Affinity For heparin-Sepharose Research Articles

Mutation of the H-helix in antithrombin decreases heparin stimulation of protease inhibition

Blood clotting proceeds through the sequential proteolytic activation of a series of serine proteases, culminating in thrombin cleaving fibrinogen into fibrin. The serine protease inhibitors (serpins) antithrombin (AT) and protein C inhibitor (PCI) both inhibit thrombin in a heparin-accelerated reaction. Heparin binds to the positively charged D-helix of AT and H-helix of PCI. The H-helix of AT is negatively charged, and it was mutated to contain neutral or positively charged residues to see if they contributed to heparin stimulation or protease specificity in AT. To assess the impact of the H-helix mutations on heparin stimulation in the absence of the known heparin-binding site, negative charges were also introduced in the D-helix of AT. AT with both positively charged H- and D-helices showed decreases in heparin stimulation of thrombin and factor Xa inhibition by 10- and 5-fold respectively, a decrease in affinity for heparin sepharose, and a shift in the heparin template curve. In the absence of a positively charged D-helix, changing the H-helix from neutral to positively charged increased heparin stimulation of thrombin inhibition 21-fold, increased heparin affinity and restored a normal maximal heparin concentration for inhibition.

Expression of the Acidic Fibroblast Growth Factor and Basic Fibroblast Growth Factor in Nasal Polyps

In order to increase our knowledge of the in vivo role of acidic fibroblast growth factor (aFGF) in the central nervous system, we have examined aFGF levels during mouse brain development. Using a specific polyclonal antibody raised against aFGF, we measured levels of aFGF-immunoreactive material (IRMaFGF) in extract of total mouse brain taken at different days of development. We found that the level of measurable IRMaFGF remained low and without significant variation during fetal brain development (0.2 ng/mg of extracted proteins). During the first 11 days postnatal (P0 to P11), IRMaFGF increased from 0.5 to 1.5 ng/mg. Between P11 and P14 IRMaFGF levels went up more rapidly, reaching 5 ng/mg. From P30 to adulthood a constant value of 2.5 ng/mg was measured. aFGF content in the different brain extracts was further characterized by its affinity for heparin-Sepharose, its elution at 1 M NaCl from this column and its capacity to induce thymidine incorporation in quiescent fibroblasts. These results were confirmed at the mRNA level. Northern blot analyses of poly A+ mRNA from brains with a specific riboprobe for bovine aFGF, revealed a major 4.5-Kb transcript and a minor 2.7-Kb transcript detectable only in postnatal brains. A similar pattern to that observed for IRMaFGF was seen with these mRNA transcripts, indicating that these aFGFmRNA are translated in the mouse brain. Our results suggest that aFGF may act in the postnatal phases of brain maturation.

The heparin-binding site of human xanthine oxidase.

The enzyme xanthine oxidase (XOD) has an affinity for heparin and can bind to cultured porcine aortic endothelial cells. We have reported that the exposure of human XOD (h-XOD) to the lysine-specific reagent trinitrobenzenesulfonic acid or the arginine-specific reagent phenylglyoxal caused it to lose its affinity for heparin-Sepharose. The heparin-binding sites in h-XOD are further studied in the present article. From a chymotryptic digest of cyanogen bromide fragmented h-XOD, two peptides with an affinity for heparin-HPLC, A-1 and A-2, were isolated. Amino acid sequence analysis showed that both peptides had lysine and/or arginine residues. The A-1 region may direct its charged side chains toward the solvent while burying its hydrophobic side chains against the hydrophobic inside, because the A-1 peptide forms a highly amphipathic structure. Peptide A-2 contains triple lysine residues and constitutes a hydrophilic region.

Binding of human xanthine oxidase to sulphated glycosaminoglycans on the endothelial-cell surface.

Much evidence has suggested that the superoxide generated by xanthine oxidase (XOD) within the endothelial cell triggers characteristic free-radical-mediated tissue injuries. Although it has been reported that XOD exists not only in the cytoplasm, but also on the outside surface of the endothelial cell membrane, it is not clear how XOD localizes on the outside of the plasma membrane. Purified human xanthine oxidase (h-XOD) had an affinity for heparin-Sepharose. The binding was largely independent of the pH over the physiological range, whereas it tended to increase at lower pH and to decrease at higher pH. Exposure of h-XOD to the lysine-specific reagent trinitrobenzenesulphonic acid or the arginine-specific reagent phenylglyoxal caused it to lose its affinity for heparin-Sepharose. The binding of h-XOD to heparin is apparently of electrostatic nature, and both lysine and arginine residues are involved in the binding. h-XOD was found to bind to cultured porcine aortic endothelial cells, and this binding was inhibited by the addition of heparin or pretreatment of the cells with heparinase and/or heparitinase. Intravenous injection of heparin into two healthy persons led to a prompt increase in plasma h-XOD concentration. These results suggest that XOD localizes on the outside surface of endothelial cells by association with polysaccharide chains of heparin-like proteoglycans on the endothelial-cell membranes. Superoxide extracellularly generated by XOD may injure the source-endothelial-cell membrane and also attract and activate closely appositional neutrophils, which themselves actually cause progressive oxidative damage.

Developmental changes of acidic fibroblast growth factor (aFGF) transcription and expression in mouse brain

In order to increase our knowledge of the in vivo role of acidic fibroblast growth factor (aFGF) in the central nervous system, we have examined aFGF levels during mouse brain development. Using a specific polyclonal antibody raised against aFGF, we measured levels of aFGF-immunoreactive material (IRMaFGF) in extract of total mouse brain taken at different days of development. We found that the level of measurable IRMaFGF remained low and without significant variation during fetal brain development (0.2 ng/mg of extracted proteins). During the first 11 days postnatal (P0 to P11), IRMaFGF increased from 0.5 to 1.5 ng/mg. Between P11 and P14 IRMaFGF levels went up more rapidly, reaching 5 ng/mg. From P30 to adulthood a constant value of 2.5 ng/mg was measured. aFGF content in the different brain extracts was further characterized by its affinity for heparin-Sepharose, its elution at 1 M NaCl from this column and its capacity to induce thymidine incorporation in quiescent fibroblasts. These results were confirmed at the mRNA level. Northern blot analyses of poly A + mRNA from brains with a specific riboprobe for bovine aFGF, revealed a major 4.5-Kb transcript and a minor 2.7-Kb transcript detectable only in postnatal brains. A similar pattern to that observed for IRMaFGF was seen with these mRNA transcripts, indicating that these aFGFmRNA are translated in the mouse brain. Our results suggest that aFGF may act in the postnatal phases of brain maturation.

Purification of basic fibroblast growth factor from rat brain: Identification of a Mr 22,000 immunoreactive form

A 18,000-dalton protein which stimulates plasminogen activator (PA) activity in endothelial GM 7373 cells has been purified from rat brain by using heparin affinity chromatography and ion-exchange chromatography. The purified molecule stimulates PA activity in a dose-dependent manner between 1 and 30 ng/ml. It also stimulates proliferation of GM 7373 cells and DNA synthesis in NIH 3T3 cells in a similar concentration range. The molecule has been identified as a bFGF-like molecule on the basis of its biological activity, its affinity for heparin-Sepharose, and its cross-reactivity with anti-human bFGF antibodies. In the final preparation of the rat brain bFGF, trace amounts (less than 5%) of a contaminant were detectable. This contaminant has a molecular weight of 22,000 and cross reacts with several anti-human placental bFGF antibodies. On the basis of its affinity for heparin-Sepharose and its immunological characteristics, this protein appears to be an high molecular weight form of bFGF.

Expression of human extracellular superoxide dismutase in Chinese hamster ovary cells and characterization of the product.

A complementary DNA clone from human placenta, encoding human extracellular superoxide dismutase (EC-SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1), has recently been isolated and characterized. An expression plasmid, based on the EC-SOD complementary DNA, was transfected into Chinese hamster ovary cells (CHO-K1). The transfected cells secreted human EC-SOD to the culture medium. The secreted recombinant (r) EC-SOD was isolated in high yield with a three-step procedure beginning with immobilized monoclonal anti-EC-SOD antibodies. The properties of the rEC-SOD were compared with native (n) EC-SOD isolated from human umbilical cords. The specific activities and amino-terminal amino acid sequences were identical. The amino acid compositions were virtually identical and very similar to the composition deduced from the complementary DNA sequence. Both rEC-SOD and nEC-SOD contained 4 Cu and 4 Zn atoms per molecule, and the presence of Zn in EC-SOD is thus now established. The rEC-SOD produced is type C, since its affinity for heparin-Sepharose was identical to that of nEC-SOD type C. Both enzymes bound to concanavalin A, lentil lectin, and wheat germ lectin and are thus glycoproteins. rEC-SOD and nEC-SOD seem to have the same subunit structure and composition as analyzed by polyacrylamide gel electrophoresis and gel chromatography.

Growth Factors in the Prostate

Certain local tissue factors, such as growth factor, in addition to androgens, are involved in the prostate growth. The prostate contains two types of growth factors capable of stimulating DNA synthesis in BALB/3T3 cells. They were divided into low affinity (LoA) type and high affinity (HiA) type by a different affinity for heparin-Sepharose. HiA-type growth factor is further classified into acidic HiA and basic HiA types. Acidic HiA type could be purified from the Dunning tumor (R 3327), a rat prostatic adenocarcinoma, and has a molecular weight of about 19,000 and a pI of about 3.8. Basic HiA type could be isolated from the tissues of human benign prostatic hypertrophy and has a molecular weight of about 12,000 and a pI of about 10.5. They are inactivated by heat and acid treatments. Acidic HiA type appears to be involved in growth of the rat prostate epithelium, and LoA type growth factor is possibly relevant to reproductive physiology because of its coexistence with "probasin," a major secretory protein in the dorsolateral prostate having a strong affinity for spermatozoa.

Purification from a human hepatoma cell line of a basic fibroblast growth factor-like molecule that stimulates capillary endothelial cell plasminogen activator production, DNA synthesis, and migration.

A 17,500-dalton protein which stimulates plasminogen activator production in cultured bovine capillary endothelial cells has been purified from a SK-Hep-1 human hepatoma cell lysate by using heparin affinity chromatography and fast protein-liquid ion exchange chromatography. The purified molecule stimulated plasminogen activator production in a dose-dependent manner between 0.01 and 1 ng/ml. It also stimulated collagenase synthesis, DNA synthesis, and motility in capillary endothelial cells in the same concentration range. This molecule was identified as a basic fibroblast growth factor-like molecule on the basis of its biological activity, its affinity for heparin-Sepharose, and its cross-reactivity with a polyclonal antibody raised against the human placental basic fibroblast growth factor.

Purification from a human hepatoma cell line of a basic fibroblast growth factor-like molecule that stimulates capillary endothelial cell plasminogen activator production, DNA synthesis, and migration.

A 17,500-dalton protein which stimulates plasminogen activator production in cultured bovine capillary endothelial cells has been purified from a SK-Hep-1 human hepatoma cell lysate by using heparin affinity chromatography and fast protein-liquid ion exchange chromatography. The purified molecule stimulated plasminogen activator production in a dose-dependent manner between 0.01 and 1 ng/ml. It also stimulated collagenase synthesis, DNA synthesis, and motility in capillary endothelial cells in the same concentration range. This molecule was identified as a basic fibroblast growth factor-like molecule on the basis of its biological activity, its affinity for heparin-Sepharose, and its cross-reactivity with a polyclonal antibody raised against the human placental basic fibroblast growth factor.

Potential proteolytic activity of human plasma fibronectin.

Evidence is presented that fibronectin (FN) polypeptide chain contains a latent proteinase. Human plasma FN was cleaved with cathepsin D into three main fragments: 140-kDa and 70-kDa single-chain and 140-kDa double-chain polypeptides. Their separation was achieved according to their affinity for heparin-Sepharose. A single-chain 140-kDa fragment (H-1) was eluted in the first peak. This peptide corresponds to the already described fragment that originates from the central part of FN; it contains a low-affinity heparin-binding site, one free SH group, and a cell-binding site. After reduction and further purification by preparative polyacrylamide gel electrophoresis, this fragment revealed a spontaneous decomposition, which could be attributed to proteolytic degradation. The subfragments, ranging from 25 to 95 kDa, yielded the same proteolytically active doublet of 28-30 kDa when tested by NaDodSO4/polyacrylamide gel electrophoresis in a gel containing copolymerized gelatin or fibrinogen. The proteolytic activity was inhibited by specific SH proteinase inhibitors. The proteinase forms a labeled complex after its incubation with 125I-labeled cystatin. Neither FN, cathepsin D, nor any products from previous purification steps were proteolytically active under the conditions of the assay. It was suggested that the same fragment may also yield an inhibitor, since structural analogies were found between the cell-binding region of FN and SH proteinase inhibitors.

Multiple forms of endothelial cell growth factor. Rapid isolation and biological and chemical characterization.

Endothelial cell growth factor (ECGF) can be rapidly purified from bovine brain to high specific activity using heparin-Sepharose affinity chromatography. Purification of the mitogen by this method results in relatively high yields of the polypeptide (10 to 100 micrograms/kg of tissue) with biological activity on murine and human endothelial cells in the picogram range. The product obtained is a mixture of two single-chain polypeptides with apparent molecular weights of 17,000 (alpha-ECGF) and 20,000 (beta-ECGF) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The two forms of ECGF can be separated by either NaCl gradient elution from heparin-Sepharose or reversed-phase high pressure liquid chromatography. The two polypeptides are related on the basis of similar: amino acid compositions, affinity for heparin-Sepharose, cyanogen bromide and trypsin-derived cleavage products, and biological activity. Furthermore, the cyanogen bromide fragments derived from the two forms of ECGF also possess similar amino acid compositions and mobilities on sodium dodecyl sulfate gels. These data suggest that there are at least two discrete molecular forms of ECGF in bovine brain and that these two molecules are structurally related.

Activation of C1, the first component of complement, the generation of C1r-C1s and C1̄ inactivator complexes in normal serum, by heparin-affinity chromatography

Investigations facilitating heparin-affinity chromatography and immunochemical procedures were utilized to study the interaction and behaviour of heparin with C1, C1 subcomponents and C1̄ IA in normal serum. Clq bound to heparin-Sepharose independent of divalent cations at 4 and 37° C at physiological pH and salt concentration requiring a high salt concentration for its complete release. MacromolecularC1 bound to heparin-Sepharose in the presence of Ca 2+ at 37°C, activating Clr and Cls in the process as demonstrated by the appearance of C1̄r-C1̄s-C1̄ IA complexes when purified C1̄ IA was added to the eluted fractions. At 4° C however, proenzyme C1r and C1s were present in the eluted fractions. Proenzyme C1r-C1s and C1̄r-C1̄s-C1̄ IA complexes did not bind directly to heparin-Sepharose. Binding of C1r-C1s in the presence of Ca 2+ was dependent on Clq bound to heparin. High levels of proenzyme C1r-C1s complexes were produced during the 4°C experiments in the presence of Ca 2+ and were collected in the void volume. C1̄r-C1̄s-C1̄ IA complexes generated during the 37°C experiments had no affinity for heparin-Sepharose. C1̄ IA did not bind directly to heparin. Recirculation of a serum sample collected after Clq was removed by heparinaffinity at 4°C in the presence of EDTA demonstrated that (1) Clr and Cls in the presence of calcium could be reassembled with heparin-bound Clq to form macromolecular Cl. (2) Clr and Cls in non-activated forms did not bind directly to heparin-Sepharose. (3) Clr and Cls contained in the reassembled Cl/heparin complex at 37°C were in active enzymic forms.