RU2451024C1 - Analysis of arg-x proteolysis and inhibition thereof in non-histone and histone proteins of supramolecular structures of plant cell nuclei - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: for analysis of Arg-X proteolysis and inhibition thereof in non-histone and histone proteins of supramolecular structures of plant cell nuclei, nuclei are separated from the endosperm at certain time intervals from the beginning of soaking. The nuclei are preserved. Nuclear fractions are extracted and histone and non-histone proteins are separated through ion-exchange chromatography with amberlite IRC-50 in discontinuous gradient of guanidine hydrochloride. Sites of Arg-X protease- and inhibitor-trypsin activity are determined in the proteins.

EFFECT: high accuracy of analysis.

Description

The invention relates to biochemistry and molecular biology of a eukaryotic cell and can be applied to the analysis of molecular genetic mechanisms of the formation of cell nucleus structures and the role of protein components in their organization, which is necessary to obtain additional information in the development and construction of computer models for the organization of gene and epigenic control networks.

A known method of producing nuclear fractions with proteinase and inhibitory activity [1], in which proteolytic and inhibitory activities were determined in the suprastructures of plant cell nuclei. The disadvantage of this method is that the analysis was carried out in total fractions of proteins, without dividing them into histone and non-histone proteins using ion exchange chromatography on amberlite IRC-50.

There is a method of preparative isolation of the main proteins of plant cell nuclei [2], which described a method for fractionation of histones using ion-exchange chromatography on a column with amberlite IRC-50. The disadvantage of this method is that the sites of Arg-X proteinase and trypsin inhibitor activity were not determined in the obtained nuclear fractions.

The above method of preparative isolation of the main proteins of cell nuclei [2] was taken as the basis in which initially, at certain time intervals from the start of soaking, 0 hours, 3 hours, 6 hours, 9 hours, 12 hours, 15 hours, 18 hours, 21 hours separation of the embryos from the endosperm followed by preservation of the embryos in 80-90% glycerol buffered at minus 25 ° C, cell nuclei are isolated, nuclear fractions are extracted with increasing concentrations of 0.14 M, 0.35 M, 2 M sodium chloride and 6 M guanidine hydrochloride with 0.1% (β-mercaptoethanol and isolated from the above RAC basic proteins by ion exchange chromatography with Amberlite IRC-50 in a discontinuous gradient of guanidine hydrochloride 6%, 8.9%, 10.6%, 13%, 40% 0.1 M potassium phosphate buffer, pH 6,8.

The purpose of the invention is a method for producing non-histone and histone proteins with Arg-X sites of protease and trypsin inhibitor activity from supramolecular structures of plant cell nuclei.

This goal is achieved by the fact that in the method for producing non-histone and histone proteins with Arg-X sites, protease and inhibitory trypsin activity from supramolecular structures of plant cell nuclei initially at certain time intervals from the start of soaking 0 h, 3 h, 6 h, 9 h 12 hours, 15 hours, 18 hours, 21 hours, the embryos are separated from the endosperm, followed by preservation of the embryos in 80-90% glycerol buffered at minus 25 ° C, cell nuclei are isolated, nuclear fractions are extracted with increasing concentrations of 0.14 M, 0 , 35 M, 2 M chloride about sodium and 6 M guanidine hydrochloride with 0.1% β-mercaptoethanol, histone and non-histone proteins are isolated from the above fractions by ion exchange chromatography with IRC-50 amberlite in a discontinuous guanidine hydrochloride gradient: 6%, 8.9%, 10.6%, 13%, 40% on 0.1 M potassium phosphate buffer, pH 6.8, and the sites of Arg-x protease and trypsin inhibitor activity were determined in them.

The invention is illustrated by the following example.

Example. The experiments were carried out on elite seeds of wheat (Triticum aestivum L.) of the varieties Artemovka, Mironovskaya 808 and Mironovskaya spring (kindly sent to us from the collection of the GNU of the All-Russian Scientific Research Institute of Plant Production named after N.I. Vavilov). Germination was carried out in the dark at 22 ± 1 ° C. At certain time intervals - 0 h (air-dry seed) and from the beginning of seed soaking: 3 hours, 6 hours, 9 hours, 12 hours, 15 hours, 18 hours, 21 hours, the embryos were separated from the endosperm. Cell nuclei were isolated according to the method of [3]. Supramolecular structures: nucleoplasm (Hp), chromatin, weakly (Chr-I) and strongly (Chr-II) bound to the nuclear matrix (NM), as well as NM were isolated from purified cell nuclei, respectively, with an increase in the ionic strength of the solution: 0, 14 M NaCl, 0.35 M NaCl, 2 M NaCl in 0.01 M Tris-HCl buffer, pH 6.8. NMs were extracted with 6 M guanidine hydrochloride (Gu · HCl) with 0.004% (β-mercaptoethanol in the same buffer. The amount of protein in the nuclei and nuclear fractions was determined by the Bradford method in our modification [1]. The obtained superstructures of cell nuclei were passed through an amberlite column IRC-50 (a polymethacrylic synthetic resin with free carboxyl groups). The resin was used in the form of powder obtained by grinding in a ball mill. The crushed resin was sieved through a 200 mesh sieve, and then repeatedly washed with water to remove the smallest particles were dried and washed with acetone. Cyclization was carried out to achieve greater chromatographic efficiency. 500 ml of 4 N NaOH was added to 100 g of the resin, stirred for 3 hours, followed by washing on the filter with water until neutral and transferred to an acid form, passing 500 ml of 4 M HCl through it, excess HCl was removed by washing with water. A 0.4 × 4.5 cm column was filled with this resin. 20-50 μg protein dissolved in 6% guanidine hydrochloride per 0.1 M potassium was applied to the column. phosphate buffer, pH 6.8. The elution rate was 6 ml / h. Preparative separation of the main proteins of the proteome of cell nuclei was carried out in an intermittent gradient of guanidine hydrochloride: 6%, 8.9%, 10.6%, 13%, 40% in 0.1 M potassium phosphate buffer, pH 6.8. Using the method described above, it was possible to separate the proteins of the superstructures of cell nuclei into 5 fractions, which correspond to the fractions according to the concentration of guanidine hydrochloride in the eluate: 0 fraction enriched in non-histone proteins and HMG proteins; fraction enriched in histone H1; fraction enriched in histones H2A and H2B; a fraction enriched in histones H3 and H4; and finally, a fraction containing residues of histones H3 and H4 mixed with proteins of the nuclear matrix. The protein content in the eluates was determined by the Bradford method in our modification [1]. In the obtained fractions of non-histone and histone proteins of plant cell nuclei, the sites of Arg-X protease and trypsin inhibitory activity were determined. Argh-x protease activity was determined by the cleavage of low-molecular-weight protamine protein (Calbiochem, USA) [1] (whose molecule consists of 33 amino acids: 22 Arg molecules; 4 Ser molecules; 3 Pro molecules; 2 Glu molecules and Val). Activity was expressed in nmol arginine · s ^-1 · mg ^-1 protein.

Analysis progress: 0.02-0.025% protamine sulfate solution was prepared on 0.1 M phosphate buffer, pH 7.0. 0.2 ml of protamine sulfate was added to 0.5 ml of the enzyme solution (in 0.1 M phosphate buffer, pH 7.0), incubated at 37 ° C for 20 min. The reaction was stopped by the addition of 0.7 ml of 20% TCA (trichloroacetic acid). Protamine sulfate was added to control samples after addition of TCA. A measure of enzyme activity was the amount of arginine-containing peptides soluble in TCA formed from protamine sulfate when it is cleaved with trypsin or trypsin-like proteins of the cell nucleus. After the addition of cold 20% TCA, the experimental and control samples were thoroughly mixed and centrifuged at 1500 g for 15 minutes. All operations were carried out at 0-4 ° C. 1.25 ml of a 0.04% solution of 8-hydroxyquinoline (Reachim) (prepared on the day of the test by five-fold dilution with cold distilled water of a 0.2% solution of 8-hydroxyquinoline dissolved in 96 ° ethanol was added to 0.5 ml of a clear centrifugate ), then 0.25 ml of 10% NaOH was added to the sample, stirred and left to stand for 2 min, after which 0.05 ml of NaBrO (prepared by dissolving 1 g of bromine in 100 ml of 5% NaOH) was added and after 15 s introduced 0.25 ml of 40% urea, after another 40 s - 2.75 ml of cold distilled water. After 5 minutes of standing, the optical density of the solutions was determined at A ₄₉₀ (FEK-56 M, USSR).

Using a calibration curve, the absorbance at A _{490 was} converted to the number of micromoles of arginine in the sample. D, L-arginine (Reanal, Hungary) was used as a standard for the calibration graph. Proteolytic activity was calculated by the formula

Designations in the formula:

V is the volume (ml) of TCA of the filtrate taken for dialysis;

ΔС is the amount of arginine in ng (in TCA filtrate);

1.4 is the total volume (ml) of the reaction mixture;

T is the incubation time (s);

V ₁ - volume (ml) of nuclear extract taken for incubation;

174.2 - molecular weight of arginine;

B - the amount of protein (mg) in 1 ml of nuclear extract.

The analysis for the determination of trypsin inhibitory activity differed from the above analysis for the determination of Arg-X proteolytic activity in that 0.0002% trypsin (SPOFA, Czechoslovakia) was added to the incubation medium in a volume of 0.2 ml, prepared on 0.1 M phosphate buffer pH 7.0. Due to the fact that the lots of trypsin differed in enzymatic activity, for each batch the concentration of the desired trypsin activity (readings of the optical density of the reaction mixture should be in the range 0.02-0.05 (FEK-56M)) was selected individually. The activity of trypsin inhibitors was expressed in nmol arginine · s ^-1 · mg ^-1 protein. The determination of the activity of trypsin inhibitors was calculated by the formula

ΔС _к - ng of arginine formed during incubation (for 20 min at 37 ° C) of protamine sulfate and trypsin; ng arginine was found by the calibration curve;

ΔC _o - ng of arginine formed during incubation (for 20 min at 37 ° C) of protamine sulfate, trypsin and an inhibitor of the nuclear trypsin fraction, the latter obtained from a nuclear extract; ng arginine was found by the calibration curve;

V is the volume (ml) of the reaction mixture;

V ₁ - volume (ml) of TCA of the filtrate taken for analysis;

T is the incubation time in s;

V ₂ - volume (ml) of nuclear extract;

174.2 - molecular weight of arginine;

B - the amount of protein (mg) in 1 ml of nuclear extract.

The effectiveness of sequential stepwise extraction of histone and non-histone proteins from the nucleoplasm having Arg-X protease and trypsin inhibitor activity is shown in Fig. 1. The ordinates show the activity of proteases (1) and trypsin inhibitors (2) expressed in nmol of arginine per 1 mg of protein / s. The abscissa axis shows the germination time of wheat germ.

Figure 2 presents the effectiveness of sequential stepwise extraction of histone and nonhistone proteins from chromatin, loosely bound to the nuclear matrix (Chromatin-I), with Arg-X protease and trypsin inhibitor activity. The ordinates show the activity of proteases (1) and trypsin inhibitors (2) expressed in nmol of arginine per 1 mg of protein / s. The abscissa axis shows the germination time of wheat germ.

Figure 3 presents the efficiency of sequential stepwise extraction of histone and non-histone proteins from chromatin firmly bound to the nuclear matrix (Chromatin-II) with Arg-X protease and trypsin inhibitor activity. The ordinates show the activity of proteases (1) and trypsin inhibitors (2), expressed in nmol of arginine per 1 mg of protein / s. The abscissa axis shows the germination time of wheat germ.

Figure 4 presents the efficiency of sequential stepwise extraction of histone and non-histone proteins from the nuclear matrix (NM) with Arg-X protease and trypsin inhibitor activity. The ordinates show the activity of proteases (1) and trypsin inhibitors (2), expressed in nmol of arginine per 1 mg of protein / s. The abscissa axis shows the germination time of wheat germ.

The analysis of Figs. 1-4 shows that during the transition of G ₁ to the S phase of the cell cycle, Arg-X proteolysis and its inhibition processes both in non-histone and histone proteins of the cell nucleus actively occur. It is known that arginine in the composition of proteins is actively involved in the processes that structure the packaging of DNA, especially with the modification of the guanidine group. Compression or stretching of the nucleoprotein superstructures of the cell nucleus can screen the hydrophobic or hydrophilic surfaces of the protein for intermolecular interactions and thereby affect the packing density of DNA and its transcriptional activity.

This invention is recommended for the analysis of molecular genetic mechanisms occurring at different levels of the spatiotemporal reorganization of interphase chromatin, which is able to play an important role in the functioning of epigenetic mechanisms that work not at the level of the triplet DNA code, but at the level of N-terminal arginine and lysine sites from the nucleosome globule.

Information sources

1. Ivanova E.A., Vafina G.Kh. A method of obtaining nuclear fractions with proteinase and inhibitory activity. Copyright certificate No. 1733471 // BI 1992. V.18, S.96.

2. Ivanova E.A. Vafina G.Kh. A method for preparative isolation of basic proteins from suprastructures of plant cell nuclei. Patent No. 2408602 // Published on January 10, 2011. Bull. No. 1.

3. Ivanova E.A., Vafina G.Kh. A method for isolating plant cell nuclei. Copyright certificate 1701747 // BI No. 48. 1991. S. 98.

Claims (1)

The method of analysis of Arg-X proteolysis and its inhibition in non-histone and histone proteins of the supramolecular structures of plant cell nuclei, in which initially at certain intervals from the start of soaking 0 h, 3 h, 6 h, 9 h, 12 h, 15 h, 18 h 21 hours, the embryos are separated from the endosperm, followed by preservation of the embryos in buffered 80-90% glycerol at minus 25 ° C, cell nuclei are isolated, nuclear fractions are extracted with increasing concentrations of 0.14 M, 0.35 M, 2 M chloride sodium and 6 M guanidine hydrochloride with 0.1% β-mer by ethanol, histone and non-histone proteins are isolated from the above fractions using ion exchange chromatography with IRC-50 amberlite in a discontinuous gradient of guanidine hydrochloride: 6%, 8.9%, 10.6%, 13%, 40% per 0.1 M potassium phosphate buffer pH 6.8 and determine the sites of Arg-x protease and trypsin inhibitor in them.

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