Protein of adipocyte plasma membrane, method of its preparation and complex for starting tyr-phosphorylation of insulin-receptor substrate proteins irs-1 and irs-2

FIELD: technological processes.

SUBSTANCE: method suggests protein of adipocyte plasma membrane, method of its preparation and complex based on this protein. Protein has molecular mass of 115 kilodaltons and has the ability to start-up tyr-phosphorylation of insulin-receptor proteins substrate in adipocyte. Method of protein preparation provides for adipocytes preparation out of rat, mouse or human tissues and plasma membranes extraction out of them. Then plenty of domains are isolated with high content of cholesterol hcDIG, which are treated with solution trypsin/NaCl. Centrifugation is done and protein fraction SDS-polyacrylamide gel is segregated with electrophoresis. Prepared protein fraction in amount of 115 kilodaltons is eluated from this gel. Complex constitutes activated protein and is formed during its combination with one of compounds from group: YCN-PIG, YMN-PIG, YCN or lcGcel.

EFFECT: protein in its activated form allows regulating glucose utilization bypassing insulin signal chain.

7 cl, 20 dwg, 1 tbl

 

The present invention relates to the protein of the plasma membrane of adipocytes, which has a specific affinity binding to postinstallation.

The role of phospholipids and phospholipases in transmembrane signal transduction firmly established. Equally well established concept zakalivanie proteins in the cell membrane with covalently attached glycosylphosphatidylinositol (GPI), and the exact chemical structure of GPI anchors are defined for multiple GPI-zayakorivaetsya proteins, such as acetylcholinesterase (AchE) of human erythrocytes, Thy-1 in rats and several envelope proteins of parasites, similar to the variant surface glycoprotein (VSG) of Trypanosoma brucei. Zakalivanie lipid occurs through the phosphatidylinositol (PI), which consists of diacyl - or etkilesimlerindeki type of phospholipid. Since the latter occurs, among others, include anchors mammals and differs from the main part of the PI presented in the membranes, it would be possible to create new types of molecules involved in the formation of secondary mediators originating from GPI. Signaling via GPI molecules is of particular interest, as these zakalivanie lipid molecules do not penetrate through the membrane, and in most cases enclosed in the outer half of the lipid bilayer. The signal-operad is radiated release of GPI molecules of the cell membrane is demonstrated for a variety of endocrine and paracrine molecules, from hormones and growth factors. Part of GPI molecules in transmembrane signal transduction and intracellular effects currently established, but less is known about the signaling pathways leading to the observed metabolic effects.

The idea that GPI-zakalivanie signaling molecules have properties that emerged from the early experiments where it was shown that the binding of insulin with its receptor activates the hydrolysis of GPI molecules. Identified low-molecular-weight substance that mimics some of the mechanisms of action of insulin on enzymes of metabolism. This substance has the structure inositolglicana and is formed in the plasma membrane as a result of hydrolysis of GPI, sensitive to insulin. Although initially thought that the GPI precursor insatalling enzyme modulator is a structural analogue of GPI-membrane protein anchors, there are clear-cut differences between the carbohydrate component between the GPI, which converts the signal, and GPI-anchored membrane proteins. GPI membrane anchor protein invariably consists of tremendosly core, followed by ethanolamine.htm, which provides the connection with the C-terminal amino acid attached protein.

Adjustable hydrolysis of GPI is not limited to insulin, but we nd the same under the action of other hormones.

In almost all cases, stimulation of the cells by hormones or growth factors leads to a temporary release of GPI-anchored proteins from the cell surface. Most of these receptors agonists are either tyrosinekinase receptors, or receptors associated with tyrosine kinases.

Many proteins involved in insulin action, identified at the molecular level. The insulin receptor is a transmembrane to tyrosinekinase which when activated as a result of binding with insulin undergoes rapid autophosphorylation and phosphorylates a number of intracellular substrates, including one or more of 50-60 kDa proteins, including Shc, 15 kDa binding protein fatty acid, and a few so-called insulin-receptor substrate proteins, IRS-1/2/3/4. After tyrosine phosphorylation of IRS-polypeptides act as "docking" proteins for several Src homology containing 2 domain adaptronic molecules and enzymes, including phosphatidylinositol-3-kinase (PI-3-K), Grb2, SHP2, Nck, and Fyn. The interaction between IRS-proteins and PI-3-K occurs through the regulatory subunit p85 of this enzyme leads to increased catalytic activity subunit p110. The enzyme PI-3-K is essential for many sensitive to insulin metabolic processes, including stimulation of TRANS is ORT of glucose and glycogen synthesis. In all cases in which there is a stimulation of tyrosine phosphorylation of IRS-proteins, and the accompanying "docking" of these proteins with the p85 subunit of the enzyme PI-3-K, and, with the exception of intersection between the signal systems of the insulin and angiotensin, this "docking" is associated with the stimulation of the activity of PI 3-K.

In addition to identifying which converts the signal paths coming directly from the insulin receptor to lower targets were delineated several intersections between the signal transmission using insulin and other hormones/growth factors or exogenous stimuli that either mimic (to some extent) or modulate a positive or negative way metabolic and/or mitogenic effect of insulin on different cell systems. Because none of these ligands are not directly activates the kinase of the insulin receptor, their signaling pathways may converge with signaling by insulin at a more distal signaling stage. This property is common to molecules postinstallscript (PIG-P) of different types, for example, PIG-P, obtained from glycosylphosphatidylinositol anchors from yeast Gce1p, which substantially mimics the metabolic action of insulin that are not accompanied by induction of kinase activity of the insulin receptor.

p> Positive mutual influence postinstallation (PIG) and PIG-peptides (PIG-P) at the cascade signal conversion insulin sensitive insulin target cells involves redistribution glycosylphosphatidylinositol (GPI)-anchored proteins in the cytoplasmic membrane (GPI-protein) and double-acylated preceptory tyrosinekinase contained in the plasma membrane, detergent-resistant, glycolipid-enriched complexes domains with high cholesterol (hcDIG) in the set of domains with low cholesterol (lcDIG).

In allocated adipocytes of rats the primary target of PIG-P localized in hcDIG. Labeled with a radioactive label, PIG-P, Tyr-Cys-Asn-NH-(CH2)2-O-PO(OH)O-6Manα1-2)-2Manα1-6Manα1-4GluN1-6lno-1,2-(cyclic)phosphate (YCN-PIG), and labeled with a radioactive label and lipolytic cleaved GPI-protein (lcGce1p) from Saccharomyces cerevisiae was obtained YCN-PIG, saturating the image associated with hcDIG, but not with lcDIG, microsomes or total plasma membranes. Linking and YCN-PIG, and lcGce1 is specific, because it is completely void of any excess chemically synthesized unlabeled YCN-PIG, or pretreatment of adipocytes with trypsin, and then NaCl or N-ethylmaleimide (NEM), which suggests that YCN-PIG is detected surface receptor cells. Related is the use of PIG-P significantly increased in hcDIG from adipocytes, pre-processed using GPI-specific phospholipases C, together with a lipolytic remove endogenous ligands, such as GPI-proteins/lipids. The binding of the affinity is greatest for YCN-PIG, followed by a combination of separate components, Tyr-Cys-Asn - NH-(CH2)2-OH(YCN) and HO-PO(H)O-6Manα1(Manα1-2)-2Manα1-6Manα1-4GluN1-6Ino-1,2-(cyclic)phosphate (PIG37), as well as the peptide variant, YMN-PIG. PIG37 and YCN separately demonstrate intermediate and low affinity. Incubation of adipocytes with YCN-PIG reduces subsequent labeling with [C14]NEM 115 kDa-polypeptide released from the cell surface consistent processing trypsin/NaCl. These data show that in adipocytes of rats mimics insulin PIG(-P) is recognized by trypsin/NaCl/NEM-sensitive 115 kDa protein from hcDIG, which acts as a receptor GPI-proteins.

In the same cell, apparently, there are several types of DIG. Pits on the surface of the terminal differentiated cells represent a special DIG, which form the flask-shaped spacelane driven by excessive expression of the marker and the structural protein caveolin is the essential component 1-3.

Pits that are in adipocytes 20% of the surface area of the plasma membrane, involved in receptor-mediated photocase (potocytosis), endocytosis, trance is itose and signal transmission. In allocated rat adipocytes lcDIG low cholesterol/caveolin is the essential component having a high buoyant density (in accordance with gradient centrifugation in sucrose density)may differ from the typical hcDIG with high cholesterol/caveolin is the essential component, characterized by low surface density. The main fraction of GPI-proteins, such as Gce1 and Nuc-acylated proteins, such as NRTK, preceptory tyrosinekinase, pp59Lynlocalized in hcDIG. In response to mimic insulin incentives, such as synthetic PIG or a sulfonylurea, glimepiride, and GPI-proteins and NRTK moved from hcDIG in lcDIG. This redistribution is not associated with the loss of their lipid modification.

Polar Central giganova head group (PIG)- or (PIG-P)-adjacent amino acids on carboxylic GPI-protein polypeptide component provide a molecular basis of the distribution of GPI-proteins between hcDIG and lcDIG in the ground state and their redistribution in response to mimic insulin incentives.

GPI-proteins are antigens on the cell surface, ectodermally, receptors or cell adhesion molecules expressed in eukaryotes from yeast to humans, and zakalivanie in the outer leaflet of the plasma membrane with covalently attached glycosylphosphatidylinositol (GPI) is epenoy component. Despite the absence of a transmembrane domain, they are involved in signal transmission through this plasma membrane.

The establishment of the fact that GPI-proteins associated with a variety of specialized lipid domains, the so-called detergent-insoluble rich in glycolipids mass, DIG, and not with specific transmembrane binding/linker proteins, suggests the possibility of lipid-lipid interactions as the main binding mechanism of signal transmission, mediated by the GPI-proteins.

The main structural element of the DIG is a lateral set of (Glyco)sphingolipids and cholesterol, which takes the form of an ordered liquid (loin contrast to adjacent parcels disordered liquid (ldin the lipid bilayer of this membrane. The plasma membrane of mammalian cells contain cholesterol (30-50 mol.%) the mixture of lipids, mainly in the ld-domains (for example, phosphatidylcholine with unsaturated tail fractions), and lipids bearing saturated acyl chains predominantly in the lo-domains (e.g., [Glyco]sphingolipids and GPI-lipid). Believe that cholesterol contributes to the dense packing of lipids in lo-domains by filling interstitial spaces between the lipid molecules and the formation of lodomains mentioned is carried out only at certain concentrations of cholesterol.

Insulin is a vital hormone that has a significant impact on the metabolism in the body. In General, it enhances the anabolic processes and inhibits catabolic processes. In particular, it increases the rate of synthesis of glycogen, fatty acids and protein, and inhibits the degradation of protein and glycogen. The vital action of the hormone is to stimulate the cells of the liver, muscles and fat cells to remove from the bloodstream glucose and some other sugars and amino acids.

Bovine insulin consists of two polypeptide chains of the polypeptide And containing 21 AK, and polypeptide, containing 30 AK, which are connected two-S-S (disulfide bridges). The same character patterns observed for insulin many mammals, including humans.

Its structure is compact, similar to a cylinder with only the carboxyl end of the b-chain, acting from the rest of the protein. There are many hydrophobic residues that interact with the formation of the Central hydrophobic core, and vneshnetorgbanke are polar residues on either side, which additionally stabilize the protein. Three disulfide bridges, two intrachain and one miaocheng, fasten this structure.

A common characteristic of the biosynthesis of many proteins, especially proteins, the former is orderway of cells, is that a protein produced in the form of a precursor, then modified, taking final shape in the retention period and before release. Insulin is synthesized by the group of cells in the pancreas called the islets of Langerhans, is stored in granules and, if necessary, is released into the bloodstream.

When insulin is synthesized for the first time, it consists of 100 AK single polypeptide chain consisting of 16 AC signal sequence, In-circuit,-circuit 33 AK, referred to as the connecting chain and a-chain. This structure is called pre-proinsulin (PPI). Suppose that the signal region is responsible for the direction of the PPI from the site of synthesis in the ER (endoplasmic reticulum) of this cell, which accumulates and packs insulin deposited in the form of granules. When the premises specified in ER signal peptide is removed using proteasome enzyme.

Diabetes mellitus is a chronic disease that requires treatment by a doctor for a long time, to limit the development of its devastating complications and how to treat them when they occur. Diabetes is associated with acute and chronic complications, such as hypoglycemia, diabetic ketoacidosis and hyperosmolar Nenetsky syndrome.

Type 1 diabetes occurs in young, lean patient and characterized by a clear inability of the pancreas to secrete insulin due to autoimmune destruction of beta cells. In patients with type 1 diabetes when canceling insulin observe the development of ketosis and ultimately the development of ketoacidosis. Therefore, these patients are dependent on exogenous insulin, which supports their lives.

Type 2 diabetes usually occurs in individuals older than 40 years who have a family history of diabetes. Type 2 diabetes is characterized by peripheral insulin resistance with impaired insulin secretion, which varies in severity. These defects lead to increased gluconeogenesis in the liver, which causes hyperglycemia on an empty stomach. The majority of patients (90%)who had developed type 2 diabetes, obese, and obesity is associated with insulin resistance, which impairs the diabetic condition.

Various other types of diabetes, previously called "secondary diabetes", caused by other diseases or drugs. Depending on the source of the process in question (namely, the destruction of the beta cells of the pancreas or the development of peripheral insulin resistance), these types of diabetes behave like type 1 diabetes or type 2. The most common are diseases of the pancreas, which destroys the beta cells (e.g., hemochromatosis, pancreatitis, cystic fibrosis, malignant is Pujol pancreas), hormonal syndromes, which inhibit insulin secretion (eg, pheochromocytoma), or caused by peripheral insulin resistance (e.g., acromegaly, Cushing's syndrome, pheochromocytoma), and diabetes induced drug (e.g., phenytoin, glucocorticoids, estrogens).

Diabetes mellitus is characterized by improper regulation of glucose level in the serum. When type 1 diabetes is an autoimmune attack on the endocrine pancreas leads to progressive and irreversible destruction of the secreting insulin beta cells. Loss of insulin effect on glucose uptake receptive to insulin cell-target and on the course of metabolism. Type 2 diabetes has multiple etiology, mostly affects cellular resistance to insulin, and is also accompanied by changes in the regulation of glucose levels in the serum. Insulin acts through associated with disulfide heterotetrameric receptor on the cell surface that includes extracellular alpha subunit associated with disulfide bonds with transmembrane and intracellular beta-subunit. When type 1 diabetes the absence of ligand with normal cell receptor structure and function in most cases, causes subsequent metabolic defects. Hormone replace the other therapy in the form of daily insulin injections provides the body with ligand with the action on the receptor, although not necessarily normal physiological way. When type 2 diabetes resistance to the action of insulin often underlies the disease with some resistance due to impaired receptor action.

It is known that in the case of insulin resistance requires more insulin to make use of the insulin receptor to start insulin signaling cascade. The present invention relates to a protein of the cell membrane of adipocytes, which is able to stimulate glucose uptake in bypass signaling pathways triggered by the insulin receptor. It provides effective solution in the absence of a screening tool to identify compounds that could act as alternatives to insulin.

Therefore, the present invention relates to a protein of the plasma membrane of adipocyte, which, apparently, is stabilized by the simultaneous presence of plasmatic membranes and/or lipid vesicles, and/or multiple domains with high cholesterol and/or lipid vesicles, and which has a specific affinity binding to phosphoinositides or phosphoinositide-peptide that is different

and] the ability to run in adipocyte tyr-phosphorylation of the substrate 1 or 2 insulin receptor after the special is practical binding phosphinothricin or phosphoinositide-peptide with the protein and

b] the ability to stimulate in adipocyte glucose uptake after specific binding phosphinothricin or phosphoinositide-peptide with the protein.

The quantity of protein compared to other proteins and/or stabilizing components and/or other compounds (e.g. salts, ion spray) is in the range between 0.01 to 10% based on wet weight.

The amount of this protein is preferably in the range from 0.1 to 5% (based on wet weight, but most preferably is in the range from 0.1 to 1% based on wet weight.

Under natural conditions, the number of the specified protein in plasma membranes is in the range of less than 10-6% in terms of wet mass.

In preferred embodiments of the present invention of phosphinothricin or phosphoinositide-peptide consists of at least one of the following connections:

YCN-PIG, YMN-PIG, PIG37, YCN or lcGce1.

Linking phosphinothricin or phosphoinositide-peptide with the protein occurs, preferably, with a binding constant (KDfrom 0.001 to 10 microns.

The binding constant is a thermodynamic ordered the quantitative description of the equilibrium between disocyanate and medicationoveruse forms complexes between data Belko is and postinstallation or phosphoinositide-peptide.

The binding constant is expressed by the ratio of rate constants of forward and reverse reactions. High values of the binding constant (for example, more than 10 mm) show weak and nonspecific binding, and low values (for example, not more than 100 μm) indicate a strong and specific binding.

The binding constants can be defined in different ways, for example, using equilibrium dialysis, spectroscopy or graphical methods (Scatchard-Plot).

With regard to the plasma membrane of adiposity, it is preferably derived from rat, mouse or human.

The molecular mass of this protein is between 100-120 kDa, preferably between 110-120 and most preferably equal to 115 kDa. It should be noted that the determination of molecular masses of proteins in any way, in particular by SDS-page, with uncertainty ±5-10%.

In addition, the present invention relates to the complex, which is formed from the above-mentioned protein of the present invention and at least one compound from the following group: YCN-PIG, YMN-PIG, PIG37, YCN or lcGce1.

A prerequisite for the formation of the complex is the specific binding of this ligand with the protein. The resulting complex can be stabilized, creating ionic or covalent linkage between ligand and protein.

Now the image is the group applies also to obtain the protein of the present invention, where:

a] adipocytes derived from tissue of the rat, mouse or human,

b] from p. a] allocate plasma membrane of adipocytes,

c] a set of domains with high cholesterol (hcDIG) derived from plasma membranes of p. b]

d] hcDIG of p. c] is treated with a solution of trypsin/NaCl,

e] the incubation mixture of p. d] centrifuged and proteins obtained supernatant separated by electrophoresis in SDS-page (polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate),

f] of this gel elute the protein fraction of the size of 100-120 kDa and solubilizing solution or suspension containing detergent or biological membranes.

In addition, the present invention relates to a method of identifying a compound that specifically binds with the protein of the present invention, in which:

a] get cell fraction, which contains the protein of the present invention,

b] get connection

c] cell fraction from p. a] is brought into contact with the connection point b],

d] determine the binding of this compound with the fraction of cells of p. a]

e] on the binding specificity conclude by comparing the results from p. d] with the results of an experiment in which the same connection as in point b] is brought into contact with the fraction of cells of the same species and/or the same tissue specificity, Thu and cells of p. a], but which does not contain the protein of the present invention, indicating, thus, a higher binding specificity in the case of larger amounts of this compound from p. b]that communicates with the fraction of cells that contains the protein of the present invention, compared with the fraction of cells that do not contain the protein of the present invention.

This fraction of cells taken preferably from adipocytes, skeletal muscle cells, cells of the heart muscle or liver cells. Each type of these cells can be obtained preferably from a mouse, rat or human. This fraction of cells is preferably from cell membranes of all cells, or more preferably from the set of domains with high cholesterol (hcDIG). This compound, which is used for implementing the method of identifying a compound that specifically binds with the protein of the present invention, it is possible to mark a radioactive isotope (for example, With14N3, R32,J121and others) or a fluorescent marker.

Further, the present invention relates to a method of identifying a compound that specifically binds with the protein of the present invention, in which:

a] get the cell, transporting glucose, which contains the protein of the present invention,

b] get connection

p> c] the cell of p. a] is brought into contact with the connection point b],

d] determine the binding of this compound with the cell, transporting glucose,

e] the conclusion that the binding specificity doing on the basis of the comparison results from p. d] with the results of an experiment in which the same connection as in paragraph b], lead in contact with the transporting glucose by cells of the same species and/or the same tissue specificity, and that cells of p. a], but which does not contain the protein of the present invention, indicating, thus, a higher binding specificity in the case of a larger number of connections from point b], communicating with transporting glucose into the cell, which contains the protein of the present invention, compared from transporting glucose into the cell, which does not contain the protein of the present invention.

Transporting glucose into the cell, which does not contain the protein of the present invention, can be obtained from transporting glucose to cells, which contains the protein of the present invention by treatment of the cells that contain the protein of the present invention, a solution of trypsin/NaCl and/or glycosidases.

Transporting glucose into the cell preferably is an adipocyte, a skeletal muscle cell, a cardiac muscle cell or a liver cell. These cells are preferably taken from the culture tkane the or human cells, murine or human origin.

This connection is preferably mark radioactive isotope or a fluorescent marker.

In addition, the present invention relates to a method of identifying a compound that is an agonist or antagonist of the protein of the present invention, in which

a] get transporting glucose into the cell, which is represented by the protein of the present invention,

b] get natural ligand protein of the present invention,

c] get a chemical compound

d] transporting glucose into the cell (a] is brought into contact with the ligand of paragraph b] and chemical compound from p. c]

e] determine the uptake of glucose transport glucose by cells of p. d]

f] determine the uptake of glucose transport glucose by cells of p. d], in which stimulation of the uptake of glucose involves agonistic activity, and inhibition of absorption of glucose involves the antagonistic activity of the compounds p. c].

The ligand of the above method of identifying agonist or antagonist of the protein of the present invention preferably is a YCN-PIG, YMN-PIG, PIG37, YCN or lcGce1. Transporting glucose into the cell in the method of identifying agonist or antagonist of the protein of the present invention preferably is an adipocyte,a skeletal muscle cell, the cell of the heart or liver cell, and preferably originate from species - human, mouse or rat.

The present invention relates to a medicinal product containing the compound, which was identified by the method of identifying a compound that binds to the protein of the present compounds, or which is an agonist or antagonist of the protein of the present invention, and auxiliary connections to create medicines. This medicinal product contains, in a preferred embodiment of the present invention, at least one compound from the following group: YCN-PIG, YMN-PIG, PIG37, YCN or lcGce1.

This drug may also contain a portion or derivative of at least one compound from the following group: YCN-PIG, YMN-PIG, PIG37, YCN or lcGce1.

In addition, the present invention relates to the use of a compound that was identified as a binding protein of the present invention, either as an agonist or antagonist of the protein of the present invention, for obtaining a medicinal product for the treatment of insulin resistance or diabetes.

This connection can preferably be a YCN-PIG, YMN-PIG, PIG37, YCN or lcGce1, or part or derivative of one of these compounds.

EXAMPLES

Chemical is intes PIG(-P): synthesis YCN-PIG (General method see the Fig. 1, 2, 3)

Synthesis of product 2 (Fig. 4; i, ii), 1 product (8.0 g, to 20.6 mmol) from Bachem (Hedelberg, Germany) was dissolved in 200 ml of pyridine, was added 5 g (81,8 mmol) of ethanolamine and 5 ml of N-ethylmorpholine. After settling (16 h, room temperature) with stirring and under 5°was added dropwise 50 ml of acetic anhydride. This reaction mixture was stirred (2 h, room temperature), and then concentrated under high vacuum. The concentrated residue was dissolved in 150 ml of hot methanol, and the resulting solution was concentrated. Then was added 100 ml of methylene chloride/methanol (15/1) and 200 ml ofn-heptane/ethyl acetate (2/1), and this product has led. Output 2: 6,1 g (84%) of white crystals with so pl. 175°C. TLC (thin layer chromatography): methylene chloride/methanol (9/1), Rf=0,7. MS: (M + Li)+=358,2, the calculated value for C16H21N3O6M=351,36.

Synthesis of product 3 (Fig. 4; and (iii), 2.0 g of palladium on coal (10% Pd) were introduced into a solution of the product 2 (12.0 g, 34,0 mmol) in 200 ml of methanol/acetic acid (1/1), and this mixture was first made (2 h, room temperature). The resulting solution was filtered on silica gel and concentrated, and the residue was isolated by purification via flash chromatography (methylene chloride/methanol/concentrated ammonia 30/5/1). Output 3: 7,3 g (98%) of a yellowish oil. TLC: methylene chloride/methanol/koncentrirane the hydrated ammonia (30/5/1), Rf=0,5. MS: (M + Li)+=224,2, the calculated value for C8H15N3O4M=217,23.

Synthesis of product 4 (Fig. 4; iv), 1.5 g (4.5 mmol) of 1(o-(cyano(etoxycarbonyl)-methylidene)amino-1,1,3,3-tetramethylethylenediamine (TOTU), 0.64 g (4.5 mmol) of ethyl-(hydroxyimino)-cyanoacetate (oxime) and 1.7 ml (13.5 mmol) of N-ethylmorpholine were made at 0°With stirring, a solution of 0.8 g (3.7 mmol) of the product 3 and 2.8 g (4.5 mmol) TrtCys(Trt) - OH in dimethylformamide, and the mixture was stirred (2 h, 0°). After adding 200 ml of ethyl acetate the mixture is washed three times with saturated solution of NaHCO3was obezvozhivani over MgSO4and concentrated. The obtained residue was ground in an-heptane/ethyl acetate (6/1) and this product has led. Output 4: 2.2 g (74%) of white crystals with so pl. 185°C. TLC: methylene chloride/methanol (15/1), Rf=0,4. MS: (M + Li)+=811,7, the calculated value for C49H48N4O5S, M=805,0.

Synthesis of 6 product (Fig. 4; v, vi), 4.0 g (5.0 mmol) of the product 4 was dissolved in 200 ml of methylene chloride. Was added 4 ml of water and 3 ml triperoxonane acid. After 15 min the mixture was washed three times with saturated solution of NaHCO3was obezvozhivani over MgSO4and concentrated with a 99%yield of the crude product 5. This crude product was dissolved in 50 ml of methanol and then drops were added 0.5 ml of 1 M solution of methanolate sodium. Che the ez for 15 min was added 50 ml of methylene chloride, and this mixture was filtered on silica gel. After concentrating the filtered solution of the residue was isolated by purification using flash chromatography (methylene chloride/methanol (9/1)). Output 6: 2.2 g (85%) of an amorphous solid. TLC: methylene chloride/methanol (5/1), Rf=0,7. MS: (M + Li)+=527,3, the calculated value for C28H32N4O4S, M=520,6.

Synthesis of product 7 (Fig. 4; vii), 2.7 g (5.2 mmol) of the product 6, 4,2 g (10.4 mmol) Ztyr(Bn) - OH, 3.4 g (10.4 mmol) of TOTU, 1.5 g (10.4 mmol) of oxime and 2 ml of N-ethylmorpholine in 50 ml of dimethylformamide similarly subjected to the interaction with the drug product 4. Output 7: 4,2 g (89%) of white crystals. TLC: methylene chloride/methanol (15/1), Rf=0,25. MS: (M + Li)+=914,8, the calculated value for C25H53N5O8S, M=908,1.

Synthesis of product 8 (Fig. 5; viii), 6.0 g (73 mmol) of phosphorous acid were subjected to four-fold concentration with pyridine, and then placed in 180 ml of anhydrous pyridine. At 10°With dropwise added 13 ml of pivaloate. This reaction solution was left for 45 min at room temperature. This reaction solution was introduced to 16.4 g (to 18.1 mmol) of the product 7 as described above. After 5 hours it was diluted with 200 ml of toluene and 150 ml of methylene chloride/methanol/33% NH3(30/10/3). After concentration the residue of pyridine three times recrystallized from 200 ml that is wala. This residue is suspended in 200 ml of methylene chloride/methanol (20/1). Any insoluble components were filtered and washed twice with 50 ml of methylene chloride/methanol (20/1). The obtained filtrate was concentrated and isolated by purification using flash chromatography. Output 8: 11.6 g (66%) of white crystals. TLC: methylene chloride/methanol/33% NH3(30/5/1), Rf=0,25. MS: (M + Li)+=978,4, the calculated value for C52H54N5O10SP, M=972,08.

Synthesis of product 10 (Fig. 6; ix, x), 4.5 g of product 8 (4.6 mmol) and 6.0 g of the product 9 (2.3 mmol; synthesis was carried out as described previously in reference 47), was dissolved in 80 ml of anhydrous pyridine. After 30 min at room temperature the reaction was cooled to 0°and added 5 ml of water and 1.3 g of iodine. This reaction mixture was stirred (30 min, 10°) and then diluted with 500 ml of methylene chloride, 150 ml of saturated NaCl solution and 30 ml of saturated solution of thiosulfate and stirred for 5 minutes the Organic phase was obezvozhivani over MgSO4and concentrated. The obtained residue was isolated by purification using flash chromatography with methylene chloride/methanol/.NH3(30/5/1-30/10/3). The yield of 10: 8.0 g in the form of amorphous solids. TCS: methylene chloride/methanol (20/1), Rf=0,5. MS: (M + Li)+=3583,6, the calculated value for C207H214N8O42SP2M=3580,0.

Synthesis of p is oduct 11 (Fig. 6; xi), 300 ml of concentrated ammonia at -78°C. it was dissolved 2.1 g (91 mmol) of sodium. This solution was diluted in 150 ml of anhydrous tetrahydrofuran, and then 8.0 g of product 10 (2.2 mmol) of protected final product, dissolved in 50 ml of anhydrous tetrahydrofuran, is slowly dropwise added to the reaction mixture with a temperature of -78°C. After 15 min of flow of the reaction (blue color should not fade), the mixture was carefully treated with 5 g of ammonium chloride. With the disappearance of the blue color of this mixture was carefully diluted with 50 ml of water and 150 ml of methanol. Gave her the opportunity to melt, and then concentrated to about 100 ml of This solution was diluted with 500 ml of methylene chloride/methanol/33% NH3(3/3/1) and was made in flash silicagel column (500 ml silica gel). It consistently suirable with 1 l each, methylene chloride/methanol/33% NH3(3/3/2) and (3/3,5/3). Then this eluruumis product chromatographically usingn-butanol/ethanol/water/33% NH3(2/2/2/1). Output 11: 2.4 g (67% of the product (9) in the form of a white solid. TCS:n-butanol/ethanol/water/33% NH3(2/2/2/1), Rf=0,5. MS: (M + NH3)+=1572,6; the calculated value for C54H88N6O40P2S, M=1555,31. P31NMR (D2O)=15,3 ppm for cyclophosphate and 0.3 for ether phosphoric acid Data for H 1and C13NMR is presented in Table 1.

The synthesis product YCN (Fig. 7; xii), and 11.0 g (11.3 mmol) of the product 7 was subjected to unprotect the same way as the drug product 11. Output YCN: 4.5 g (90%) of white crystals. TLC: methylene chloride/methanol/concentrated ammonia (30/15/5), Rf=0,25. MS: (M + Li)+=448,3, the calculated value for C18H27N5O6S, M=441,51.

The synthesis product YMN-PIG, YMN-PIG synthesized using the same sequence of reactions, which is shown in Fig. 2. Use BocMetOH instead TrtCys(Trt) - OH gave YMN-PIG with similar output in the form of a white solid. TCS:n-butanol/ethanol/water/33% NH3(2/2/2/1), Rf=0,5. MS: (M + NH3)+=1600,6; the calculated value for C56H92N6O40P2S, M=1583,38. P31NMR (D2O)=15,3 ppm for cyclophosphate and 0.3 for ether phosphoric acid.

Getting radioactively labeled and lipolytic split Gce1p (lcGce1p)

Gce1p with intact GPI anchor were isolated by purification from growing on lactate yeast cells that were metabolically labeled with myo-[C14]Inositol and then enzymatically converted into spheroplasts. Plazmaticeski membrane was obtained by selection purification using centrifugation in a gradient of ficoll, solubilizers using 0,35% β-aminocaproate and subjected to TX-114-division. Gce1p, with the holding in rich detergent phase, were isolated by purification by gel-filtration chromatography on Sephadex S-300, chromatography on affinity on Sepharose N6-(2-amino-ethyl)-cAMP and chromatography on phenyl-Sepharose. Elution from the column was accompanied by continuous monitoring of H3-radioactivity. Partially selected cleaning Gce1p besieged (12% polyethylene glycol 6000), then resuspendable in buffer G (25 mm Tris/acetate, pH 7.4, 144 mm NaCl, 0.1% β-aminocoumarin, 0.5 mm DTT, 0.2 mm EDTA, 5% glycerol, 0.1 mm PMSF, 5 μm leupeptin, 1 mm todatetime, 10 μg/ml trypsin inhibitor from soybeans) at a concentration of 0.2 mg protein/ml and subsequently incubated (3 h, 25° (C) in the presence of 6 units/ml PI-specific PLC (B. cereus). After adding ice-cold solution consisting of 2% Triton X-114, 10 mm Tris/HCl (pH 7.4), 144 mm NaCl and separated phases (incubation for 2 min at 37°and centrifugation at 12000×g for 1 min at 25°C)lcGce1p extracted from the upper poor detergent phase. After two additional excerpts from the bottom rich detergent phase by adding an equal volume of 10 mm Tris/HCl, 144 mm NaCl, reconstitution on ice and consistent separation of the phases, United poor detergent phase precipitiously (12% polyethylene glycol 6000).

Radiolabelled lcGce1p suspended in buffer in the absence of β-aminocaproate at 200-1000 decays the minute/µl.

Getting radiolabelled YCN-PIG

Radiolabelled YCN-PIG was prepared from Gce1p by successive treatment with V8 protease (S. aureus) and PI-PLC (B. cereus). YCN-PIG was removed from the poor detergent phase after TX-114-distribution, and then were isolated by purification using cation exchange chromatography (Dowex 50W-X8), gel filtration on BioGel P4, anion-exchange chromatography on SAX-HPLC column, two thin-layer chromatographic razgonom on Si-60-HPTLC plates using different solvent system and, finally, gel filtration on BioGel P4. Elution of the material during each chromatographic separation was accompanied by the measurement of the H3-radioactivity, UV absorption (A220) and insulin-like activity in the stimulation of glucose transport in isolated rat adipocytes. To demonstrate the radiochemical purity of the final drug YCN-PIG was subjected to anion-exchange HPLC on a Dionex CarboPac PA-1, calibrated on a Dionex-complexes by including a standard mixture of glucose oligomer. Internal standards were detected using a pulsed amperometric detector. The emergence of14labeled fragments were monitored continuously monitoring radioactivity using Raytest Ramona. To determine the concentration YCN-PIG hydrolyzed (6 M HCl, 16 h, 110°and determined the amount of inorganic phosphate (2 mol/molecule) and tyrosine (1 mol/molecule). Dehydrated YCN-PIG kept at -80°before use, and then suspended in H2O, containing 2 mm DTT in a final concentration of 100 ám.

Getting rat adipocytes and their incubation with PIG(-P)/YCN

From the fat bodies of the epididymis of male rats Sprague Dawley (140-160 g, fed to satiety) by treatment with collagenase were isolated adipocytes, and incubated them in KRH-buffer (0.14 mm NaCl, of 4.7 mm KCl, 2.5 mm CaCl2, 1.2 mm MgSO4, 1.2 mm KH2PO4, 20 mm Hepes/KOH, pH 7.4)containing 1% (wt./about.) BSA, 100 μg/ml gentamicin, 100 mm 1-methyl-2-phenylethylamine, of 0.5 units/ml adenozindezaminazy, 0.5 ml of nutriiveda and 5 mm D-glucose, in the presence of the PIG(-P)/YCN (dissolved in 20 mm Hepes/KOH, pH 7.4, 2 mm DTT) at 37°on the rocking chair with a water thermostat at a constant bubbling with 5% CO2/95% O2during these periods.

Treatment of rat adipocytes using trypsin/NaCl or NEM

For processing trypsin/NaCl-Ohm, 2 ml suspension of adipocytes (3.5 x 106cells/ml) in KRH buffer containing 5 mm glucose, and incubated (20 min, 30° (C) in the presence of 100 μg/ml of trypsin. Was added trypsin inhibitor from soybeans (final concentration 100 μg/ml) and 2 ml of KRH containing 1 M NaCl and 0.5% BSA, and continued incubation (10 min, 22°). When NEM-treatment of 1 ml suspension of adipocytes (3.5 x 106cells/ml) in KRH containing 5 mm glucose, incubated (30 min, 2° C) together with NEM (1.5 mm final concentration), and then with DTT (15 mm final concentration, 5 min). After these treatments, the cells were centrifuged (1500 xg, 5 min, bucket-rotor), and the bottom layer was removed by suction. In the remaining cell suspension was added 10 ml of KRH containing 0.5% BSA, and then again centrifuged (500 xg for 1 minute, bucket-rotor). After two additional washing stages of the final suspension of these cells was brought to 25 ml with KRH containing 0.5% BSA, 50 mm glucose and 1 mm nutriiveda. Portions of 0.2 ml were analyzed for lipogenesis to control the loss of response to PIG41. Control cells were subjected to the same centrifugation and washing operations, as treated cells, but with H2O instead of trypsin/NaCl. For labelling adipocytes with [C14]NEM this cell suspension was centrifuged (500 xg, 1 min), and the lower layer was removed. Portions of 50 μl (7 x 106cells/ml) were incubated (10 min, 30° (C) with 2.5 µci [C14]NEM in the total volume of 60 ál. After adding 5 μl of 10 mm DTT and 55 μl KRH containing 10 mm glucose, was carried out by trypsin/NaCl-processing, as described above, in a total volume of 200 μl. Portions of 50 μl was carefully layered on top of 200 ál-type layers of oil, consisting of dinnerplate, 0.4 ml-new centrifuge tubes. After centrifugation (5000 xg, 15 sec) these tubes cut through the oil layer. Proteins of this is Reda, contained in the lower part of the centrifuge tubes, besieged (10% THU, two washing with acetone), suspended in buffer laemmli's method for samples and analyzed using SDS-page.

Obtaining plasma membrane, total cell lysates and microsomes

Of the selected rat adipocytes, which are described earlier, received a post-nuclear the bottom layer. For obtaining of plasma membranes 1 ml-new portions were layered on top of 5 ml-new pillows, consisting of 38% (wt./about.) sucrose, 25 mm Tris/HCl (pH 7.4), 1 mm EDTA, and centrifuged (110000 xg, 1 h). Membrane in the interphase between the two layers (0.5 ml) was removed by suction, was diluted with four volumes of buffer for homogenization, and layered on top of 8 ml-purpose pillow, comprising 28% of Percoll, 0.25 M sucrose, 1 mm EDTA, 25 mm Tris/HCl (pH 7.0). After centrifugation (45000 xg, 30 min) plasma membrane from the lower third of the gradient was removed using a Pasteur pipette, diluted with 10 volumes of buffer for homogenization and centrifuged (200000 xg, 90 min). To study the binding obtained washed precipitate suspended in the buffer to bind to a concentration of 1-2 mg protein/ml To obtain total cell lysates post-nuclear the bottom layer was added dezoksiholatom and Nonidet P-40 (final concentration, respectively, 0.3 and 0.2%), incubated (1 h, 4°C) and finally centrifuge is ovale (100000 xg, 1 h, 4°). The obtained supernatant was used for thus. To obtain microsomes were centrifuged post-nuclear supernatant (100000 xg, 1 h, 4°). Received centrifuge the precipitate suspended in the buffer to bind to a concentration of 1-2 mg protein/ml

Getting hcDIG/lcDIG

Dedicated cleaning precipitated by centrifugation of the plasma membrane (0.5-1 mg) suspended in 1.5 ml of ice-cold 0.5 M Na2CO3(pH 11,0)containing 50 mm NaF, 5 mm natriumpyrofosfaatti, 10 μm okadaic acid, 1 mm matriarchate, 20 μm leupeptin, 5 µm pepstatin, 1 μm Aprotinin, 5 mm udacyate, 20 μm PMSF, 1 mm EDTA, and incubated (1 h, 4°With circular stirring, shaking and suction with a pipette). Then this suspension was mixed with an equal volume of 85%sucrose in 15 mm MES/KOH (pH 6.5), 75 mm NaCl and layered it on a 1.5 ml-new pillows, each of which consists of 42,5, 35, 28, 22, 15 and 5% sucrose in the same environment, and centrifuged (230000 xg, rotor SW41 Beckman, 18 h). Light-diffusing opalescent band flocculent substance in 15-22% (fractions 4 and 5) and 28-35% (fractions 8 and 9) the sucrose interfase and flocculent substance in 42,5% pillows (fractions 12-15) were collected as hcDIG, lcDIG and solubilizing plasma membrane proteins, respectively, using needles No. 19 and syringe (0.75 ml per fraction). Density determine the Yali by measuring the refractive index of the indicated fractions. hc/lcDIG characterized enrichment/loss of detectable markers, as described earlier. To study the binding hc/lcDIG suspended in the buffer for binding (15 mm Mes/KOH, pH 6.5), 0.25 M sucrose, 75 mm NaCl, 2 mm MgCl2, 0.5 mm EDTA, 0.5 mm DDT, protease inhibitors).

Binding of radiolabelled YCN-PIG or lcGce1p with subcellular fractions

10 μl of radiolabelled YCN-PIG or lcGce1p (60000-80000 disintegrations per minute/nmol, final concentration 5 μm) was added to 40 μl of suspended plasma membranes, microsomes or hc/lcDIG (40-80 μg protein) in buffer for binding in the absence or in the presence of unlabeled competitor (as indicated in the legend to this figure) in a total volume of 100 μl, and incubated (30 min, 4°). To separate the membranes from the incubation medium 45 ál-purpose aliquot was carefully layered on top of 200 ál-type layers of oil, consisting of dibutyl phthalate and dioctylphthalate (1/1 by vol., the final density 1,012), in the case of plasma membrane/microsome or consisting of dibutyl phthalate and dinnerplate (1/9 on about., the final density 9,863) in the case of hc/lcDIG in 0.4 ml-new pre-cooled (4° (C) centrifuge tubes (microtube No. 72.700, Sarstedt, Germany). After centrifugation (at 48,000 xg, 2 min), these tubes with closed caps cut down on the oil layer on the bottom and the top part (with udalen the mi caps), containing precipitated by centrifugation plasma membrane/microsome assay and the floating hc/lcDIG, which respectively penetrate or not penetrate below the surface layer of oil was transferred into a 10 ml-purpose scintillation vials containing 1 ml of 10%SDS. After thorough shaking (16 h, 25°C)read the radioactivity in 9 ml ACSII-scintillation cocktail (Beckman). Under these conditions, adhering to the walls of the tube and dispersed in the oil layer radioactively labeled YCN-PIG and lcGce1p gave 50-120 disintegrations per minute (i.e. less than 0.5% of the total radioactivity used for incubation) and therefore were not taken into account when calculating data binding. Typically, in accordance with the data defining protein, plasma membranes and microsomes were extracted, respectively, 78-85; and 65-80%, and hcDIG and lcDIG respectively, 83-92% 70-78%.

Chemical synthesis PIG(-P)

Hydrophilic GPI structure can be obtained from natural sources using two experimental methods: (i) PIG-release with GPI-specific PLC/D free GPI lipids in the form of their polar Central piganovich head groups, and therefore not having any amino acid, and (ii) PIG-P, obtained using combined lipolytic and proteolytic cleavage of the GPI-protein, giving a polar Central pianoboy head group together with one or more amino acid is Tami, extracted from carboxylic remaining GPI protein. And GPI GPI lipid and protein balance in the outer leaflet of the plasma membrane of eukaryotic cells together with the Central piganovym head groups are stored in organisms from yeast to humans. To determine the binding of the Central pianoboy head GPI group used the synthesis of radioactive label authentic PIG(P)-structure, which is described in "Müller et al., Endocrinology 138, 3459-3475, 1997"; YCN-PIG received from radiochemically pure GPI-protein, Gce1p, the plasma membrane of S. cerevisiae, which were metabolically labeled with myo-[14With]Inositol by sequential proteolytic and lipolytic cleavage in vitro. To determine the correlation of structure and activity in binding used chemically synthesized YCN-PIG and its derivatives. (Fig. 1: YCN-PIG; Fig. 2: YMN-PIG; Fig. 3: PIG37; 4: YCN).

Synthesis of Tripeptide YCN-PIG was carried out by means known in the art of peptide synthesis. Hexagrid synthesized using trichloroacetimidate method, which is described in "Frick et al., Biochemistry 37, 13421-13436, 1998". Key stage synthesis PIG-P ended with the formation of the phosphodiester bond. From different proven methods most productive H-phosphonate method.

To unprotect a final compounds were carried out under sodium in liquid N 3strengthened by the presence of cysteine (hydration with palladium impossible) and kislotoneustoichiwami cyclophosphate. All compounds were characterized by means of mass, H1NMR, C13NMR and P31NMR-spectroscopy.

Specific binding PIG(-P) with hcDIG

Total plasma membrane obtained with unstimulated adipocytes by differential centrifugation, were enriched (as opposed to total cell lysates) specific marker enzymes of this plasma membrane. Ouabain-sensitivepair-nitrophenylphosphatase (corresponding to the catalytic subunit of Na+/K+-ATPase) was enriched by 9.5 times, and Nuc 10.9 times (in accordance with enzymatic activities), β1-integrin - 13.9 times and syntaxin-1 - 16.4 times (in accordance with immunoblotting), and Gce1 - 7.8 times (in accordance with photoaffinity a tagging). Simultaneously, this drug plasma membrane was depleted (as opposed to total cell lysates) marker sarcoplasmatic reticulum, EGTA-sensitive Ca2+-adenosinetriphosphatase, 5.7 times, and endosomal marker, SCAMP, ("Secretary-vector"/ membrane protein) 37/39, 8.5 times, and GLUT4 (glucose Transporter 4), 16.9 times (in accordance with immunoblotting). Microsome assay of unstimulated is depozitul were enriched, in contrast, total cell lysates, GLUTA4 14.4 times, SCAMP 37/39 8.5 times, the transferrin receptor in 6.9 times and IGFII-receptor 9.7 times, but were depleted, in contrast to the total cell lysates,para-nitrophenylphosphatase in 24.6 times, Gce1 - 12.5 times Nuc - 15.8 times, β1-integrin - 39.5 times and syntaxin-1 - 48.5 times, in accordance with immunoblotting, and Ca2+-adenosinetriphosphatase activity - 19.9 times. This indicates that this fraction represented the primary endoplasmic reticulum and endosome patterns and that it is practically devoid of fragments of plasma membrane and sarcoplasmatic reticulum. hsDIG and lcDIG were obtained from unstimulated adipocytes on the basis of their insolubility in 0.5 M Na2CO3(pH 11,0) and low buoyant density by centrifugation in a density gradient of sucrose. They were characterized by the loss of their (as opposed to total plasma membranes) GLUT4 and β-subunit of the insulin receptor. hcDIG and lcDIG differed from each other at a much higher enrichment of caveolin is the essential component, pp59Lynand Gce1 in hcDIG compared to lcDIG.

Selected subcellular membrane fractions were incubated with increasing amounts of radiolabelled YCN-PIG, and the incubation was completed rapid separation from the incubation medium price is trifoliorum through the layer of oil proper density.

Membrane-associated YCN-PIG was mainly extracted with hcDIG, and to a lesser extent with lcDIG, concentration-dependent and satisfying way, while the cytoplasmic membrane and microsome assay were virtually no radioactive label (Fig. 5). In the linear range of the nonspecific binding YCN-PIG with hcDIG was less than 20%, which is estimated at 500-fold excess of its synthetic YCN-PIG or other competitors (Fig. 5). Subsequent experiments were performed using concentrations YCN-PIG, corresponding to the end of the binding in the linear range.

Other ways of determining the receptor-ligand interaction, such as rapid filtration and centrifugation on the basis of sedimentation and density did not allow to detect specific binding YCN-PIG with any membrane sub-fractions of adiposity (data not shown), probably due to the binding framework for the affinity and/or high speed dissociation. Scatchard-graphical analysis showed Kdin the range of 50 nm-500 nm and Bmax50-200 pmol per mg protein hcDIG. The binding specificity YCN-PIG with hcDIG demonstrated by significantly reduced the efficiency of peptide variants, YMN-PIG and PIG37 not containing peptidylarginine-component, as well as a very low level one only peptidylarginine-component, YCN, competitive the analysis (6).

Combining unlabeled YCN and PIG37 (equimolar ratio) shifted the binding of radioactively labeled YCN-PIG with hcDIG only slightly less efficient than its YCN-PIG, and much more than just a PIG or peptidylarginine-component, and YMN-PIG. This fact demonstrates the simultaneous and synergistic recognition PIG and peptidylarginine-components. IC50competition was only 3-4 times higher for YCN and PIG37 compared with covalently attached YCN-PIG (Fig. 6). Additionally clarified the question of whether the identified binding site for PIG(-P) has relcovaptan nature. hcDIG pre-treated trypsin/NaCl or NEM, and then incubated with increasing concentrations of radioactively labeled YCN-PIG in the absence or in the presence of excess unlabeled synthetic YCN-PIG (to assess nonspecific binding).

Sequential treatment with trypsin and 0.5 M NaCl or treatment with NEM completely annulled specific binding of radioactively labeled YCN-PIG with hcDIG, and trypsin or NaCl alone or NEM in the presence of DTT had no significant effect (Fig. 7). An identical example of inactivation was observed at a lower interaction by affinity YCN-PIG with lcDIG. These data suggest the existence of sensitive to the action of trypsin/NaCl and NEM binding protein PIG(-P) with DIG cell the surface adipocyte. Preference YCN-PIG in the binding hcDIG compared to lcDIG was confirmed as a result of their transformation in the process of depletion of cholesterol in the plasma membrane of adipocytes using m-βCD and subsequent analysis hc/lcDIG on the specific binding of radioactively labeled YCN-PIG. In control adipocytes main part of the YCN-PIG was extracted with hcDIG, compared with 20%balance associated with lgDIG (Fig. 8). However, treatment of intact rat adipocytes using m-βCD (1-10 mm) were found concentration-dependent decrease in the number YCN-PIG associated with hcDIG, accompanied by a corresponding increase lcDIG. Treatment of adipocytes trypsin/NaCl or NEM after depletion of cholesterol, but before obtaining DIG, significantly worsened the specific binding YCN-PIG and hcDIG, and lcDIG (data not shown). These data indicate that the vast placement of the PIG(P)-receptor in hcDIG rat adipocytes, which formation is highly dependent on cholesterol.

Lipolytic cleavage of the GPI-protein, specifically associated with hcDIG

PIG-component, -NH-(CH2)2-O-PO(OH)O-6Manα1(Manα1-2)-2Manα1-6Manα1-4GluN1-6Ino-1,2-(cyclo)-phosphate, from YCN-PIG, YMN-PIG and PIG37 (Fig. 1, 2 and 3), is identical to the polar pianoboy head group in all eukaryotic GPI-proteins. So to find out whether b is loopology the binding site for PIG-P interacts with lcGPI-proteins, namely, whether or not he recognizes PIG(P)-component in accession to complete the polypeptide part of the GPI-protein. In order to obtain radiolabelled lcGPI-protein, Gce1p from metabolically labeled cells of S. cerevisiae were treated with PI-specific PLC (B. cereus), and hydrophilic split the product was isolated by purification to radiochemical homogeneity. Using the same method centrifugation in oil for PIG(-P), found that lcGce1p concentration-dependently associated with a DIG of the selected rat adipocytes and satisfying way - with hcDIG that 11-15 times more effective than lcDIG. The nonspecific binding in the presence of 200-fold molar excess of unlabeled lcGce1p was less than 15% of the total lcGce1p learned to DIG in nanasawa concentrations lcGce1p. According to Scatchard-graphical analysis, Kdfor lcGce1p-binding hcDIG is in the range of 0.1 to 1.0 μm and Bmaxwith 70-200 pmol per mg protein hcDIG. Total plasma membrane and microsome assay did not detect specific binding lcGce1p. Thus, hcDIG plasma membrane of adipocytes, obviously, included specific binding sites for lcGce1p of yeast. Further analysis of the identity of the binding sites for PIG(-P) and lcGPI-proteins, as shown by the similar values of Kdand Bmaxrelative Wed the rotary synthetic PIG(-P)connections for lcGce1p-binding site in hcDIG, compared to competitive research (Fig. 9).

The binding of radioactively labeled lcGce1p with hcDIG was offset by an excess (more than 500-fold) labeled synthetic YCN-PIG, YMN-PIG and YCN plus PIG37, more than 75% of the total lcGce1p binding, confirming the specificity of the interaction lcGce1p and hcDIG. Competition binding lcGce1p with PIG37 and YCN was significantly less effective. The relative ranking of the various PIG(-P), which reflects the average value of the IC50push lcGce1p of hcDIG amounted YCN-PIG>YCN+PIG37>YMN-PIG>PIG37>YCN and, thus, identical to that inhibits the binding YCN-PIG (6). In addition, the average values of the IC50very similar to competition lcGce1p - YCN-PIG-linking, indicating that in both cases recognized by the same determinants, and residual protein component of the GPI-protein (except carboxykinase tributyltinchloride-residue) do not contribute to the binding. Further, the sensitivity of the interaction lcGce1p c hcDIG to trypsin/NaCl - and NEM-treatment of intact rat adipocytes was studied in conditions that are almost completely disrupt the binding of the labeled isotope YCN-PIG (Fig. 7). hcDIG of trypsin/NaCl-and NEM-treated adipocytes showed a relationship with radioactively labeled lcGce1p, not exceeding the nonspecific binding in the presence of 500-fold excess unlabeled YCN-PIG (which accounts for about the olo 30% of the total Gce1p, retrieved from hcDIG of untreated control cells) (Fig. 10). In contrast, incubation of adipocytes with NEM in the presence of an excess of DTT (Fig. 10), or separately with trypsin or NaCl (data not shown), did not impair the binding of radioactively labeled lcGce1p and its competition with 3 μm YCN-PIG37, 5 μm PIG37 and 10 μm YCN compared with untreated cells. Taken together, the sites that specifically binds YCN-PIG and lcGec1p, showed very similar characteristics regarding localization in hcDIG the plasma membrane of adipocytes, absolute and relative affinity (with structural derivatives), the level of expression and sensitivity in relation to the trypsin/NaCl and NEM.

Endogenous ligands for receptor PIG(-P) and lcGPI-proteins

Candidates for physiological ligands undoubtedly identical binding sites for the PIG(-P)and lcGPI-proteins are darassalam GPI structure, namely GPI lipids and/or GPI-anchor proteins. To test this assumption, the selected rat adipocytes were subjected to treatment with various GPI-specific PL and subsequent salt wash (0.5 M NaCl) to obtain hcDIG order to specifically remove the estimated endogenous GPI molecules that interact with the receptor and thereby masked binding sites for YCN-PIG/lcGce1p.

Incubation of rat adipocytes with increasing the concentration of the through publications PI-specific PLC from B. cereus or GPI-specific PLD from human serum gave a concentration-dependent increase in the number of radioactively labeled YCN-PIG and Gce1p that are specifically associated with hcDIG (Fig. 11). The effectiveness of lipolytic treatments demonstrated in parallel, by loss Gce1p and Nuc have hcDIG.

Their loss, respectively, 75% and 65%, correlated with increased binding YCN-PIG or lcGce1p with hcDIG up to 200% and 260%. The specificity of GPI-splits demonstrated an inability PC-specific PLC (B. cereus) and PLD from cabbage (which do not attack the GPI structure) to a significant displacement Gce1p or Nuc from hcDIG, as well as to stimulate binding YCN-PIG (lcGce1p) hcDIG (Fig. 11, 12). Scatchard-graphical analysis of specific binding with hcDIG of PI-specific pre-treated PLC adipocytes (nonspecific binding was not changed) showed that increased Association of radioactively labeled YCN-PIG/lcGce1p due mainly 2-3-fold increase in Bmaxand almost invariant Kd. These data indicate that about 50% of the binding sites in PIG(-P)or lcGPI-proteins in hcDIG in selected rat adipocytes in the ground state, busy endogenous GPI structures, split through (G)PI-specific PLC/D. it is Noteworthy that insulin at physiological concentrations imitated to some extent the effect of GPI-specific the PLC/D processing in the rat adipocytes, causing moderate, but still significant decrease Gce1p and Nuc in hcDIG. Insulin-induced loss of GPI-proteins from hcDIG leads to a distinct increase in binding abilities YCN-PIG or lcGce1p (Fig. 11, 12).

In addition, we could demonstrate that the receptor for PIG(-P)and lcGPI-protein identical to 115 kDa - protein, sensitive to the action of trypsin/NaCl and NEM, which is called CIR.

Linking PIG-P with this receptor affects its availability for subsequent covalent modification with NEM and/or to cleavage and release from the cell surface adipocyte using trypsin/NaCl.

Rat adipocytes were incubated with PIG(-P) and then were subjected to labeling with [C14]NEM and treatment with trypsin/NaCl. Using phosphorescent image and SDS-page analysis released radioactively labeled polypeptide and (Fig. 13), found that PIG(-P) reduced the stitching 115 kDa-polypeptide with [C14]NEM, and/or remove it from the lower layer of adipocytes after trypsin/NaCl-processing. Reduction using YCN-PIG or PIG37 3 μm and YCN 30 μm were, respectively, 83, 65, and 28%, compared with control cells. This protein was only the main NEM-labeled component, which was released from plasma membranes with trypsin/NaCl, but not by using any one treatment (Fig. 13), and Eden is icen CIR. In accordance with the experimental proof of the existence of endogenous ligands (e.g., GPI-proteins) and their removal from the corresponding binding site lipolytic cleavage (see Fig. 11, 12), treatment of adipocytes with exogenous PI-specific PLC (B. cereus) or insulin slightly, but reproducibly stimulated trypsin/NaCl-dependent release of [C14]NEM-labeled CIR, respectively, 30% and 20% (Fig. 13). As the relative ratio of the released CIR from the cell surface adipocyte using trypsin/NaCl-treatment, compared to tripsinova treatment with NaCl treatment (100/20/10), was roughly comparable in the control PIG(P)-stimulated PLC and/insulin-treated cells, the binding of PIG(-P) and endogenous GPI-ligands with hcDIG apparently weakens the tagging CIR using NEM, not trypticase splitting. This is due to conformational changes CIR detected in the result of the interaction of ligands with PIG(P)-receptor in hcDIG the plasma membrane of adipocyte.

Table 1

H1and C13chemical shifts of the signal [ppm] for YCN-PIG in D2O, pD=8,1 (ncorr.)
BalancePositionH1[ppm]C13[ppm]
Tyrosine CO-?
α4,1255,18
β2,99, 3,0337,05
γ-125,90
δ7,05131,20
ε6,77116,57
ξ-155,30
CysteineCO-?
α4,56not defined
β2,64, 2,7137,35
AsparagineCO-not defined
α4,58not defined
β2,89, 3,0537,05
γ--?
Ethanolamine1not definednot defined
2not definednot defined
Mannose-I1is 4.93102,84
23,9670,83
3to 3.7371,05
4of 3.64to 6.19
5to 3.6773,91
6not definednot defined
Mannose II15,18101,40
24,0179,10
3a 3.8770,60
43,7067,12
53,76to 72.87
6not definednot defined
Mannose III1to 4.9899,07
23,8979,69
33,5973,45
4not definednot defined
5not defined70,85
6not definednot defined
Mannose IV15,08102,62
23,9570,91
33,6871,08
43,5167,60
5to 3.7373,21
6not defined67,15
14,86100,12
23,0057,00
33,7572,89
4to 3.5877,88
5of 3.4675,99
63,65, 3,7861,68
Inositol14,3578,52
2to 4.6278,09
33,6270,24
4of 3.5672,60
53,3772,57
63,9682,39

List of Figures

Fig. 1: General scheme of the synthesis of the PIG, part 1

Fig. 2: General scheme of the synthesis of the PIG, part 2

Fig. 3: General scheme of the synthesis of the PIG, part 3

Fig. 4: Synthesis YCN-PIG, part 1

Fig. 5: Synthesis YCN-PIG, part 2

Fig. 6: Synthesis YCN-PIG, part 3

Fig. 7: Synthesis YCN

Fig. 8: Chemical formula YCN-PIG

Fig. 9: Chemical formula YMN-PIG

Fig. 10: Chemical formula PIG37

Fig. 11: Chemical formula YCN

Fig. 12: Specific binding PIG(-P) with hcDIG. The increased amount of radioactively labeled YCN-PIG allocated from S. cerevisae, incubated (1 h, 4° (C) with hcDIG (6.5 µg protein), lcDIG (6.5 µg), plasma membrane (47,5 g) and microsomes (68 g) of the separation is controlled rat adipocytes.

The membrane fraction/DIG was subjected to centrifugation in the oil layer was extracted with/from the sediment / upper oil layer was solubilizers and read radioactivity. Specific binding was calculated as the difference between the radioactivity measured in the absence and in the presence of 10 μm unlabeled YCN-PIG. Each point corresponds to the average ± standard deviation (SD) of the three incubations using at least 4 different membrane preparations.

Fig. 13: Specic binding of PIG-P with hcDIG:

Radioactively labeled YCN-PIG (18000-22000 disintegrations per minute) were incubated (1 h, 4° (C) with hcDIG (6.5 µg protein) in the absence or in the presence of increasing amounts of unlabeled YCN-PIG, YCN+PIG37, YMN-PIG, PIG37 and YCN (competition). The membrane fraction/DIG was subjected to centrifugation in the oil layer was extracted with/from the sediment / upper oil layer was solubilizers and read radioactivity.

Fig. 14: characterization of the binding site for PIG-P hcDIG

An increasing number of radioactively labeled YCN-PIG secreted from S. cerevisiae were incubated (1 h, 4° (C) with hcDIG (6.5 µg protein) of the selected rat adipocytes that were pre-treated with trypsin/NaCl, trypsin, NEM+DDT, NaCl or NEM or left untreated (Control). DIG was subjected to centrifugation in a layer of oil, extracted from the upper oil layer was solubilizers and MF is Tivoli radioactivity. Specific binding was calculated as the difference between the radioactivity measured in the absence and in the presence of 10 μm unlabeled YCN-PIG. Each point corresponds to the average ± standard deviation (SD) of the three incubations using at least 3 different adiposity pre-processing.

Fig. 15: characterization of the binding site for PIG-P hcDIG

Radioactively labeled YCN-PIG (12000-18000 disintegrations per minute) were incubated (1 h, 4° (C) certain (proportional) quantities hcDIG and lcDIG obtained from selected rat adipocytes, which was pre-treated (50 min, 30° (C) increasing concentrations of m-βCD, or left untreated. DIG was subjected to centrifugation in a layer of oil, extracted from the upper oil layer was solubilizers and read the radioactivity measured in the absence and in the presence of 10 μm unlabeled YCN-PIG. Each point corresponds to the average ± standard deviation (SD) of the three incubations using at least 3 different pre-treatments of adipocytes.

Fig. 16: Specific binding lcGce1p c hcDIG

Radioactively labeled Gce1p obtained from S. cerevisiae and treated with PI-specific PLC (B. cereus), incubated (1 h, 4° (C) with hcDIG (6.5 µg protein)isolated from untreated rat adipocytes, in the absence or in prisutstvuyuschee PIG-P. hcDIG was subjected to centrifugation through a layer of oil, was solubilizers and read radioactivity.

Each point corresponds to the average ± standard deviation (SD) of four incubations using at least 3 different hcDIG drugs and pre-treatment of adipocytes, respectively.

Fig. 17: Specific binding lcGce1p c hcDIG.

Radioactively labeled Gce1p obtained from S. cerevisiae and treated with PI-specific PLC (B. cereus), incubated (1 h, 4° (C) with hcDIG (6.5 µg protein)isolated from adipocytes that were pre-treated with trypsin/NaCl, NEM, NEM+DTT or left untreated (Control), in the absence or in the presence of its YCN-PIG (final concentration 3 μm), YCN+PIG37 (3 μm), PIG37 (5 μm) and YCN (10 µm). hcDIG was subjected to centrifugation through a layer of oil, was solubilizers and read radioactivity. Each point corresponds to the average ± standard deviation (SD) of four incubations using at least 3 different hcDIG drugs and pre-treatment of adipocytes, respectively.

Fig. 18: the Impact of PL and insulin treatment of adipocytes binding YCN-PIG and lcGce1p c hcDIG. Selected rat adipocytes (h7cells/ml) were incubated (30 min, 30° (C) with the specified quantities of PI-specific PLC (B. cereus), PC-specific PLC (B. cereus), GPI-specific PD (human serum) or PLD (cabbage) or human insulin in a total volume of 2 ml with a weak shaking under 5% CO 2/95% O2. After adding 2 ml of 1 M NaCl, the adipocytes were washed by flotation. Allocated hcDIG, and 6.5 ml-purpose aliquots were incubated (1 h, 4° (C) using radioactively labeled lcGce1p derived from S. cerevisiae, and YCN-PIG (15000-25000 disintegrations per minute) in the absence or in the presence of unlabeled YCN-PIG (final concentration 10 μm)were subjected to centrifugation through a layer of oil, extracted from the upper oil layer was solubilizers and read radioactivity. Specific binding was calculated as the difference between the absence and presence of YCN-PIG.

Each point corresponds to the average ± standard deviation (SD) of the three incubations using at least two different hcDIG drugs.

Fig. 19: the Impact of PL and insulin treatment of adipocytes binding YCN-PIG and lcGce1p c hcDIG. Selected rat adipocytes (h7cells/ml) were incubated (30 min, 30° (C) with the specified quantities of PI-specific PLC (B. cereus), PC-specific PLC (B. cereus), GPI-specific PLD (human serum) or PLD (cabbage) or human insulin in a total volume of 2 ml with a weak shaking under 5% CO2/95% O2. After adding 2 ml of 1 M NaCl specified adipocytes were washed by flotation. Allocated hcDIG, and 6.5 ml-purpose aliquots were incubated (1 h, 4° (C) using radioactively labeled lcGce1p obtained from S. cerevisiae, and YCN-PIG (15000-25000 disintegrations per minute) in the absence or in the presence of elchinova YCN-PIG (final concentration 10 μm), was subjected to centrifugation through a layer of oil, extracted from the upper oil layer was solubilizers and read radioactivity.

Fig. 20: Effect of PIG(-P), PI-specific PLC and insulin on NEM-tagging CIR. Selected rat adipocytes were incubated (30 min, 37° (C) in the absence (Control) or in the presence of PIG37, YCN-PIG, YCN, PO-PLC (B. cereus) or insulin in this concentration and then were labeled with [C14]NEM. After processing, the trypsin/NaCl, as indicated, the adipocytes were separated from the incubation medium by centrifugation through a layer of oil.

Proteins extracted from the environment (below the layer of oil) and were separated in SDS-page.

Phosphorescent image presented from the usual experiment with three repetitions with similar results. Quantitative evaluation of four different incubations of adipocytes with three dimensions is given as independent units (average±SD) number of CIR released from trypsin/NaCl-treated control cells taken as 100.

1. Protein of the plasma membrane of adipocyte, which has

affinity specific binding with postinstallation or postinstallation characterized

a) molecular weight of 115 kDa,

b) the ability to run tyr-phosphorylation of the substrate insulinretseptorny substrate proteins IRS-1 and IRS-2 in adipocyte p the following specific binding phosphinothricin or postinstallscript with this protein and

C) the ability to stimulate the consumption of glucose by adipocyte after specific binding phosphinothricin or postinstallscript with this protein.

2. The protein according to claim 1, in which phosphinothricin or postinstallscript consists of at least one compound from the following group: YCN-PIG, YMN-PIG, PIG37, YCN or IcGcel.

3. The protein according to claim 1, in which phosphinothricin or postinstallscript associated with a given protein with a binding constant of 0.001-10 μm.

4. The protein according to claim 1, in which phosphinothricin or postinstallscript associated with a binding constant of 0.001-1 μm.

5. The protein according to claim 1, in which the adipocyte comes from rat, mouse or human.

6. Complex to run tyr-phosphorylation insulinretseptorny substrate proteins IRS-1 and IRS-2, formed by the protein according to claim 1 and at least one compound from the following group: YCN-PIG, YMN-PIG, PIG37, YCN or IcGcel.

7. A method of obtaining a protein according to claim 1, comprising the following stages:

(a) adipocytes derived from tissue of the rat, mouse or human,

b) from adipocytes secrete plasma membrane

c) from plasma membranes have many domains with high cholesterol (hcDIG),

d) hcDIG treated with a solution of trypsin/NaCl,

e) centrifuged incubation mixture n.d], and proteins, and the obtained supernatant separated using SDS-page,

f) protein fraction size 115 kDa elute from the gel and, if possible, solubilizers with a solution or suspension containing detergent or biological membranes.



 

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