of biochemistry, molecular biology, medicine and can be used in studies of the function of human sphingomyelin synthase.

BACKGROUND OF THE INVENTION

In studies of the biological role of sphingomyelin synthases 1 and 2 (SMS1 and SMS2), their expression level is usually determined at the level of mRNA and the total enzymatic sphingomyelin synthase activity of cell and tissue lysates [Qureshi A, Subathra M, Gray A, Schey K, Del Poeta M, Luberto C. Role of sphingomyelin synthase in controlling the antimicrobial activity of neutrophils against Cryptococcus neoformans. Plos one. 2010 Dec 28; 5 (12): e15587, Hsiao JH, Fu Y, Hill AF, Halliday GM, Kim WS. Elevation in sphingomyelin synthase activity is associated with increases in amyloid-beta peptide generation. Plos one. 2013 Aug 20; 8 (8): e74016, Giussani P, Tringali C, Riboni L, Viani P, Venerando B. Sphingolipids: key regulators of apoptosis and pivotal players in cancer drug resistance. Int J Mol Sci. 2014 Mar 12; 15 (3): 4356-92, Tafesse FG, Huitema K, Hermansson M, van der Poel S, van den Dikkenberg J, Uphoff A, Somerharju P, Holthuis JC. Both sphingomyelin synthases SMS1 and SMS2 are required for sphingomyelin homeostasis and growth in human HeLa cells. J Biol Chem. 2007 Jun 15; 282 (24): 17537-47, Ding T, Li Z, Hailemariam T, Mukherjee S, Maxfield FR, Wu MP, Jiang XC. SMS overexpression and knockdown: impact on cellular sphingomyelin and diacylglycerol metabolism, and cell apoptosis. J Lipid Res. 2008 Feb; 49 (2): 376-85, Separovic D, Semaan L, Tarca AL, Awad Maitah MY, Hanada K, Bielawski J, Villani M, Luberto C. Suppression of sphingomyelin synthase 1 by small interference RNA is associated with enhanced ceramide production and apoptosis after photodamage. Exp Cell Res. 2008 May 1; 314 (8): 1860-8]. Evaluation of the content of any enzyme is necessary to determine its specific activity. Determining the expression level of SMS1 or SMS2 is necessary to assess the role of two SMS isoforms and the mechanisms of regulation of their activity. Immunoblotting is a common method for detecting and quantifying the level of protein expression in cells and tissues. However, in studies of the function of sphingomyelinsynthases it is practically not used. An exception is the work in which cells were transfected with genetically engineered constructs encoding a protein crosslinked with antigenic peptides such as V5 or flag, antibodies to which are used to detect protein [Huitema K, van den Dikkenberg J, Brouwers JF, Holthuis JC. Identification of a family of animal sphingomyelin synthases. EMBO J. 2004 Jan 14; 23 (1): 33-44, Yeang C, Varshney S, Wang R, Zhang Y, Ye D, Jiang XC. The domain responsible for sphingomyelin synthase (SMS) activity. Biochim Biophys Acta. 2008 Oct; 1781 (10): 610-7]. The usual method for evaluating the expression level of SMS1 protein in cells and tissues by immunoblotting may be difficult due to the relatively low level of expression. For example, in the enriched fraction of Golgi apparatus membranes from tissues of the small and large intestine of a pig [Guillén N, Navarro MA, Surra JC, Arnal C, Fernández-Juan M, Cebrián-Pérez JA, Osada J. Cloning, characterization, expression and comparative analysis of pig Golgi membrane sphingomyelin synthase 1. Gene. 2007 Feb 15; 388 (1-2): 117-24] and in the total fraction of membrane proteins of HeLa cells [Burns TA, Subathra M, Signorelli P, Choi Y, Yang X, Wang Y, Villani M, Bhalla K, Zhou D, Luberto C. Sphingomyelin synthase 1 activity is regulated by the BCR-ABL oncogene. J Lipid Res. 2013 Mar; 54 (3): 794-805] SMS1 was detected as a weak band, and in the total fraction of membrane proteins of cell lines U937 and HL-60 SMS1 was not detected at all [Burns TA, Subathra M, Signorelli P, Choi Y, Yang X, Wang Y , Villani M, Bhalla K, Zhou D, Luberto C. Sphingomyelin synthase 1 activity is regulated by the BCR-ABL oncogene. J Lipid Res. 2013 Mar; 54 (3): 794-805].

To analyze the expression of membrane proteins, which include sphingomyelin synthases, in cells and tissues, before total detection, the total protein is extracted using buffers containing detergents such as SDS, Triton X-100 or NP-40 [Weissman AM. Solubilization of lymphocytes. Curr Protoc Immunol. 2003 Nov; Chapter 8: Unit 8.1A, Bonifacino JS, Dell′Angelica EC, Springer TA. Immunoprecipitation. Curr Protoc Immunol. 2001 May; Chapter 8: Unit 8.3, Abeam, Western blotting - a beginner′s guide, http://www.abcam.com/ps/pdf/protocols/WB-beginner.pdf]. Commonly used solutions include RIPA buffer ( R adio I mmuno P recipitation A ssay) containing 50 mM Tris-HCl, [pH 7.5; 150 mM NaCl; 1% NP-40; 0.5% sodium deoxycholate; 0.1% SDS; 5 mM EDTA) [MacPhee DJ. Methodological considerations for improving Western blot analysis. J Pharmacol Toxicol Methods. 2010 Mar-Apr; 61 (2): 171-7, Ngoka LC. Sample prep for proteomics of breast cancer: proteomics and gene ontology reveal dramatic differences in protein solubilization preferences of radioimmunoprecipitation assay and urea lysis buffers. Proteome Sci. 2008 Oct 24; 6:30]. However, obtaining a protein extract using RIPA buffer or complete tissue lysis with a buffer containing 4% SDS did not allow the authors of the present invention to study the expression of SMS1 in human tissues by immunoblotting, since the detected bands in the samples deposited on phoresis were very weak.

Formerly Wassler M. et al. showed that the extraction of rat hepatocyte cell cultures with solutions with different concentrations of Gypsophila paniculata saponin causes differential permeabilization (permeability) of cell membranes [Wassler M, Jonasson I, Persson R, Fries E. Differential permeabilization of membranes by saponin treatment of isolated rat hepatocytes. Release of secretory proteins. Biochem J. 1987 Oct 15; 247 (2): 407-15]. At a concentration of saponin of 0.04 mg / ml, the plasma membrane is permeabilized and cytosol is extracted, at 0.2 mg / ml, the Golgi apparatus and endoplasmic reticulum are permeabilized, as well as the extraction of lumen content, in particular, secretory proteins. Moreover, even large concentrations of saponin (5 mg / ml) solubilized only a small part, less than 10%, of the endoplasmic reticulum membrane [Wassler M, Jonasson I, Persson R, Fries E. Differential permeabilization of membranes by saponin treatment of isolated rat hepatocytes. Release of secretory proteins. Biochem J. 1987 Oct 15; 247 (2): 407-15], while upon cell extraction with 0.5% Triton X-100, cell membranes were almost completely dissolved [Fey EG, Wan KM, Penman S. Epithelial cytoskeletal framework and nuclear matrix-intermediate filament scaffold : three-dimensional organization and protein composition. J Cell Biol. 1984 Jun; 98 (6): 1973-84].

Methods for processing biological samples with lysing solutions containing saponin of various origins and in various concentrations for the analysis of proteins and other biological molecules are well known in the art. For example, international patent publication WO 2006/117557 (publication date 11/09/2006) discloses a method for detecting cells of interest in a sample by treating it with a lytic reagent, which is preferably saponin, followed by measuring the activity of intracellular enzymes. International patent publication WO 2012/162133 (publication date 11/29/2012) discloses a method for the selective lysis of endogenous cells in a biological sample, comprising contacting the biological sample with a lysing solution and exposing the mixture to ultrasound; as well as a lysing solution containing saponin and proteinase. From US patent No. 8617839, publ. December 31, 2013, there is a known method of treating a biological sample with saponin with methyl-6-O-CN-heptylcarbamoyl) alpha-D-glucopyranoside.

Closest to the present invention, technical solutions are described in British patent No. 1533835, publ. 11/29/1978, in which a method for obtaining a sample from the patient’s respiratory tract for the detection of pathogens is disclosed, comprising mixing the sample with saponin as a lysing agent, wherein the concentration of saponin in solution with various reagents is from 1% to 5%; as well as in international patent publication WO 2007/090862 (publication date 08/16/2007) regarding a method for diagnosing and / or controlling a disease by altering the expression of sphingomyelin synthase 3 (SMS3), for which it is proposed to obtain a biological sample from a subject and identify it the amount and / or activity of SMS3 or the nucleic acid encoding it, while 0.1% saponin in PBS is used for cell lysis.

However, despite the existence of numerous methods for the preparation of proteins and their extracts, including using saponin, the problem of analyzing the quantity and / or activity of sphingomyelin synthases 1 and 2 in biological samples remains unresolved for further study of the pathogenesis of a number of diseases, as well as for their diagnosis. The purpose of the present invention is to overcome this problem.

Brief Description of the Drawings

The accompanying drawings provide a comparison of the extraction efficiency of SMS1 from tissues using increasing concentrations of saponin and RIPA buffer.

In FIG. 1 shows the results of immunoblotting of 80 μg of protein obtained from the human kidney cortex using RIPA buffer with the rabbit polyclonal antibody PAB10379 to the full-length protein SMS1. Upon incubation of the membrane with the antibody overnight, a weak band of the expected size was observed (molecular weight calculated from the band mobility was about 48 kDa).

In FIG. Figure 2 presents the results of immunoblotting of 50 μg of protein obtained from the human kidney cortex using a buffer containing increasing concentrations of saponin 0.05 mg / ml, 0.1 mg / ml and 1 mg / ml, as well as using RIPA buffer.

In FIG. Figure 3 presents a comparison of the SMS1 content of 75 mg protein in extracts of the renal cortex obtained using RIPA buffer and 5 mg, 10 mg and 15 mg of protein of the same tissue obtained by sequential extraction with a buffer containing saponin at concentrations of 0.05 mg / ml and 1 mg / ml.

In FIG. 4 presents the results of the analysis of SMS1 content in the lung and testes using extraction of 50 μg of protein using RIPA buffer and saponin-containing buffer.

Disclosure of the present invention

Sphingomyelin synthases 1 and 2 catalyze the biosynthesis of membrane phospholipid sphingomyelin, which is 2-15% of mammalian cell phospholipids [Tafesse FG, Ternes P, Holthuis JC. The multigenic sphingomyelin synthase family. J Biol Chem. 2006 Oct 6; 281 (40): 29421-5, M. Koval, R.E. Pagano, Intracellular transport and metabolism of sphingomyelin. Biochim. Biophys. Acta 1082 (1991) 113-125]. The synthesis occurs by reversibly transferring phosphocholine from phosphatidylcholine to ceramide to form sphingomyelin and diacylglycerol [Tafesse FG, Ternes P, Holthuis JC. The multigenic sphingomyelin synthase family. J Biol Chem. 2006 Oct 6; 281 (40): 29421-5]. Sphingomyelin synthase 1 (SMS1) is localized in the membrane of the Golgi apparatus and synthesizes most (up to 90%) of sphingomyelin cells [Holthuis JC, Luberto C. Tales and mysteries of the enigmatic sphingomyelin synthase family. Adv Exp Med Biol. 2010; 688: 72-85]. Sphingomyelin synthase 2 is localized mainly in the plasma membrane and to a lesser extent in the Golgi apparatus [Huitema K, van den Dikkenberg J, Brouwers JF, Holthuis JC. Identification of a family of animal sphingomyelin synthases. EMBO J. 2004 Jan 14; 23 (1): 33-44, Tani M, Kuge O. Sphingomyelin synthase 2 is palmitoylated at the COOH-terminal tail, which is involved in its localization in plasma membranes. Biochem Biophys Res Commun. 2009 Apr 10; 381 (3): 328-32]. Sphingomyelin forms membrane microdomains, and it is assumed that its synthesis in the Golgi apparatus and its further transport to the plasma membrane, where it is concentrated in the exoplasmic leaf, ensures the formation of compositional differences between the endoplasmic reticulum, Golgi apparatus and the plasma membrane [Tafesse FG, TuisCernes Pernes, . The multigenic sphingomyelin synthase family. J Biol Chem. 2006 Oct 6; 281 (40): 29421-5, Holthuis JC, Pomorski T, Raggers RJ, Sprang H, Van Meer G. The organizing potential of sphingolipids in intracellular membrane transport. Physiol Rev. 2001 Oct; 81 (4): 1689-723]. The synthesis of sphingomyelin is accompanied by the expenditure and formation of important regulators of the cellular processes of ceramide and diacylglycerol, respectively, which connects the SMS1 function with such processes as membrane vesicular transport, signal transduction of receptors, cell proliferation and apoptosis [Tafesse FG, Ternes P, Holthuis JC. The multigenic sphingomyelin synthase family. J Biol Chem. 2006 Oct 6; 281 (40): 29421-5, Holthuis JC, Luberto C. Tales and mysteries of the enigmatic sphingomyelin synthase family. Adv Exp Med Biol. 2010; 688: 72-85].

The biological role of sphingomyelinsynthases in the cells and organism of animals and humans has been actively studied in recent years. Research results show that impaired function may be involved in the pathogenesis of a number of diseases. In particular, a decrease in the activity or expression of SMS1 or SMS2 in HL-60 cells of human promyelocytic leukemia differentiated by the neutrophilic pathway disrupted their killer activity against cryptococci by secretion of antimicrobial factors such as defensins [Qureshi A, Subathra M, Gray A , Schey K, Del Poeta M, Luberto C. Role of sphingomyelin synthase in controlling the antimicrobial activity of neutrophils against Cryptococcus neoformans. Plos one. 2010 Dec 28; 5 (12): e15587]. An increase in the expression of SMS1 or SMS2 in Huh7 human hepatoma cells increased the level of sphingomyelin, cholesterol and ApoA-1 alipoprotein in them, which indicates the effect of sphingomyelin synthesis on cholesterol metabolism [Yan N, Ding T, Dong J, Li Y, Wu M. Sphingomyelin synthase overexpression increases cholesterol accumulation and decreases cholesterol secretion in liver cells. Lipids Health Dis. 2011 Mar 21; 10: 46]. In humans, elevated plasma sphingomyelin levels are a risk factor for coronary atherosclerosis [Jiang XC, Paultre F, Pearson TA, Reed RG, Francis CK, Lin M, Berglund L, Tall AR. Plasma sphingomyelin level as a risk factor for coronary artery disease. Arterioscler Thromb Vase Biol. 2000 Dec; 20 (12): 2614-8, Slotte JP. Biological functions of sphingomyelins. Prog Lipid Res. 2013 Oct; 52 (4): 424-37]. Alzheimer's disease, which is characterized by the formation of plaques containing β-amyloid peptide in certain brain structures, is also accompanied by a disturbance in the balance of phospholipids in brain tissues, in particular, a decrease in sphingomyelin level and an increase in ceramide level [Haughey NJ, Bandaru VV, Bae M, Mattson MP . Roles for dysfunctional sphingolipid metabolism in Alzheimer′s disease neuropathogenesis. Biochim Biophys Acta. 2010 Aug; 1801 (8): 878-86]. In Alzheimer's disease, the expression of the SMS1 gene has been significantly increased in human hippocampus. In addition, the inhibition of sphingomyelin synthase activity in CHO-APP cells using D609 reduced the secretion of β-amyloid peptide by these cells [Hsiao JH, Fu Y, Hill AF, Halliday GM, Kim WS. Elevation in sphingomyelin synthase activity is associated with increases in amyloid-beta peptide generation. Plos one. 2013 Aug 20; 8 (8): e74016]. Increased ceramide levels and the initiation of apoptosis mediate the effects of many chemotherapeutic drugs. Various ways to reduce the level of ceramide in cells can determine the resistance of tumors to chemotherapy. One such pathway may be an increase in sphingomyelin synthase activity [Taniguchi M, Okazaki T. The role of sphingomyelin and sphingomyelin synthases in cell death, proliferation and migration-from cell and animal models to human disorders. Biochim Biophys Acta. 2014 May; 1841 (5): 692-703, Itoh M, Kitano T, Watanabe M, Kondo T, Yabu T, Taguchi Y, Iwai K, Tashima M, Uchiyama T, Okazaki T. Possible role of ceramide as an indicator of chemoresistance: decrease of the ceramide content via activation of glucosylceramide synthase and sphingomyelin synthase in chemoresistant leukemia. Clin Cancer Res. 2003 Jan; 9 (1): 415-23]. On the other hand, a decrease in sphingomyelin synthase activity in tumor cells can lead to their resistance to apoptosis, which is induced by cells of the immune system through Fas, TNF and TRAIL receptors [Taniguchi M, Okazaki T. The role of sphingomyelin and sphingomyelin synthases in cell death, proliferation and migration-from cell and animal models to human disorders. Biochim Biophys Acta. 2014 May; 1841 (5): 692-703, Morad SA, Cabot MC. Ceramide-orchestrated signalling in cancer cells. Nat Rev Cancer. 2013 Jan; 13 (1): 51-65].

Sphingomyelin synthase in the cells is represented at a very low level, and therefore for many years this protein could not be isolated, although the activity of this enzyme was determined. Attempts to obtain a protein extract of tissues by known methods and subsequent immunoblotting with antibodies to SMS gave a weak band, and even then not in all tissues. However, the authors of the present invention noted that in the presence of saponin, the protein leaves the membrane structures into a lysate. Therefore, to obtain an enriched SMS extract of biological tissues, a saponin buffer was used.

Saponins, also called saponosides, are glycosides of plant origin with surface-active properties. Saponins are widespread in nature, found in various parts of plants - leaves, stems, roots, flowers, fruits. As you know, saponins have the ability to cause cell lysis.

Thus, it is an object of the present invention to provide a method for producing an SMS-rich human tissue extract by sequentially extracting a biological sample with a saponin-containing buffer to further study the level of SMS protein expression in human tissues.

This goal is achieved by the fact that saponin is included in the buffer, the concentration and the sequence of processing of biological samples are selected in which SMS is effectively extracted from the tissues.

In accordance with the present invention, the method comprises several stages. In the first stage, the fabric is mechanically crushed in liquid nitrogen to a fine powder consistency and homogenized on ice with 10 volumes (1 ml per 100 mg of tissue) of a buffer containing 50 mM Tris-HCl, pH 7.5.150 mM NaCl, 2 mM EDTA, 2, 4 mg / ml of a mixture of protease inhibitors (Complete protease inhibitor cocktail, Santa Cruz Biotechnology, USA) and 0.05 mg / ml of saponin (BioClot GmbH, Germany) in a Downs homogenizer with a weakly ground piston. The homogenate is centrifuged at 4000 g, 4 ° C for 10 minutes, the supernatant is removed, and the precipitate is suspended in the same volume of a buffer with 0.05 mg / ml saponin, stirred by rotation (14 rpm, 4 ° C, 30 minutes) and centrifuged at 13000 g, 4 ° C for 10 minutes. Then, the resulting precipitate is suspended in the same volume of the above solution with an increase in saponin concentration to 1 mg / ml. After stirring by rotation in the above mode, the homogenate is centrifuged for 15 minutes at 13000 g, 4 ° C, the supernatant is taken without touching the precipitate. All manipulations are carried out on ice with solutions cooled to 0 ° C. The resulting supernatant is a tissue protein extract enriched with SMS. Thus obtained extracts of different tissues contain from about 1.5 to 3 μg / μl of total protein. For further immunoblotting, it is convenient to obtain a more concentrated protein solution. For this, the protein can be precipitated with a mixture of chloroform and methanol according to the Wessel-Flugge method [Wessel D, Flügge UI. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem. 1984 Apr; 138 (1): 141-3], wash the precipitate with acetone, dry and store at -20 ° C until use. The resulting precipitate was washed with acetone, dried and stored at -20 ° C. At the next stage, SMS is detected by immunoblotting using the standard method. Samples of protein extracted from tissues are dissolved in a small amount of a solution of 2% SDS, 15 mM NaOH at a temperature of 37-45 ° C, neutralized by adding 1/10 volume of 150 mM HCl (controlling the pH of the solution using indicator paper). After determining the protein concentration using the RC DC protein Assay kit (Bio-Rad, USA), the samples were separated in a denaturing 10% polyacrylamide gel according to the Laemmli method [Gallagher SR. One-dimensional SDS gel electrophoresis of proteins. Curr Protoc Mol Biol. 2012 Jan; Chapter 10: Unit 10.2A] and transferred by electrotransfer to a PVDF membrane (0.2 μm, Bio-Rad, USA).

For immunodetection, you can use the ECL Advance Western Blotting Detection Kit (GE Healthcare, USA) and Lumigen TMA-6 reagents (Beckman Coulter, USA). Immunodetection is carried out according to the recommendations of manufacturers with small modifications. The membrane is blocked with 5% skim milk in TBS buffer (20 mM Tris-HCl, pH 7.6; 137 mM NaCl) containing 0.1% Tween 20 for 1 hour at room temperature. The membrane is then rinsed with TBS with a buffer containing 0.06% Tween 20 (TBS-T buffer) and incubated with polyclonal rabbit anti-SMS antibody PAB10379 (Abnova, Taiwan) at a dilution of 1: 30,000 in a TBS buffer containing 0.05% Tween 20 and 0.5% skim milk, overnight at 4 ° C. The membrane is then washed with TBS-T buffer and incubated with goat peroxidase conjugated goat anti-rabbit immunoglobulins pGAR-Iss (IMTEC, Russia) at a dilution of 1: 500000 in TBS buffer containing 0.05% Tween 20 and 0.5% skim milk, for 1.5 hours at 4 ° C. The membrane was washed as described above, rinsed twice with TBS buffer and the signal was detected using a Lumigen TMA-6 chemiluminescent substrate and Hyprefilm ECL film (GE Healthcare, USA).

The SMS content in tissue extracts was estimated by densitometric scanning of immunoblots using ImageQuant 5.2 (Molecular Dynamics, USA) in the linear range of the integral optical density of immunoblot bands versus the amount of protein in the immunoblotted sample. The amount of SMS is normalized by the amount of tissue extract protein. The result is expressed as the mean ± S.D. of three independent experiments.

Thus, the present invention relates to a method for producing a protein extract enriched in sphingomyelinsynthase using a saponin-containing buffer, characterized in that the biological sample is sequentially extracted with a buffer with a different concentration of saponin.

According to one preferred embodiment, the biological sample is sequentially extracted with a buffer containing saponin in concentrations of 0.05 and 1 mg / ml.

According to one preferred embodiment, the sphingomyelinsynthase is sphingomyelinsynthase 1.

In one preferred embodiment, the saponin is saponin derived from Quillaja saponaria.

Examples of the implementation of the present invention

Example 1

According to an embodiment of the present invention, sphingomyelinsynthase 1 was detected by immunoblotting in human kidney cortex tissue using a polyclonal rabbit antibody to a full-sized protein (Abnova, Taiwan). To this end, to enrich the tissue lysate with membrane proteins, the kidney cortex tissue was extracted with a buffer containing increasing concentrations of Quillaja saponaria saponin 0.05 and 1 mg / ml. To reduce the volume of the extraction buffer, the tissue was pre-homogenized in a buffer containing 0.05 mg / ml saponin, and the homogenate was precipitated without incubation with the buffer. This washing to some extent balanced the tissue sample with the buffer and removed a significant portion of the protein without noticeable SMS1 loss. The kidney cortex extract obtained by this method contained SMS1 12 times more than the extract obtained in RIPA buffer (Fig. 1).

Example 2

50 μg of protein was extracted from a sample of human kidney cortex using a buffer containing increasing concentrations of saponin 0.05, 0.1 and 1 mg / ml, as well as using RIPA buffer. Immunoblotting of extracts with polyclonal rabbit antibody RAB10379 on the full-length SMS1 protein showed that a buffer containing 0.05 mg / ml saponin extracts approximately the same amount of SMS1 as RIPA buffer. A buffer containing 0.1 mg / ml saponin recovered a slightly larger amount of SMS1. Tissue extraction with a buffer containing 1 mg / ml saponin gave a significantly greater SMS1 yield. Tissue extraction with saponin at a concentration of 1 mg / ml after preliminary extraction with saponin at a concentration of 0.05 mg / ml gave the highest yield of SMS1 (Fig. 2).

Example 3

The SMS1 content was compared in 75 mg of protein in extracts of the cortex of the kidney obtained using RIPA buffer and 5 mg, 10 mg and 15 mg of protein of the same tissue obtained by sequential extraction of saponin at concentrations of 0.05 mg / ml and 1 mg / ml Densitometric analysis of RIPA immunoblots and saponin extracts of the renal cortex showed that the saponin extract contained 12.65 ± 0.16 times more SMS1 than the RIPA extract. From the presented example it can be seen that the inventive method of extracting protein using a saponin-containing buffer is more effective than extraction with RIPA buffer (Fig. 3).

Example 4

According to the method in accordance with the present invention, an analysis was made of the content of SMS1 in the lung and testes using extraction of 50 μg of protein with RIPA buffer and saponin-containing buffer. The results showed that sequential extraction with saponin at concentrations of 0.05 mg / ml and 1 mg / ml allowed to increase the relative content of SMS1 in the total lysate protein of the studied tissues (Fig. 4).

Thus, the method in accordance with the present invention allows the detection of sphingomyelin synthase in biological samples of various tissues without prior isolation of the membrane fraction, which makes it less time-consuming and costly, and also significantly increases the information content and effectiveness of further analysis carried out using sphingomyelins synthases.

Claims (1)

The method of obtaining protein extract from human tissues enriched with sphingomyelin synthase 1 by sequential extraction of tissue selected from the tissue of the human cortex of the kidney, lung or testes, with a buffer containing saponin in concentrations of 0.05 and 1 mg / ml, where saponin is obtained from Quillaja saponaria.

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