SU1763488A1 - Method for preparation of c@/m@-dependant endonuclease inhibitor - Google Patents

Abstract

Usage: biochemistry, preparation of inhibitor of Ca / Mg-dependent lymphocyte endonuclease. SUMMARY OF THE INVENTION: Lymphocytes of peripheral blood or spleen of a human or cattle are used as an object for malignant lymphoproliferative diseases, most preferably for chronic lymphocytic leukemia, cellular nuclei are isolated from lymphocytes in solutions of sucrose of increasing density (from 0.25 to 2.0 M), the cell cores are extracted with 0.3-0.6 M NaCl or KCI solutions in the presence of 0.075-0.1% Triton X-100, the extracts are salted out with ammonium sulfate in the amount of 80-85% saturation and desalted an inhibitor was isolated from the precipitate by high performance liquid column chromatography.

Description

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WITH

This invention relates to biochemistry, in particular to the preparation of nuclease inhibitors.

Nuclease inhibitors can be used to differentially suppress the activity of individual enzymes in incubation mixtures containing several nucleases. They can also be used to prepare intact nucleic acid preparations and for affinity chromatography of nucleases. The study of nuclease inhibitors is important for understanding the mechanisms of regulation of these enzymes in the cell.

The Ca / Mg-dependent endonuclease inhibitor is not known in the literature and has been obtained for the first time. Natural inhibitors of bacterial endonucleases are known. In particular, Actynomyces levoris 2789, an extracellular DNase inhibitor, was obtained. The inhibitor was isolated from culture mycelium extracts, fractionated with ammonium sulphate and chromatographic on Sephadex G-100 and KM-cellulose. The inhibitor was characterized as a labile protein with a ph of 7.8-8.2, specific for A. lazor 2789 DNAse 1 (1). Natural endonuclease inhibitors derived from animal tissues are also known. For example, an DNase 1 (pancreatic) inhibitor was isolated from the spleen of the spleen. The inhibitor was isolated, fractionating the ammonium sulfate extract and chromatography with active fractions on alumina gel and DEAE-cellulose. The inhibitor was identified as G-actin or a closely related protein (2). This inhibitor does not act on the Ca / Mg-dependent endonuclease of cellular nuclei, in particular, from lymphoid tissues. Natural inhibitors of Ca / Mg-dependent endonuclease are not described in the literature and we obtained it for the first time.

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About CO N CO 00

The purpose of the invention is to obtain a natural inhibitor of the Ca / Mg-dependent endonuclease of cellular nuclei.

The goal is achieved by using lymphocytes of peripheral blood or spleen of cattle or humans in malignant forms of lymphoproliferative diseases — most preferably in chronic lymphocytic leukemia (CLL), in order to obtain an inhibitor, and the inhibitor is not isolated from whole cells, but from isolates. rovaine cell der. For this, the cell cores are extracted with a solution containing 0.3-0.6 M KCI with 0-0.1% Triton X-100, the extract is fractionated with ammonium sulfate to 80-85% saturation, and the active fractions after dissolution and dialysis are desalted. on columns with G-25 and high-performance liquid column chromatography on Mono S cation-exchange columns (Pharmacia, Sweden), with elution with a gradient of sodium chloride from 0 to 0.5 M. The method allows to obtain a drug with a pronounced inhibitory activity against Ca / Mg-dependent cellular endonucleosis p lymphocytes. The inhibitory activity is specific because the drug does not inhibit DNase 1 and micrococcal nuclease and is associated with protein.

The invention is illustrated by the following examples.

PRI me R 1. Peripheral blood lymphocytes obtained from a cow with large chronic lymphocytic leukemia were suspended in a solution containing 10 mM Tris-HCl, pH 8.0, 3 mM MgCI2, 0.9 M sucrose, and 0.002% Triton X -100 (TMS-0.9); 107 lymphocytes were used with 10 ml of medium. The suspension was homogenized in a Zweeeim-Potter homogenizer and layered on 10 mM Tris-HCl, pH 8.03 mM MgCl2, 1.2 M sucrose in the ratio 3: 1. Samples were centrifuged at 1500-2000 g for 10 min at 4 ° C. 10 ml of TMS-0.9 was added to the sediments, suspended thoroughly and re-centrifuged in the same mode. The sediments were represented by intact cellular dramas. Cell nuclei were suspended in 10 mM Tris-HCl, pH 7.4-7.6, to a final concentration of 2 mg / ml (DNA). 3 volumes of 0.4 M KCI, 0.1% Triton X-100 in 10 mM Tris-HCl, pH 7.4-7.6 were added to the suspension (final concentrations of KCI and Triton X-100 were 0.3 M and 0.075%, respectively) and stirred in an ice bath for 30-60 minutes. After this, the samples were centrifuged at 24,000 g for 15 min, 4 ° С, the precipitates were discarded. Supernatants (extracts) contained nonhistone chromatin proteins and inhibitory factors. Special studies regarding extraction conditions showed the following. When the final concentration of KCI in

extracting mixture of 0.3 M (see above); the degree of extraction of nuclear proteins is 21% (the degree of extraction was determined by the formula (a / a + b) 100, where a is the amount of protein in the extract, b is the amount of protein in

0 draft). With a decrease in the final concentration of KCI (to 0.2 and 0.1 M), the degree of extraction decreased significantly, and the yield of the target product also decreased. With increasing concentration of KCI to 1.0 M degree of extraction

5 proteins increased to 45-50%, however, the activity of the inhibitor in the extract did not increase after a concentration of 0.6 M. The introduction of triton X-100 into the extraction mixture at a final concentration of 0.05-0.075% increased the degree of protein extraction in the presence of 0.3 M KCI to 46%, while the activity of the inhibitor increased by 20-25%. Other investigated conditions for the extraction of nuclear proteins - ultrasonic processing

5 nuclei, extraction with guanidine chloride in the presence of dithiothreitol, using only Trion - X-100 - gave the worst results.

The resulting extracts were fractionated by salting out with ammonium sulfate. For this, samples were desalted on Sephadex G-25 and dry ammonium sulfate was added up to 80% saturation with continuous stirring. The precipitate formed was divided by centrifugation at 100,000 g, 1 h, 4 ° C; the supernatant was discarded. The use of salting out with ammonium sulfate ensured the concentration of the samples, the release of part of the ballast proteins and

0 inhibitor stabilization. The resulting precipitates could be stored at 4 ° C without loss of activity for 1 month. Reducing the concentration of ammonium sulfate to 70% led to a decrease in the yield of the inhibitor by 10-15% and

5 reduce the stability of the drug. Increasing the concentration of ammonium sulfate to 85% and 90% did not lead to a change in the results.

The precipitate obtained after salting out was dissolved in a minimum volume of 10 mM Tris-HCl, pH 7.4-7.6, and desalted on Sephadex G-25 columns. A desalted sample containing 1-1.5 mg of protein in 1-2 ml was applied to a cation exchange column.

5 Mono S (FPLC system, Pharmacy, Sweden), pre-equilibrated with the same buffer. The application rate was 0.1-0.2 ml / min. After applying the sample, the column was washed with 5-7 ml of the same buffer at a rate of 1 ml / min, after which

The elution of sorbed proteins with a linear NaCl gradient from 0 to 0.5 M in the same buffer was started. The elution rate is 1 ml / min, the pressure is 1.0-1.5 MPa, the gradient volume is 10 ml. Fractions of 0.5 or 1.0 ml were collected and analyzed for inhibitory activity.

The activity of the inhibitor is determined as follows. Incubation samples were prepared containing 40 μl of 10 mM Tris-HCl, pH 7.6, 10 mM MgCI2, 1 mM CaCfc, 10 rrjM 2-mercaptoethanol, and 10 units Ca / Md-dependent endonuclease of the cell lymphocytes of healthy animals. Samples were prepared in the wells of 96-well microplates. An amount of enzyme activity was taken to be sufficient for complete cleavage of 0.1 µg of A-phage DNA in 2 hours of incubation at 37 ° C in media containing 10 mM MgCIa, 1 mM CaCJ2. Complete cleavage was monitored with 0.8% agarose gel electrophoresis.

To the incubation samples, 10 µl of each fraction of the gradient was added and incubated on ice for 5-10 minutes. Then, the samples were made with 1 μg of DNA And -phage and incubation was continued for 2 hours at 37 ° C. After that, 5 μl of 50% glycerol with 2.5% bromophenol blue was added to the samples and applied to a horizontal block of 0.8% agarose gel. Electrophoresis was carried out at a voltage of 150 V; The gels were stained with ethidium bromide and photographed in reflected ultraviolet light with a wavelength of 254 nm.

In the fractions corresponding to the NaCI concentration from 0.25 to 0.3 M, preservation of intact (undigested) A-phage DNA was found, which indicates the presence of a Ca / Md-dependent endonuclease inhibitor in them.

Per unit of the activity of the inhibitor drug, such an amount was taken that ensured inhibition of 10 units of the Ca / Mg-dependent endonuclease by 1%. The inhibitor yield was 12000 units / mg of DNA of the initial nuclei, specific activity — 600 units / µg of protein, purification — 100 times with respect to the extract.

According to the electrophoresis in polyacrylamide gels of sodium xdodecyl sulfate and the treatment of the preparation with various proteases, the inhibitory activity of the preparation is associated with polypeptides ranging in size from 60 to 67 kO.

PRI mme R 2. All operations were carried out analogously to example 1, except that they used the peripheral blood lymphocytes of people with chronic

lymphocytic leukemia, and the inhibitor activity was determined directly to cell dermal extracts.

Froze The spleen was used for preparative isolation of the inhibitor.

Cattle, a patient with spontaneous CLL,

All procedures were performed as described above, except that the cell cores were isolated as follows. In the experiment took 0.2 kg of the spleen. The sample was ground

0 in m cutter. The resulting mass was transferred to a 5-liter flask and filled with 2 l of cooled buffer A (0.9% NaCl, 0.5% Na-citrate, 10 mM Tris-HCl, pH 7.6). The flask was transferred to an ice bath and the contents were stirred, intensively rotating the flask for 30 minutes. The resulting suspension was filtered through 4 layers of gauze and the filtrate was centrifuged at 5000 g for 30–40 min. To the precipitate was added 1 L of buffer B (0.25 M sucrose, 0.4% non-distilled

0 P-40, 5 mM CaCl2, 50 mM Tris-HCl, pH 8.5) and homogenized in 60 ml portions in a Potter homogenizer. The homogenate was filtered through 4 layers of gauze and centrifuged at 5000 g for 30 min. By

According to the microscopy, the sediment consisted of more than 90% lymphocytes. 500 ml of buffer C (1.0 M sucrose, 5 mM CaCI, 50 mM Tris-HCl, pH 8.5, 0.4% nonidet P-40) were added to the precipitate and were intensively homogenized. The homogenate was centrifuged at 50,000 g for 30 min. The sediments were suspended in buffer C and layered in 100-ml centrifuge tubes in 20 ml of buffer E (2.0 M sucrose, 50 mM Tris-HCl, pH 8.5, 5 mM CaCi2) and centrifuged at 70,000 g for 2 hours.

EXAMPLE 4 Cell nuclei derived from the peripheral blood lymphocytes of a healthy animal were used to isolate an inhibitor. All stages were performed

0 as in example 1. Inhibitory activity could not be detected.

EXAMPLE 5 All the procedures were carried out analogously to Example 1, except that the effect of the inhibitor on the

5 Ca / Mg-dependent endonuclease of cellular dermal lymphocytes of the spleen of healthy animals and humans. 120 units of the drug inhibited the activity of the bovine spleen enzyme by 100%. and the human spleen is 80%.

PRI me R 6. The effects of the inhibitor on commercial preparations of DNase I (pancreatic), DNase II (pig's spleen) and micrococcal nuclease were determined.

5 The amounts of the enzyme were selected so that the electrophoretic pattern of DNA cleavage of the A -phag was similar to that for the amounts of Ca / Mg-dependent endonuclease used. The corresponding amounts were 0.15 µg DNA for DNase I, 2 U / Mg DNA for DNase II and 2 U / µg DNA for micrococcal nuclease. 120 units of the drug did not affect the activity of DNase 1 and micrococcal nuclease, but inhibited DNase by 40% (see table).

Thus, the method allows to obtain an inhibitor of Ca / Mg-dependent endonuclease of cellular dermal lymphocytes from cellular lymphocytes of patients with chronic lymphocytic leukemia. The inhibitor is absent in the cellular fractions of lymphocytes of healthy individuals and exhibits specificity for the Ca / Mg-dependent endonuclease, does not act or inhibit to a lesser extent some other nucleases - DNase I, DNase I and micrococcal nuclease. This allows it to be used for differential inhibition of Ca / Mg-dependent endonuclease, and can also be used in the diagnosis of chronic lymphoma.

The effect of the inhibitor on various nucleases.

leukemia For the inhibitor obtained, the term leukosin is suggested.

Claims (1)

The invention The method for producing a Ca / Md5 dependent endonuclease inhibitor is that the cellular nuclei of peripheral blood lymphocytes and spleen of a human or cattle in malignant forms of lymphoproliferative diseases are separated from lymphocytes by differential centrifugation in solutions of sucrose of increasing density from 0, 25 to 2.0 M, extracted with a solution of potassium chloride or chloride 15 sodium in a concentration of from 0.3 to 0.6 M in the presence of Triton X-100 in a concentration of from 0.075 to 0.1%, the resulting extract is salted out with ammonium sulfate in an amount of 80-85% saturation, the precipitate is desalted 20 on Sephadex G 25 and the inhibitor is purified by high performance liquid chromatography.