DE19810998C1 - New metal-containing ribonucleotide proteins with nuclease and phosphatase activities, used e.g. for cell-selective inhibition of protein synthesis and inhibition of tumors - Google Patents

Abstract

Metal-containing ribonucleotide-proteins (A) comprise a protein (I) of the S-100 family, a copper ion and one of four RNAs, designated clones 4-1b, 238/1, 238/2 or 238/3 of about 340, 130, 120 and 290 nucleotides, respectively (all sequences given in the specification). An Independent claim is also included for preparation of (A).

Description

The present invention relates to metal containing Ribonucleotide polypeptides (RNP) and methods for their Manufacture, their use and means, the Ribonuk suffering polypeptides or their molecular biological Equivalent structures and / or parts and / or derivatives contain.

For the investigation and elucidation of nuclein acid molecules or nucleosides or nucleotides despite a large number of existing connections with Nuclease activity or deaminase activity or Dephosphorylation activity of other compounds needed, for example, single-stranded RNA and DNA substrates can degrade to 3 'nucleotides and at the elucidation and investigation of nucleic acids are useful.

The object of the present invention is therefore to provide Ver to provide bonds with such activity. These tasks are solved by the objects of Claims.

It has been found by the inventors that there are compounds based on nucleic acid with a defined sequence, the specifically single-stranded RNA and DNA substrates can hydrolyze and nuclease activity as well as the same show early phosphatase activity.

The nucleases found by the inventors are in Form of a bioactive metal ribonucleotide peptide (RNP) before. This is called angiotropin.

The structure of angiotropin can make the ribonucleo tide proteins (RNP). It consists of a protein part (ARP = Angiotropin Related Protein) and an RNA part (ARNA = angiotropin RNA). For the Formation of the complex from ARP and ARNA is to eat Cu (II) tially. In addition to the copper ion, angiotropin contains a Ca (II) ion.

For the diverse biological and biochemical Functions of angiotropin are also Mg (II) ions useful.

The protein part (ARP) consists of a protein belonging to the family of S100 proteins (Dell'Angelica et al., J. of Biological Chemistry, Vol. 269, No. 46, pp. 28929-28936 (1994)) can be assigned and is preferably 91 amino acids long. The primary structure of this preferred ARP is as follows:

It has two EF hand motifs and a binding site for zinc (II) ions. The dissociation constants K _{D of} the metal ion complexes are 10.0 µM for Ca (II) ions and 0.1 µM for zinc (II) ions and 1.0 µM for copper (II) ions.

In this context it should be mentioned that the protein part according to the invention according to the usual, on the Methods known in the art can be modified, without losing biological activity. to these modifications include exchanges, insertions or deletions of amino acids, the structure of the Modify the protein part, with its biological Ak activity is essentially retained. To the exchange preferentially count "conservative" exchanges of Amino acid residues, d. H. Exchange for biological similar residues, e.g. B. the substitution of a hydrophobic one Residues (e.g. isoleucine, valine, leucine, methionine) against another hydrophobic residue, or the substitution of one polar residue against another polar residue (e.g. arginine versus lysine, glutamic acid versus asparagine acid etc.). Deletions can lead to the generation of moles kulen that are much smaller in size wise, d. h., For example, amino acids at the N- or C-terminus missing.

The RNA part (ARNA) has the following sequences:

(a1) ARNA II

or

(a2) ARNA III

or

(a3) ARNA IV

or

(a4) ARNA V

The specified ARNAs also include molecular biology Equivalence structures, d. H. nuc linic acids (preferably under stringent conditions, like 20 ° C below the melting point of the RNA) or above the degenerate genetic code related nucleic acids.

The RNPs according to the invention are characterized by the following properties:

• - Melting point: about 200 ° C
• - Solubility: soluble in aqueous media, including 20% ethanol a pH value of 4-10.
• - In the presence of Mg ²⁺ , angiotropin hydrolyzes single-stranded RNA and DNA substrates to 3 'nucleotides (nuclease activity - phosphodiesterase). There is a preference for hydrolysis behind purine residues, with preference for adenosine (A <G). In the absence of Mg ²⁺ , angiotropin cannot catalyze the hydrolysis of phosphodiester bonds on pyrimidine nucleotides. In addition, angiotropin catalyzes the dephosphorylation of 5 'and 3' nucleotides and deoxynucleotides (phosphatase activity - phosphomonoesterase). ^{Mg 2+ is also} essential for this activity. The activities mentioned can neither be stimulated by ATP nor by GTP nor by 5'-GMP.
• - Angiotropin reversibly inhibits the Translation of β-globin mRNA and TMV RNA both in the rabbit reticulocyte lysate system as well as in the wheat germ extract system. the Inhibition is achieved through the addition of ATP and through a decrease in methionine levels partially canceled again in the reaction mixture.

The functioning of angiotropin is shown in FIG .

The components of angiotropin alone also have activity:

• - ARNA-free ARP is able to cell-stranded nucleic acids to hydrolyze sate (nuclease activity - Phosphodiesterase). However, it does arise exclusively oligonucleotides by Hy drolysis behind purine residues, with a clear preference for adenosine residues (A. << G) can be observed. It won't Hydrolysis of the phosphodiester bonds Pyrimidine nucleotides observed. the Hydrolysis of Single-Stranded Nuclein acids by ARP is not metal ions pending. The hydrolysis induced by ARP but runs slower than that of An giotropin (ARP + ARNA) induced Hydrolysis. ARP itself shows no or only a very weak one Phosphatase activity.
• - Furthermore, ARP is able to quantitatively deaminate adenosine and deoxyadenosine to inosine and deoxyinosine, respectively. The resulting deamination products (inosine, deoxyinosine) are then depurinated by ARP. Ribose or deoxyribose and hypoxanthine are formed. 3'-AMP is also deaminated and depurinated more slowly than adenosine. Interestingly, cAMP is recognized as a substrate by ARP and deamination takes place, but no depurination. In contrast, adenine is not recognized as a substrate (see FIG. 6).
• - RNA substrates (e.g. tRNA ^Phe ) are deaminated by ARP only in the absence of metal ions.
• - In the absence of inosine will also Guanosine to Guanine and Ribose by ARP hydrolyzed. In about the same amount as Guanine creates xanthine, that Deamination product of guanine.
• - ARP reversibly inhibits the cell-free Protein synthesis in reticulocyte lysate and wheat germ extract.

In the case of RNPs according to the invention, the protein content is through Interactions bound to the RNA part.

The invention also provides a method for Production and extraction of the bioactive RNPs, thereby characterized in that the cells, e.g. B. cells or Leukocytes or inflammatory tissue or homogenized Cells or leukocytes cultured and the resulting RNPs from the homogenates or from the supernatants of the Culture solution wins by standard methods. For example, the supernatants can be serum-free Mass cell cultures of Concanavalin-A activated Pig blood leukocytes and leukocytes from ischemis Chen / infarct myocardial cells angiotropin isolated and cleaned up to homogeneity.

The invention also provides a method for Production and extraction of the bioactive RNP morphogens the cells or leukocytes and the inflammatory tissue, which is characterized in that cells or Leukocytes of the reticulo-endothelial system of the Leukocytes and the inflammatory tissue that causes them is characterized in that the cells or leukocytes homogenized or leukocytes cultured and the resulting RNP morphogens from the homogenates or from the excess culture solution wins.

In principle, it is also possible to use the cells, e.g. B. Leukocytes on mediators directly without culture work.

The culture of the cells (leukocytes) can basically in each medium received the cells (leukocytes) be performed.

To culture cells, like leukocytes, the culture is used media with a planned duration of culture over 1 Hour mostly serum, for example calf serum or horse deserum, added as the serum components for the Maintenance of the vital functions of the cells are favorable. However, if the serum-containing culture solution on proteins (Mediators) should be dealt with by the Culture are generated, the extraction of the mostly prepares contained only in low concentrations Product proteins because of the multitude of those from the serum foreign proteins originating in great difficulty. In addition, it cannot be determined with certainty whether a certain mediator is humoral or zel is of lular origin and what species it is from; d. H. whether he is a mediator of the species whose cells cultivated, or the species from which the ver applied (mostly heterologous) serum.

The fully synthetic which is preferably used according to the invention table cell culture medium contains the usual group of substances pen, such as salts, sugars, amino acids, nucleosides and nuc leosidbasen, vitamins, vitaminoids, coenzymes and Steroids in aqueous solution. It is characterized by it net that there is also one or a mixture of meh contains other substances that are particularly valuable for leukocyte viability and growth and prove their ability to produce mediators. to these substances include unsaturated fatty acids, Flavonoids, Ubiquinones, Vitamin C and Mevalolactone.

The cell culture medium is used for longer-lasting cell or Leukocyte culture preferably without the addition of serum ver turns. Instead, it receives at least one defining tes protein, which in a particularly preferred embodiment form of ultra-pure, molecularly uniform serum bumin is.

In further preferred embodiments he can Fully synthetic serum-free, used according to the invention Cell culture medium still further, for the culture of Leukocytes favorable compounds from the substance classes of polyhydroxy compounds and sugars, amino acids, Nucleosides, anionic compounds and / or vitamins, their use in the known culture media is not is common to include. The components of the inven duly used medium are in their quantities ver conditions so aligned that the Kon concentration of the components in the medium largely the adapted to natural concentration ranges of the plasma are; see Ciby-Geigy AG (editor) (1969) in Documenta Geigy, Scientific Tables, 7th edition Geigy S.A., Basel.

The cell culture medium is preferably free of surfactants, Heavy metal salts and dyes that damage cells and the recovery of the desired cell products from the Can interfere with culture solution.

Is particularly preferred for the culture of the leukocytes im Method of the invention of the cell culture medium with the in Composition given in Table 1 below.

To produce the medium, water is used with the ATM-1- Quality used; see ASTM D-1193-70 standard Specification for Reagent Water 1970; Annual Book of ASTM Standards, Easton Maryland, ASTM 1970. It is in addition, of possible endotoxin contamination by ultrafiltration on surfactant-free membranes with the Exempt from exclusion limit of 10,000 Daltons. The finished one Medium is filter-sterilized on surfactant-free membranes with ≦ 0.2 µm pore size.

Table 1

Depending on the type of products you want, either mixed cell or leukocyte populations or one cultivated individual cell or leukocyte types. the Production and culture of the cells or leukocytes must take place under sterile conditions. The culture will performed long enough to a to obtain a satisfactory mediator yield. For this has a duration of about 10 to 50 hours is suitable proven. For shorter times, the mediator yield is too low, so that the process is uneconomical. If the culture lasts more than 50 hours, it is different on the other hand, the medium is exhausted and the cells begin die out, so that there is no longer any increase in the yield is to be expected.

The culture of the cells or leukocytes is in a Temperature from about 30 to 42 ° C, preferably from about 37 ° C. At lower temperatures is the Culture process unsatisfactory while at temperatures above 42 ° C the leukocytes are damaged.

The culture is carried out at a concentration of about 10 ⁶ to 5 × 10 ⁸ cells / ml, preferably up to 10 ⁷ to 10 ⁸ cells / ml. At lower cell concentrations, the yield per unit volume of the culture solution is too low. The process is uneconomical due to excessive culture volumes. At cell concentrations above 5 × 10 ⁸ cells / ml, the medium rapidly becomes depleted in nutrients.

The culture can be carried out in the atmosphere. Preferably there is an increased coal over the culture Dioxide parital pressure is maintained until about 10 % By volume, in particular up to about 2% by volume. from The supply of oxygen to the Kul is of great importance door. You can, for example, by introducing air be ensured. About contamination of the culture the air supplied is preferred to avoid sterilized and heat decontaminated, d. H. from En free of dotoxins and other organic components. The solution can be stirred or poured during culture be telt. The preferred cell stimulant is Con A used.

To end the culture, the cells or The leukocytes are centrifuged off from the culture solution then worked up on the resulting angiotropins is being worked on. To damage the cells and thus contamination of the culture solution by cell contamination avoiding constituent parts is the culture of proportion moderately low acceleration, d. H. about 300 to 400 × g, centrifuged. After cutting off most of the Cells from the supernatant are expediently added to this centrifuged again to higher acceleration remove remaining suspended particles. The severed ones Leukocytes can either be cultured again, cryopreserved or put to another use will.

Besides by culture of leukocytes the bioactive RNP morphogens of the invention also from inflammatory tissue be won. There they arise through the accumulation of leukocytes as a result of tissue damage triggered inflammatory process. The inflammatory tissue can be obtained in the usual way and used for preparation the RNP can be used. To do this, the inflammation tissue is homogenized in buffer solution and the soluble Components (exudate) are from the insoluble Struk ture components of the tissue separated.

Preferably inflamed heart muscle is infarcted tissue used, which by ligation of the left anterior descending branch of the left coronary artery using a transfemoral catheter technique during 24 Hours is formed. The leukocyte containing, inflamed myocardium part will not be at 0 to 4 ° C severed, infarcted, healthy tissue.

For the isolation and recovery of the bioactive RNPs Invention is the processing of a very large culture solution volume required. At the beginning of Purification method, it is therefore out of practical Reasons necessary, a reduction that is as effective as possible of the volume to be treated. The culture solution contains in addition to the small amounts of generated Substances, including mainly proteins, the mixture the components of the medium. Advantageously, will therefore a separation in the first step of the cleaning process the resulting proteins from the components of the Medium and at the same time from the large volume of the aqueous solution carried out. This can be done through a selective salting out of the proteins from the culture solution be effected, for example, by adding a Sulfates or Phosphates is achieved. Below is the precipitation of proteins using the example of salting out by adding ammonium sulfate to the culture solution described.

By saturating the culture solution with ammonium sulfate it becomes the majority of the resulting proteins together with possibly contained serum albumin precipitated. To the separation of the substance precipitate, for example by centrifugation, this can be done in the following in its individual components separated and the contained bioactive RNP obtained will. The supernatant obtained contains in addition to the sol union components of the medium also those in saturated Ammonium sulphate solution soluble part of the substances, among which there are also some of the bioactive RNPs det. The supernatant is concentrated and the contained Substances are made from it in the following way won. When the protein-containing culture solution is mixed with Am Monium sulfate is added to saturation, the falls larger part of the accompanying proteins. To this Way, a protein mixture is obtained that consists of a Number of different proteins consists and their Tren As a result, it is tedious to break down the individual components is. In a preferred embodiment of the method of the invention is contained in the culture solution Protein mixture therefore already in the precipitation stage in separated several fractions. This separation into multiple protein fractions is possible as the individual Proteins at different concentrations of ammonium sulfate be failed. Preferably the culture solution in the process of the invention therefore stepwise with Am monium sulfate up to certain degrees of saturation ver sets, being in each fraction the part of the proteins precipitates, the solubility product of which is below the respective degree of saturation. By appropriate choice the saturation limits of the individual fractions can be set in Method of the invention a rough separation into group pen of proteins already achieved during the precipitation the.

For example, the culture solution is initially up to a saturation of 35% with ammonium sulfate. The protein precipitate obtained is separated off. After that, the degree of saturation of the supernatant solution increased to 45%. A protein is formed again precipitation that is separated. Then the supernatant solution to a degree of saturation of 90% set. The resulting protein precipitate is also separated. The supernatant solution this precipitation is, for example, by dewatering Dialysis or ultrafiltration concentrated.

The salt precipitation of the proteins is just like that subsequent cleaning preferably at one temperature from about 0 to 10 ° C, in particular about 0 to 4 ° C carried out. The solutions used for cleaning have a pH value between 5 and 9, in particular between 6 and 8. To get a pH-constancy of the solution reach, is preferably a before the salt precipitation strong buffer, e.g. B. 0.1 mol / l phosphate buffer, added. To maintain the redox potential of the Proteins is preferably cysteine in a solution Amount of 0.001 mol / l added. Sterile conditions for protein purification is not required.

The proteins obtained during the salt precipitation can after Dissolve in a protein non-damaging medium directly to the cleaning described below and Separation are fed. The overhang of the last Precipitation stage is concentrated, for example by Dehydration dialysis or ultrafiltration. Be there all compounds with a molecular weight greater than than about 300 to 500 daltons, i.e. H. also the proteins and Peptides of this fraction, obtained quantitatively as retentate th.

Those obtained in the step described above Protein fractions contain the bioactive RNPs of the Er found in a mixture with numerous foreign proteins (other secreted proteins may be serum albumin and possibly CON). The foreign proteins are in the Mixtures in the vast majority of quantities. By a Series of purification steps must be the bioactive RNP enriched and freed from the foreign proteins so far that these are their molecular biological specificity don't bother anymore. The bioactive RNPs themselves are flat if a substance class, which is divided into its individual, specifically acting individuals is separated.

Generally there are cleaning procedures for protein bodies (Proteins) and other natural substances from a sequence combined separation processes, which molecular size, Charge, shape, structural stability and Molecular surface texture differences between the active substance sought and the accompanying foreign substance exploit fencing for separation. Accordingly, you can numerous combinations of different separation methods drive to purify a protein to be elaborated. For the handling properties, technical execution availability, automation and economy a cleaning process as well as for the quality of the The sought-after natural product is therefore not the only one Type of separation steps used is important, but especially their optimized design and their sensible combination in a cleaning sequence within the framework of structural stability and the other structural parameters of the drug sought. That also means that even the use of the same or similar separation principles (e.g. molecular sieve filtration, dialysis, ion exchange adsorption, etc.), but in different combinations, for the Manageability and economy of the Cleaning process can be crucial. In esp in certain cases is the use or omission of a single technique (e.g. hydroxylapatite chromatography, Zone precipitation chromatography, etc.) on a bes at the same point in the cleaning sequence, or within a limited partial sequence, of decisive importance Significance for the quality of the active ingredient sought as well as for the manageability and the Economy of its cleaning process. Clear These general relationships and Basic principles of natural product cleaning, for example on the well-known fact that in one economically reasonable and technically a manageable natural product cleaning process Column chromatographic purification step or a Lyophilization step doesn't make sense before that Total starting volume or the starting concentration of the accompanying foreign constituents of the raw active ingredient extract not to at least 1/500 to 1/1000 by other ver driving steps was reduced.

Offer for the purification of the individual protein fractions a plurality of in themselves individually in biochemistry known cleaning steps. Examples of such Purification steps are: preparative and analytical Molecular sieve filtration, anion and cation removal cherchromatography or one-pot adsorption process, Chromatography on hydroxyapatite, zone precipitation chromatography and circular or cascade molecular sieve filtration.

Already by performing one of the above Cleaning procedures can take a considerable amount Accompanying proteins separated from the bioactive RNP who the. However, those contained in the political groups hold Substances despite their different molecular weights often very close to each other. You will for example in molecular sieve filtration by not existing ideal equilibria in protein polyelectrolytes incomplete according to their molecular weight separated. It is therefore advisable to use at least two of the to carry out the mentioned separation processes one after the other. Preferably, the bioactive RNP activity is ent holding protein fractions at least three of the specified nth cleaning steps one after the other.

All combinations of the separation steps mentioned are Subject of the method according to the invention. Included it belongs to the knowledge of the skilled person that certain Sequences of separation steps are less useful than other combinations. For example, the person skilled in the art aware that when performing a preparative Molecular sieve filtration after an analytical Molecular sieve filtration besides the cumbersome process Rensweise also a worse overall result in terms of on the separation effect is obtained than the reverse Series.

The molecular sieve filtration causes a separation of the Proteins according to their molecular weight. There a predominant part of the accompanying foreign proteins different molecular weight than the bioactive RNP their separation can be achieved in this way will. For the separation of substances according to theirs Molecular weight becomes a hydrophilic, swellable in water lendes molecular sieve used. Examples of suitable Molecular sieves are cross-linked with epichlorohydrin Dextrans (Sephadex), agaroses crosslinked with acrylamide (Ultrogel) and room-linked acrylamides (biogels), whose exclusion limits are greater than those for separation used separation limits.

The molecular sieve filtration is, if several separation levels are used, preferably as one of the First performed. Depending on the length-diameter Ratio of the column used and the part diameter of the gel matrix, which is the theoretical Affecting the number of plates on the column will be the molecular sieve filtration as "preparative" or "analytical" designated. It is referred to as "preparative" if the chromatography on columns with a dimension ver Ratio length: diameter up to 10: 1 and one load of up to 1/3 of the column content or under full off use of the entire, typical matrix Separation volume is carried out. "Analytically" means a length-to-diameter ratio over 10: 1, preferably about 50: 1, and a maximum load of 3% of the column content, also with HPLC versions.

In preparative molecular sieve chromatography Gel matrices with the largest possible particle size used, fast flow rates of the often somewhat viscous ones Protein solutions at the lowest possible pressures aim. In analytical molecular sieve filtration the particle size of the gel matrix becomes as small as possible chosen to have a maximum theoretical number of trays the pillar for technical and safety ver evaluable pressure and a flow rate of the mobile phase of 2 to 4 cm / h. These Parameters depend on the gel matrix structure and varies from gel to gel.

If there are several preparative molecular sieve filtrations are carried out one after the other, the Separa can be selected in stages. After that can use an analytical molecular sieve filtration carried out according to graded separation limits will. The exclusion limit of the gel used must in any case greater than about 10,000 Daltons, by one Volume distribution of angiotropins between the stationary gel matrix phase and the mobile aqueous Allow buffer phase.

The "exclusion limit" denotes the hydrodynamic parameter of a dissolved particle, which corresponds to the pore size of the gel matrix. Particles with larger hydrodynamic parameters can no longer penetrate the gel matrix (volume distribution coefficient K _D = 0). The "separation limit" denotes a hydrodynamic parameter which is expediently established for the separation of dissolved particles and which lies between a volume distribution coefficient K _D = 0 and K _D = 1.

The substances are dissolved for molecular sieve filtration in a liquid that does not damage the substances applied to the molecular sieve. A more special one An example of a suitable solvent is 0.003 mol / l sodium potassium phosphate solution with a content of 0.3 mol / l NaCl and 0.001 mol / l cysteine and a pH value of 7.4. After filtration, the RNT will be containing Fractions in the manner described below kon centered, and possibly another Subjected to cleaning step.

The anion exchangers for cleaning the substances are suitable, for example, crosslinked with epichlorohydrin Dextran (Sephadex) or cellulose matrices to which functional groups with anion exchange capacity are coupled. You can go through after use Regeneration are needed repeatedly. Is preferred a pre-swollen in a buffer solution and equilibrated weak anion exchanger in the Cl⁻- Form, such as DEAE-Sephadex-A 50, used and the Treatment carried out at a pH of 8 to 10. A specific example of such a buffer solution is 0.01 mol / l Tris-HCl, which is 0.04 mol / l NaCl and 0.001 mol / l contains cysteine and has a pH of 8.0.

When using the anion exchanger, the Substance fraction of such an amount of anion exchanger added, the complete adsorption of the An giotropine and the positively adsorbing companion proteins are sufficient. Usually two are sufficient Parts by volume of swollen anion exchanger per volume concentrated protein fraction. The reaction can either as a chromatographic method or as an easier one manageable one-pot adsorption process who designed the. In the one-pot process, the supernatant liquid becomes ability with the negatively adsorbed proteins of the with the positively adsorbed RNP and other substances loaded anion exchanger, for example by Filter (in the chromatography column), decant or centrifugation (in a one-pot process). The loaded anion exchanger is washed with Water or a saline solution, which is 0.04 mol / l NaCl has equivalent maximum ionic strength, preferably at most about 15 ° C of adhering, negatively adsorbed th connections. A specific example of a saline solution suitable for washing out is er mentioned Tris-HCl buffer solution with a pH value of 8.0.

The freed from negatively adsorbed compounds, with RNP and other substances loaded anion exchangers is now with a protein non-damaging aqueous Saline solution eluted which has a greater than 0.04 mol / l NaCl corresponding ionic strength and a has a pH value between 4.0 and 10.0. Preferably will a salt solution of high ionic strength with a pH of 5.0 to 7.0 used. A specific example of one such a salt solution is a 2.0 mol / l NaCl solution, which with 0.01 mol / l piperazine HCl to pH 6.5 is buffered and which contains 0.001 mol / l cysteine.

The anion exchange reaction is called Chromatogra phieverfahren designed, so the elution of the RNP and other substances also by a linear NaCl-Kon centering gradients take place.

As cation exchangers are suitable for cleaning the protein fraction, for example with epichlorohydrin cross-linked dextran (Sephadex) or cellulose matrices which functional groups with cation exchange shear capacity are coupled. You can use it can be used repeatedly through regeneration. A weakly acidic cation exchanger is preferred in the Na + form, such as CM-Sephadex C-50, used, and the treatment at a pH value of 4 to 6 carried out. The substance fractions can be used adjustment of the setting of the load equilibrium the treatment with the cation exchanger with a Proteins are not diluted in a non-damaging saline solution, which is a maximum equivalent to 0.04 mol NaCl / liter Has ionic strength. They can be used at the same time to recruit serve the ph value. A specific example of one such saline solution is a 0.001 mol / l potassium phosphate acetate buffer solution with a content of 0.04 mol / l NaCl and a pH value of 4 to 6. This Cation exchange reaction can be used as a chromato graphical process as well as being technically easy to handle one-pot process can be permitted.

The cation exchanger is the substance fraction in added in an amount sufficient to make the protein adsorb fraction. Usually this is enough 2 parts by volume of swollen ion exchanger per volume part protein fraction. Then the overhanging river liquidity of that loaded with the substances Cation exchangers, for example by decanting or centrifugation, separated. The loaded one Cation exchanger is made by washing with water or a saline solution, which is one in 0.04 mol / l NaCl has equivalent maximum ionic strength, preferably at one pH of about 4 to 6 and a temperature of before preferably at most about 15 ° C of adhering, not freed adsorbed compound. A specific example for a saline solution suitable for washing out is the Mentioned potassium phosphate acetate buffer solution from the pH value 5.0.

The freed from negatively adsorbed compounds, with The cation exchanger loaded with the substances is now with one that does not damage proteins and nucleic acids aqueous saline solution eluted. This is preferably done a high ionic strength saline solution with a pH of around 4 to 10 used. Specific examples an aqueous 0.5 mol / l is for such salt solutions Potassium phosphate solution with a pH value of 6.5 to 7.5 or a 2 up to 5 mol / l sodium chloride solution with the same pH value.

For the chromatography on hydroxyapatite are poss Certainly existing from previous steps Salts, e.g. B. ammonium sulfate and above all phosphates, preferably by dialysis or ultrafiltration on a Membrane with an exclusion limit of 500 Daltons removed after application to the hydroxyapatite. From seen from the increase in viscosity due to external additives but is on for the success of the chromatography Hydroxyapatite only the phosphate concentration of the Protein solution critical. The elution of the substances he followed by a potassium phosphate concentration gradient th, which is preferably linear. The RNP containing Fractions are collected and listed below imagined way focused.

The use of hydroxyapatite is for the structure gentle net recovery of the RNP is essential Meaning. For technical and economic reasons but it is associated with considerable difficulties larger volumes of substance on hydroxylapatite columns chromatograph. On the one hand, namely tends Hydroxyapatite is very strong for larger volumes of substance to blockage and becomes unusable as a result. Other on the other hand, hydroxyapatite is expensive, which makes its use in opposed to a larger scale. For these reasons it is it is preferred in the process according to the invention, from the Substance fractions in which the bioactive RNP as Traces are included even before the chromatography in hydroxyapatite a large part of the accompanying Foreign proteins through suitable process steps to separate and thereby the protein volume, which is on the Hydroxylapatite column must be applied, crucial to zoom out.

In zone precipitation chromatography (cf. J. Porath, Nature, 1962: 47-48 (1962)) Pro tein contamination of the bioactive RNP by Aus salt fractionation of proteins using a salt con centration gradients separated.

Basic principle of protein separation by means of the zones precipitation chromatography are different, structure-given reversible solubility properties of proteins. They are among the most sensitive mo lecular separation parameters and were often called Kri terium for demonstrating molecular uniformity of a protein is used. Temperatures and pH value, dimension of the column, type of salt, shape of the Gradients and loading of the column in a proportion moderately wide range can be varied.

The temperature in the zone precipitation chromato graphy can be around 0 to 40 ° C. Is preferred a temperature range of about 0 to 10 ° C, esp especially around 4 to 6 ° C. The pH value can be between approx 4 and 10 lie; a pH value in the range is preferred from 6 to 8, especially about 7. The ratio of Length: The diameter of the column used should be greater than about 10: 1, a ratio of 30 is preferred up to 100: 1, especially about 50: 1. Come as salts all salts which do not damage proteins and nucleic acids with impact in question. Examples of such salts are Sodium potassium phosphate, ammonium sulfate and sodium sulfate. Ammonium sulfate is preferably used.

The salt concentration gradient can take any shape as long as the salting out points of the proteins are separated according to the route. Linear ones are preferred Concentration gradient, especially an increasing one linear concentration gradient from 25 to 100% Am monium sulfate saturation. The loading of the column is at most about 5%, preferably about 1%.

Circular or cascade molecular sieve filtration can be carried out under the conditions that above for analytical molecular sieve filtration are described. The same molecular sieves can be used and the same column conditions are used.

Sephadex G 50 is preferred for a length-diameter Ratio of the column of at least about 50: 1 and a load of no more than about 3% of the column content tes. As a solvent and for elution are in front of preferably that of the analytical molecular sieve Filtration used solvent used.

In the case of circular molecular sieve filtration, the eluate is at the specified separation limits in the circuit fed back into the same column. In this way the path of the proteins is differentially ver long. In another embodiment, the Kas kadenolekularsiebfiltration, the eluate is with the defined separation limits on a new column with the same or similarly defined parameters.

Between the cleaning steps explained above can contain the bioactive RNP obtained Substance solutions for subsequent separations of the Proteins purified from unwanted salts and con be centered. This concentration (cutting off the Most of the aqueous salt solution from the proteins) can be done in several ways. For example can the bioactive RNP and the accompanying substances by ultrafiltration or dehydration dialysis on a Membrane with the exclusion limit 500 Daltons or through Lyophilize to be concentrated. A Molecular sieve filtration by choosing the appropriate one mobile phase applied modified in the usual way will. In the case of molecular sieve filtration, the Substance solution preferably about 0.4 mol / l ammonium sulph fat added. In contrast to higher concentrations the ammonium sulphate has one in this concentration strong salt effect on proteins. Through these measures will be hence the proteins during molecular sieve filtration kept in solution. Ammonium sulfate also prevents this bacterial growth and inhibits certain enzymes. Through this it contributes to the stabilization of the bioactive RNP especially if the chromatography at higher temperatures (over about 20 °) and under non-sterile conditions is carried out.

The temperature and pH conditions are at the through Execution of the cleaning steps is not particularly critical. If the preservation of the native conformation of the Substan zen is intended, compliance is recommended a temperature in the range of about 0 to 8 ° C preferably about 0 to 4 ° C. Furthermore, the separation and Purification stages below essentially physiological Ph and salt conditions are carried out. A essential advantage of the method according to the invention is that compliance with these conditions is easily possible for the first time. To prevent oxidation the substance solution is preferably also with about 0.001 mol / l cysteine added.

The bioactive RNP obtained can be stored in a buffer th physiological saline solution, for example in 0.0015 mol / l sodium potassium phosphate solution, which is 0.15 mol / (0.9%) Contains NaCl and 0.001 mol / l cysteine and has a pH value of 7.4, after conventional filter sterilization (0.2 µm pore size) native and biologically active even in space temperature (for at least 200 hours) or frozen be stored at -25 ° C (for at least 5 years). This stability of the bioactive RNP can among other things as one of the criteria for their high purity condition be considered.

The RNPs according to the invention can also be used chemically or biologically synthesized partial sequences or parts and homologous sequences thereof made will. It is preferred that one chemically or biologically synthesized oligonucleotide or An tisensenucleotide sequences in vivo or in vitro, which which encode partial sequences given according to claim 1, starts with at least 6 bases in the PCR reaction or employs antisense bioprocess technology.

The examples illustrate the invention. In the examples is the production of RNP morphogens starting from Pig blood leukocytes described. The invention however, it is not limited to this embodiment. It can also contain cells of the reticulo-endothelial system or inflammation, wound tissue or fluid (Exudate) of other mammals can be used.

The invention is further based on the figures described which show:

Fig. 1 Energy-minimized secondary structure of ARNA II,

Fig. 2 Energy-minimized secondary structure of ARNA III,

Fig. 3 Energy-minimized secondary structure of ARNA IV,

Fig. 4 Energy-minimized secondary structure of ARNA V,

Fig. 5 functional model of angiotropin with ARNA and ARP-specific functions,

Fig. 6 deaminase and Nukleosidaseaktivitäten of ARP,

Figures 7-19 Nucleoside Analysis.

The invention is further illustrated by an example:

example 1 Nucleoside analysis

In a buffer with the composition 50 Mm Tris / HCl, 50 Mm NH ₄ Cl, 10 Mm MgCl ₂ , Ph 7.6, 7 μg ARP or angiotropin with 0.0334 A ₂₆₀ units of tRNA ^Phe from yeast, 0.0399 A ₂₆₀ units of ssDNA (primary structure: AT-TACCGCGCATGATAACATTCATAAAGAATAGTAATAAG) or 10 MM substrate nucleoside (nucleotide) incubated for 17 hours at 37 ° C. The reaction mixture optionally contained 5 units of alkaline phosphatase from E. Coli (EC 3.1.3.1). The volume of the batch was 150 μl in each case.

devices

High-resolution liquid chromatography system: System Gold with integrated diode array flow detector 168 (Beckman company).
Chromatography column: Supercosil LC18S 25 cm × 4.6 mm (Supelco)
Chromatography conditions (Davis et al., 1979):
Mobile phase A: potassium phosphate 50 mM, pH 4.4
Mobile phase B: methanol

The detection of the nucleosides took place at the wavelength gen 254 and 280 nm. The diode array detector allowed the synchronous recording of UV spectra. Of this Possibility was made use of as needed.

The results of the nucleoside analysis are shown in Figures 7-19.

Claims (19)

1. Metal-containing ribonucleotide proteins containing a protein from the family of S100 proteins, an RNA and copper as a metal ion in the form of a ternary complex, characterized in that the RNA of the ternary complex contains the following sequence:
(a1) ARNA II or
(a2) ARNA III or
(a3) ARNA IV or
(a4) ARNA V 2. Process for the production of a metal-containing Ribonucleotide protein according to claim 1, characterized characterized by having leukocytes or inflammation dung tissue homogenized or leukocytes cul activated and the resulting RNPs from the Homogenates or from the supernatants of the culture solution through standard methods wins. 3. Using the ribonucleotide proteins according to Claim 1 and / or molecular biological Equivalent structures and / or fragments and / or Derivatives for structural analysis of nucleic acids, Nucleosides or nucleotides. 4. Use according to claim 3 for the deamination of Adensosine and deoxyadenosine. 5. Use according to claim 3 for the hydrolysis of Inosine and deoxyinosine. 6. Use according to claim 3 for the hydrolysis of Guanosine. 7. Use according to claim 3 for the deamination of Guanine. 8. Use according to claim 3 for the hydrolysis of Phosphoric acid diesters. 9. Use according to claim 3 for the hydrolysis of Phosphoric acid monoesters. 10. Use according to claim 3, also in the presence of Mg ²⁺ ions, for the hydrolysis of nucleic acid substrates. 11. Use of the ribonucleotide proteins according to Claim 1 and / or molecular biological Equivalent structures and / or fragments and / or Derivatives for inhibiting protein biosyn thesis. 12. Use according to claim 11 for cell-selective, reversible inhibition of protein biosynthesis in Endothelial cells. 13. Use of the ribonucleotide proteins according to Claim 1 and / or molecular biological Equivalent structures and / or fragments and / or Derivatives for inhibiting blood endothelial cells len. 14. Use of ribonucleotide proteins according to Claim 1 and / or molecular biological Equivalent structures, and / or fragments and / or derivatives for specific influencing the formation of blood vessels (angio morphogenesis) in vivo and in vitro, especially for heart and vascular diseases, in wound healing, in the graft station surgery or the inhibition of the Growth of tumors. 15. Use of the ribonucleotide proteins according to Claim 1 and / or molecular biological Equivalent structures and / or fragments, and / or derivatives thereof, for cell-selective transmission of genetic In formation. 16. Use of the ribonucleotide proteins according to Claim 1 and / or molecular biological Equivalence structures and / or parts and / or Fragments used to diagnose changes in the Blood vessel growth. 17. Use of the ribonucleotide proteins and / or molecular biological equivalent structures and / or fragments and / or derivatives thereof, for the production of adenosine, deoxyadenosine, Guanosine, deoxyguanosine, inosine, adenine, guanine, Hypoxanthine or xanthine. 18. Use of the ribonucleotide proteins and / or molecular biological equivalent structures and / or fragments and / or derivatives thereof, for the production of nucleic acids in vivo and in vitro.