Heparin inhibits EcoRI endonuclease cleavage of DNA at certain EcoRI sites.

Studies presented here demonstrate that heparin inhibits EcoRI endonuclease cleavage of DNA whereas related proteoglycans show no effect. The inhibition occurs at particular EcoRI sites that are near or overlap with palindromic sequences in the murine lambda 5 and Lyt-2 genes. Endogenous heparin from peritoneal mast cells co-isolates with DNA and inhibits digestion of peritoneal cell DNA at the inhibitable sites. Digestion of spleen DNA is inhibited at the same sites when commercial heparin is added prior to digestion. In both cases, the inhibition is abolished by pre-treating the DNA with heparinase. Thus, potential artifacts in restriction fragment length analyses could occur with DNA isolated either from cells that are naturally rich in heparin or from cells to which heparin has been added, e.g., as an anticoagulant.     

An inhibitor of DNA polymerases alpha and delta in calf thymus DNA.

Most, although not all, samples of commercial calf thymus DNA were strongly inhibitory to DNA polymerase alpha; the inhibition made the DNA useless as a template for this enzyme. In a pre-assembled DNA polymerase assay mixture (minus enzyme but including activated DNA) the inhibition tended to diminish with time but at a rate that was not predictable, and some inhibition usually persisted. It was concluded that the inhibition was the result of contamination of the DNA by a heparin-like material on the basis of the following: 1) the inhibition could be reversed by treatment of the DNA with heparinase; 2) both the endogenous inhibitory effect of calf thymus DNA as well as the inhibitory effect of heparin on DNA polymerase alpha are reversed by protamine (which is known to prevent the antithrombin activity of heparin); 3) both the endogenous inhibition and inhibition by heparin are also reversed by ampholyte (which also prevents the antithrombin activity of heparin); and 4) both the endogenous and the heparin-induced inhibitory effects display the same spectrum of activity against mammalian DNA polymerases, i.e. both DNA polymerases alpha and delta are extremely sensitive whereas, DNA polymerases beta and gamma are resistant. The last result also suggests the use of heparin as a specific inhibitor of purified mammalian DNA polymerases alpha and delta, similar to the use of aphidicolin.     

Binding of Escherichia coli RNA polymerase to T7 DNA. Displacement of holoenzyme from promoter complexes by heparin.

Escherichia coli RNA polymerase holoenzyme bound to promoter sites on T7 DNA is attacked and inactivated by the polyanion heparin. The highly stable RNA polymerase-T7 DNA complex formed at the major T7 A1 promoter can be completely inactivated by treatment with heparin, as shown by monitoring the loss of activity of such complexes, and by gel electrophoresis of the RNA products transcribed. The rate of this inactivation is much faster than the rate of dissociation of RNA polymerase from promoter complexes, and thus represents a direct attack of heparin on the polymerase molecule bound at promoter A1. Experiments employing the nitrocellulose filter binding technique suggest that heparin inactivates E. coli RNA polymerase when bound to T7 DNA by directly displacing the enzyme from the DNA. RNA polymerase bound at a minor T7 promoter (promoter C) is much less sensitive to heparin attack than enzyme bound at promoter A1. Thus, the rate of inactivation of RNA polymerase-T7 DNA complexes by heparin is dependent upon the structure of the promoter involved even though the inhibitor binds to a site on the enzyme molecule.     

DNA inhibits catalysis by the carboxyltransferase subunit of acetyl-CoA carboxylase: implications for active site communication

Acetyl-CoA carboxylase (ACC) catalyzes the first committed step in the synthesis of long-chain fatty acids. The crystal structure of the Escherichia coli carboxyltransferase component of ACC revealed an alpha(2)beta(2) subunit composition with two active sites and, most importantly, a unique zinc domain in each alphabeta pair that is absent in the eukaryotic enzyme. We show here that carboxyltransferase binds DNA. Half-maximal saturation of different single-stranded or double-stranded DNA constructs is seen at 0.5-1.0 muM, and binding is cooperative and nonspecific. The substrates (malonyl-CoA and biocytin) inhibit DNA:carboxyltransferase complex formation. More significantly, single-stranded DNA, double-stranded DNA, and heparin inhibit the reaction catalyzed by carboxyltransferase, with single-stranded DNA and heparin acting as competitive inhibitors. However, double-inhibition experiments revealed that both DNA and heparin can bind the enzyme in the presence of a bisubstrate analog (BiSA), and the binding of BiSA has a very weak synergistic effect on the binding of the second inhibitor (DNA or heparin) and vice versa. In contrast, DNA and heparin can also bind to the enzyme simultaneously, but the binding of either molecule has a strong synergistic effect on binding of the other. An important mechanistic implication of these observations is that the dual active sites of ACC are functionally connected.     

Inhibition and dissociation of mammalian polymeric DNA polymerase by heparin

The activity of polymeric DNA polymerase isolated from the cytoplasm of baby hamster kidney cells is sensitive to inhibition by heparin, whereas that of nuclear DNA polymerase is resistant. Under conditions of partial inhibition, heparin brings about a change in both the K_m and V values and a twofold decrease in the ability to utilize DNA templates. Heparin effectively blocks DNA synthesis irrespective of the time of addition or conditions of preincubation with the reaction components. Upon interaction with heparin, polymeric DNA polymerase undergoes dissociation accompanied by the formation of a new species of active enzyme sedimenting at 5.3S which is partially resistant to inhibition by excess heparin. High ionic strength (0.45 m) dissociates the polymeric enzyme into active dimers and monomers irrespective of the presence of heparin. The differential susceptibility of polymeric DNA polymerase into monomeric enzyme, which resembles nuclear DNA polymerase, probably reflects a conformational change during dissociation.     

Self-assembled terplexes for targeted gene delivery with improved transfection

To improve transfection efficiency and to incorporate target ligands to the gene delivery systems, heparin and heparin-biotin were introduced to complexes of polyamidoamine dendrimer and DNA (PAMAM/DNA) via electrostatic interactions to form self-assembled PAMAM/DNA/heparin and PAMAM/DNA/heparin-biotin terplexes, respectively. The self-assembled terplexes were characterized by agarose gel electrophoresis and particle size analysis. The MTT assay indicated that, after incorporation of heparin and heparin-biotin, the terplexes exhibited decreased cytotoxicity. In addition, as compared with PAMAM/DNA and PAMAM/DNA/heparin complexes, the PAMAM/DNA/heparin-biotin complexes exhibited much higher cellular uptake into HeLa cells due to the specific interactions between biotin and biotin receptors on HeLa cells, which led to the enhanced transfection activity. The PAMAM/DNA/heparin-biotin complexes would be a promising targeting gene delivery system.     

Influences of anionic polysaccharides on DNA synthesis in isolated nuclei and by DNA polymerase α: Correlation of observed effects with properties of the polysaccharides

The basis of the differential effect of anionic polysaccharides on replicative DNA synthesis in liver and hepatoma cell nuclei was investigated. The differential effect of heparin was lost when more than 40% of its sulfate was removed. DNA synthesis in liver nuclei was optimally stimulated by heparin of molecular weight 22 600 and sulfate to hexosamine ratio 2.42, but inhibited by heparin of molecular weight 4300 and sulfate to hexosamine ratio 2.35. A heparin fragment (molecular weight 2800 and sulfate to hexosamine ratio 1.81), prepared by partial nitrous acid treatment was a potent inhibitor of DNA synthesis in hepatoma nuclei. There was no significant difference in the rate of entry of heparin or its subfractions into either liver or hepatoma nuclei. In both cases less than 15% of added polysaccharide entered the nuclei and only about 4.5% was found associated with the chromatin. The influence of the anionic polysaccharides on DNA synthesis was correlated with their ability to complex with histones as determined by relative light scattering in a laser nephelometer. The relative light scattered on mixing with histones (H1, H2A + H3, H4) was high for DNA synthesis stimulators (heparin, dextran sulfate); medium for DNA synthesis inhibitors (chondroitin 4- and 6-sulfates, heparan sulfate) and low for non-effectors (keratan sulfate, hyaluronic acid). Heparin and chondroitin sulfate H, which at low concentrations stimulate DNA synthesis in liver nuclei, inhibited DNA synthesis by calf thymus DNA polymerase α at all concentrations. This inhibition was not simply due to electrostatic interactions.     

Interaction of heparin with human basic fibroblast growth factor: protection of the angiogenic protein from proteolytic degradation by a glycosaminoglycan

Fibroblast growth factors (FGF) are a family of heparin-binding angiogenic polypeptide mitogens. In the presence of heparin, recombinant human basic fibroblast growth factor (bFGF) is fully protected from tryptic digestion and partially protected from chymotryptic digestion. Complete protection of bFGF by heparin is achieved at ratios of growth factor:heparin of approximately 10 or less (w/w). The protection requires bioactive bFGF because inactivated bFGF is rapidly degraded by trypsin or chymotrypsin in the presence of heparin. The bFGF-heparin interaction forms hydrophobic complexes which become insoluble in aqueous buffers at bFGF:heparin ratios of 8 to 10 (w/w). The heparin was found to bind up to a tenfold excess of bFGF on a weight basis. bFGF in the presence of heparin is as active as bFGF alone in inducing 3H-thymidine incorporation into Swiss 3T3 fibroblast DNA.     

Studies on isolated cell components

1. The addition of heparin to rat liver, kidney, or brain nuclei has been found to bring about the release of a gel. Chemical analysis and histochemical studies on whole homogenates and isolated nuclei demonstrated that the material released by heparin contained desoxyribonucleic acid (DNA) and protein. The action of heparin on nuclei is interpreted as the result of a combination with the basic proteins of the nucleus with a consequent displacement of DNA. 2. The addition of heparin to a finely divided dilute liver homogenate prepared in a phosphate-sucrose solution at pH 7.1 brings about a marked increase in viscosity which reaches a maximum in 6 to 8 minutes at 23° and then declines. 3. The concentration threshold for the viscosity effect was 0.1 mg. per 100 mg. fresh rat liver, with further increases in viscosity at higher heparin concentrations. Over a period of several hours a marked decrease in response to heparin was observed in homogenates stored at 0°. 4. Fractionation of the homogenate demonstrated that the viscosity increase was due to the presence of the nuclei alone, other components showing no effect. Microscopic observation showed that the increase in viscosity was associated with the appearance of a clear gel around nuclei treated with heparin. 5. Heparin brought about the release of DNA from the nuclei of incubated rat liver, kidney, and brain homogenates. In some instances over half the DNA is found in the supernatant after high speed centrifugation (20 minutes, 21,000 x g). 6. No correlation was found between anticoagulant activity of heparin preparations and their effectiveness in causing an increase in the viscosity of liver homogenates. Desulfated heparin produced none of the results described here for heparin.     

Studies on isolated cell components III. A cytological study of the effects of heparin on isolated nuclei

The gel released from isolated rat liver nuclei in response to heparin treatment has been found to stain with methylene blue, azure A, and methyl green when the dyes were added to the salt-sucrose nuclear isolation medium.Azure A and methylene blue caused rapid nuclear shrinkage to as little as $\text{1}\text{4}$ the original nuclear volume. Subsequent treatment with heparin caused the nuclei to fade rapidly and swell to approximately $\text{5}\text{4}$ of the original volume. With methylene blue stained nuclei heparin caused the extrusion of deeply stained, slightly birefringent rods through apertures on the nuclear surface. Methyl green also caused nuclear shrinkage, but to a lesser degree.Studies with the Feulgen reaction demonstrated structural damage in isolated rat liver nuclei as a result of heparin action. The viscous material released by heparin was shown to be Feulgen positive by resort to hydrolysis without prior fixation, since after customary fixatives the presence of a Feulgen positive reaction outside the nucleus could not be clearly demonstrated. The possibility is suggested that the Feulgen reaction following the customary fixatives depends in part on the manner in which the DNA is bound.The nuclei of leucocytes with visually intact cell membranes included in the nuclear preparations failed to show structural damage due to heparin and it is suggested that either the cell membrane provides some protection against heparin action or that damaged cells are more susceptible to this action.Observations made provide additional basis for the conclusion that heparin replaces DNA in the nucleo-histone of the nucleus, resulting in the structural damage observed, and releasing DNA in the form of a soluble viscous protein containing complex.     

Heparin can liberate high molecular weight DNA from secondary necrotic cells

The borderline between necrosis and apoptosis is indistinct, but that between types of cell death is important because necrosis may lead to local inflammation, whereas apoptosis usually does not. In certain autoimmune disorders, inhibition of cell death is crucial, since macromolecules released from the dead cells may accelerate the autoimmune processes. We have used various cell death inhibitors to block cell death induced by 4HPR [N‐(4‐hydroxyphenil)‐retinamide] the BL41 and U937 cell lines. VD‐FMK, a general caspase inhibitor, inhibited DNA fragmentation induced by 4HPR, but not PI (propidium iodide) uptake and necrosis. Interestingly heparin, a serine‐protease inhibitor, lowered the PI fluorescence of the dead cell population and increased the sub‐G1 population as measured by flow cytometry. Regarding these changes, we found that heparin failed to increase DNA fragmentation, but merely liberated high molecular mass DNA fragments from dead cells. The exact mechanism is unclear, but heparin during secondary necrosis might enter the cells, bind RNPs (ribonucleoproteins), and pull them out with the attached DNA, where they would be sensitive to enzymatic degradation. Thus, the results suggest that heparin treatment helps in the clearance of cell debris and decreases the immunogenity of secondary necrotic cells.     

In Vivo Gene Transfer to the Mouse Nasal Cavity Mucosa Using a Stable Cationic Lipid Emulsion

We evaluate a new cationic emulsion as a mucosal gene carrier and elucidate the relationship between the transfection efficiency and the stability of the carrier/DNA complex. A cationic lipid emulsion was formulated with soybean oil and 1,2-dioleoyl-sn-glycero-3-trimethylammonium-propane (DOTAP) as major components and was used to transfer genes to the epithelial cells of the mouse nasal cavity via intranasal instillation. Correlation between the transfection efficiency and the stability of the carrier/DNA complex was investigated by measuring the carrier size changes and by observing the degree of DNA protection against DNase I digestion in the presence of heparin. The cationic emulsion showed at least 3 times better transfection activity than the liposomal carriers in nasal mucosae. The cationic emulsion was stable in the presence of heparin whereas the liposomal carriers became very unstable. Unlike DNA in liposome/DNA complexes, DNA in the emulsion/DNA complex was resistant to heparin exchange and DNase I digestion. The cationic emulsion was more effective in delivering DNA to nasal mucosae than commercially available liposomal carriers. The transfection activities of the lipid carriers in nasal cavity mucosae are in agreement with the stability of the lipid carriers and their complexes with DNA.     

Mechanism of inhibition of mammalian DNA topoisomerase I by heparin.

We have previously shown that heparin is a potent inhibitor of a mammalian DNA topoisomerase I. We have now investigated the mechanism of its inhibition. This was carried out first by scrutinizing the structural features of heparin molecules responsible for the inhibition. Commercial heparin preparation was fractionated by antithrombin III-Sepharose into non-adsorbed, low-affinity and high-affinity fractions, of which only the high-affinity fraction of heparin is known to contain a specific oligosaccharide sequence responsible for the binding to antithrombin III. These fractions all exhibited essentially similar inhibitory activities. Furthermore, when chemically sulphated to an extent comparable with or higher than heparin, otherwise inactive glycosaminoglycans such as heparan sulphate, chondroitin 4-sulphate, dermatan sulphate and neutral polysaccharides such as dextran and amylose were converted into potent inhibitors. Sulphated dermatan sulphate, one of the model compounds, was further shown to bind competitively to the same sites on the enzyme as heparin. These observations strongly suggested that topoisomerase inhibition by heparin is attributable primarily, if not entirely, to the highly sulphated polyanionic nature of the molecules. In a second series of experiments we examined whether heparin inhibits only one or both of the topoisomerase reactions, i.e. nicking and re-joining. It was demonstrated that both reactions were inhibited by heparin, but the nicking reaction was more severely affected than was the re-joining reaction.     

The effects of heparin on endogenous DNA polymerase activity of rat liver nuclei and chromatin fractions

1. 1. Heparin activates endogenous DNA polymerase activity in isolated rat hepatic nuclei to a synthesis rate two to three times the control values, when added in amounts as small as 10% of total nuclear DNA.

2. 2. Cations such as polylysine, polyornithine and unfractionated histones form insoluble complexes with heparin and reverse its effect in either order of addition.

3. 3. Autoradiography demonstrates many apparent new sites of DNA synthesis in heparin-treated nuclei, both in previously inactive and in previously active nuclei.

4. 4. Electron microscopy shows a dramatic change in the chromatin pattern upon treatment with heparin; fiber diameters are significantly decreased, probably indicating loss of supercoiling.

5. 5. The response to heparin of the endogenous polymerase activity in three chromatin fractions is examined. Two of the three fractions show patterns similar to whole nuclei; the third, a high specific activity fraction, has a distinctively different pattern of response to heparin.

6. 6. The kinetic data combined with the behavior of the high specific activity chromatin fraction indicate that the heparin effect on endogenous DNA synthesis may have more than one component.

     

Carboxyl-terminal structure of basic fibroblast growth factor significantly contributes to its affinity for heparin

The carboxyl-terminal sequence of basic fibroblast growth factor (bFGF) is rich in basic amino acid residues, a common characteristic amongst fibroblast growth factors, and is considered to contribute greatly to the binding to negatively charged extracellular matrixes such as heparin. To study the relationship between the affinity for heparin and the carboxyl-terminal structure of bFGF, amino- or carboxyl-terminal truncated molecules were produced in Escherichia coli using recombinant DNA techniques. These terminally truncated bFGFs were applied to a heparin-affinity HPLC column. Truncation of more than six amino acid residues from the carboxyl-terminal made the bFGF produced in E. coli markedly difficult to solubilize and weakened its affinity for heparin, though bFGF having up to 46 amino acids removed showed significant stimulation of the DNA synthesis of BALB/c3T3 cells. This stimulation of the DNA synthesis was also recognized by the bFGF having 40 amino acids removed from its amino-terminal, while the affinity of this peptide for heparin has been shown to be equal to that of the mature bFGF (146 amino acids). These results show that the affinity of bFGF for heparin depends significantly on its carboxyl-terminal structure and that the essential part for receptor binding is present between Asp41 and Ser100. Moreover, it suggests that the Phe139Leu140Pro141, present in all members of the FGF family, contributes greatly to the stable structure of the intact molecule.     

The use of heparin as a simple cost-effective means of controlling background in nucleic acid hybridization procedures.

The use of heparin as a simple, effective and cheap substitute for conventional methods of controlling background in hybridization procedures is described and illustrated with reference to the use of DNA probes in filter and in situ hybridization. Possible specific mechanisms for this heparin effect are discussed.     

Monitoring complex formation in the blood-coagulation cascade using aptamer-coated SAW sensors

Specific binding of the anticoagulants heparin and antithrombin III to the blood clotting cascade factor human thrombin was recorded as a function of time with a Love-wave biosensor array consisting of five sensor elements. Two of the sensor elements were used as references. Three sensor elements were coated with RNA or DNA aptamers for specific binding of human thrombin. The affinity between the aptamers and thrombin, measured using the biosensor, was within the same range as the value of K(D) measured by filter binding experiments. Consecutive binding of the thrombin inhibitors heparin, antithrombin III or the heparin-antithrombin III complex to the immobilized thrombin molecules, and binding of a ternary complex of heparin, anithrombin III, and thrombin to aptamers was evaluated. The experiments showed attenuation of binding to thrombin due to heparin-antithrombin III complex formation. Binding of heparin activated the formation of the inhibitory complex of antithrombin III with thrombin about 2.7-fold. Binding of the DNA aptamer to exosite II appeared to inhibit heparin binding to exosite I.