SU1733471A1 - Method for preparation of nuclear fraction, showing proteinase and inhibiting activity - Google Patents

Abstract

The invention relates to the physiology and biochemistry of plants. The aim of the invention is to increase the yield and activity of the target product. Plant tissue is preserved in glycerol medium, the cores are successively treated first with increasing concentrations of sodium chloride in the presence of 0.5% Triton X-100, then with solutions of guanidine hydrochloride with 1-mercaptoethanol and 0.5 N, with alkali sodium. Sepharose 4B affinity chromatography is carried out with immobilized either trypsin or trypsin inhibitor. 3 ill., 4 tab.

Description

The invention relates to the physiology and biochemistry of plants, can be applied in molecular developmental biology when assessing the functional state of interphase nuclei.

The purpose of the invention is to increase the activity and release of nuclear trypsin-like proteinases and their inhibitors.

The method consists in that the plant tissue is preserved prior to isolation of the cell nuclei, and the nucleus is cleaned with a solution containing 0.5% Triton X-100, followed by extraction of nuclear fractions with increasing concentrations of 0.14 M, 0.35 M, 2 M sodium chloride, 6 M guanidine hydrochloride with 0.1% / 3-mercapto-ethanol and 0.5 n, sodium hydroxide solution with concomitant affinity chromatography of the above listed nuclear fractions on sepharose 4 V with immobilized trypsin or trypsin inhibitor,

Example. The experiments were carried out on resting, 1-2 day old seedlings, 3-4 day old coleoptile tissues and leaves of Moscow-35 wheat, Mironovskaya winter and dairy. The seedlings were grown at a temperature of 25 ° C in a thermostated chamber between the layers of filter paper moistened with distilled water. The embryos, seedlings, coleoptiles and leaves, separated from the endosperm, were washed for 3 seconds with cold ether and repeatedly distilled water. Sprouts and tissues were preserved at -25 ° C in 80-90% glycerol. Then the embryos, seedlings and tissues were filled with a homogenizing medium: 20% glycerol; 0.005 M MsCl2; 0.025 M KCI; 0.003 M CaCl2; 0.005 M NaCl; 0.004 M US-mercaptoethanol; 0.02% triethanolamine (TEA) HCl pH 6.8; 0.004 M n-octyl alcohol, GoV

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Mogenization of seedlings was carried out for 7 s at 15,000 rpm (MP8-302 homogenizer, Poland). The homogenate was filtered through 1 layer of flannel, fluselin and 3 layers of nylon (pore size 70 μm). With a coarse residue of plant material, homogenization and filtration was carried out another 2 times 15 s and 30 s at 15,000 rpm. The combined homogenates were centrifuged at 500 rpm (CLR-1, USSR) for 5 min in order to free from coarse undigested tissues and whole cells, then the supernatant was centrifuged at 2700 rpm (K-23, GDR) for 20 min The precipitate was collected by suspension with homogenization medium (50 ml) and layered on a discontinuous glycene gradient consisting of 5 layers (30 ml each) of increasing glycerol concentration (50, 60.70, 80.90 w / v) prepared on TEA NA pH 6.8 with all the listed components of the homogeneous medium, excluding 20% glycerol. Gradient centrifugation was carried out at 2000 rpm for 1 h (CLR-1, USSR). The pellet was washed with 0.5% Triton X-100 on a homogenizing medium, but without glycerol, followed by centrifugation at 2,700 rpm (K-23, GDR) for 15 minutes, after which the cores were washed three times in the medium next composition: 0.005 M MsCIa; 0.025 M 0.003 M CaOfc: 0.005 M NaCl; 0.01 M Tris-HCl pH 6.8, followed by centrifugation under the indicated conditions. 1% Triton X-100 wash the cores because they swell and spill.

The degree of purity of the isolated cell dermal preparations was determined microscopically, spectrophotometrically, and by the component composition of the cell cores (protein / DNA, RNA / DNA, Table 1).

Comparison of the percent yield of nuclei and their specific enzymatic activity using sucrose-gum arabic and glycerol isolation methods is presented in Table 2.

From purified preparations, nucleus-nucleic proteins were extracted at a low ionic strength of 0.14 MNaCI, 0.01 M Tris-HCI pH 6.8 with buffer. Unstable nuclear proteins were isolated by three consecutive extractions of 0.35 M NaCl in the same buffer.

Next, the precipitate was fractionated by suspension in Tris-HCl buffer with 2 M NaCl. The fraction containing the nuclear matrix remained in the sediment. Subsequent extraction was carried out with 6 M gaunidine hydrochloride with 0.1% / 5-mercaptoethanol in Tris-HCl buffer pH 6.8 (nuclear matrix) L

the residual components were extracted with 0.5 n NaOH (nuclear matrix M). All fractions of nuclear components were obtained by three successive extractions. Sufficiency of extraction was controlled by protein yield. Nuclear fractions were stored at -196 ° in nitrogen.

Affinity chromatography was carried out on columns (1 X 0.5 cm) either with immobilized trypsin (SPOFA, Czechoslovakia), or trypsin inhibitor (Reanal, Hungary) covalently attached to CNBr-activated agarose (Institute of Chemistry, Academy of Sciences of ESSR). Proteinase activity of trypsin and

5 trypsin-like proteinases were determined by cleavage of low molecular weight protamine protein (Calbiochem, United States),

The amount of arginine released was calculated using calibration gauge.

0 schedule. D, L-arginine (Reanal, Vengry) was used as a standard. The activity of trypsin-like proteinases and their inhibitors was expressed in nmol of arginine per mg of protein / s.

5 Analysis of the completeness of nuclear fractions (Table 3) showed that all fractions are not pure protein, but nucleic acid and protein complexes.

The efficiency of sequential stepwise extraction of trypsin-like proteinases and their inhibitors, followed by identification either on columns with immobilized trypsin or trypsin inhibitor is demonstrated in FIG.

5 1, 2, The ordinate axis (Fig. 1) shows the content of proteinase and inhibitory nucleoprotein complexes in terms of μg of protein per 10,000 seeds. On the abscissa axis is indicated the age of the seedlings from the resting

0 embryos (0) up to 2-day-old seedlings; 3-4 daily coleopteel; 3-4 daily leaves; 3-4 daily roots; The following notation is used; 1-nucleoplasm, 2-unintegrated chromatin proteins, 35 main chromatin proteins, 4-core matrix I, 5-core matrix II, A-proteinase. B-proteinase inhibitors,

If we isolate the nucleoplasm from the nucleus (0.14 M NaCI) and then immediately extract them with 0.5 N NaOH (Fig. 2), then we will not get the effect shown in Fig. 1, i.e. the violation of the given extraction sequence does not have the effect of isolating trypsin-like proteinases and inhibitors.

5 In FIG. Figure 2 shows the optical density of solutions at 280 nm on the ordinate axis, and the sample numbers are shown along the abscissa. FIG. 2, the following designations are given: 1-column with immobilized trypsin inhibitor, H-column with immobilized trypsin, and - trypsin-like proteicases. b - trypsin inhibitor,

FIG. 3, the ordinate axis shows the units of specific proteolytic and inhibitory activities, and the abscissa indicates the daily age of the seedlings and leaves: 1,2 - one - two-day whole seedlings; 4 - four-day leaves. FIG. 3 — the following designations are given: 1 — the proteolytic activity of trypsin; 2 - the proteolytic activity of seedlings and leaves; 3 — inhibition of trypsin nuclear inhibitor activity; 4 - inhibition of nuclear proteinase by a nuclear inhibitor; A - proteolytic activity; B - inhibitory activity.

The study of fractions possessing proteinase and inhibitory activity includes the preliminary conservation of plant tissues and the isolation of cell nuclei from them in a glycerol medium. Table 1 shows that the structure of the cellular nuclei according to the content of the genetic material is not disturbed; tab. 2 illustrates an increase in the percentage yield of specific proteinase activity; tab. 3 illustrates the completeness of the release of nuclear fractions; tab. 4 illustrates the component composition of affinity-secreted nuclear try-syn-like proteinases and their inhibitors.

FIG. 1-2 illustrate the effect of stepwise sequence of nuclear fraction extraction on trypsin-like yields.

five

0

five

0

proteinases and their inhibitors: FIG. 3 illustrates the protease and inhibitory activity of affinity-secreted trypsyn-like proteinases and their inhibitors compared with trypsin activity.

The proposed method is recommended in the study of the molecular-genetic mechanisms of endogenous regulation of the organization of genetic, morphogenetic programs for the development of an organism at various ontogenetic stages of differentiated plant growth.

Claims (1)

The invention of the method for obtaining nuclear fractions with proteinase and inhibitory activity from seedlings of cereals by isolating cell nuclei, cleaning them with a solution containing Triton X-100 and sodium chloride, and evaluating the proteinase activity, characterized in that of the activity of the target product, the plant tissue is preserved prior to the isolation of the cell nuclei, and the nucleus cleaning is carried out sequentially with solutions containing 0.5% Triton X - 100 with increasing concentrations of 0.14 M, 0.35 M and 2.0 M sodium chloride, then a solution containing 6 M guanidine chloride with 0.1% / 3-mercaptoethanol, then 0.5N. Sodium hydroxide solution and Sepharose 4B affinity chromatography with immobilized trypsin or trypsin inhibitor. 35 table 2 Table 3 T a b l ii ts l 4 Inhibitor trypsin RNA / 7 . About 0.9 1.2 l -4 CO CO Ј -J J ch  D I -Јs Nj A ga 10H 0.1 / V-. " one t-1-rif- -i-i-g - // 1- - 3 690it 7 GO Number lrv & fig. 2  HU / ur & ma fig. 3