ABSTRACT: This paper describes a simple purification procedure for protease nexin, a serine proteinase inhibitor secreted by cultured human fibroblasts that regulates proteinase activity at and near the cell surface. The first step in the procedure takes advantage of the high-affinity binding of protease nexin to dextran sulphate-Sepharose. This step eliminates the need for prior concentration of the serum-free fibroblast-conditioned medium, since protease nexin binds to the resin in the presence of physiological saline. The use of dextran sulphate also provides an affinity resin with considerably less variability than the heparin-based resins previously used. Final purification to homogeneity involves a combination of DEAE-Sepharose in-line with dextran sulphate-Sepharose to simultaneously purify and concentrate the protein. Purified protease nexin is shown by Ouchterlony analysis and peptide mapping to be immunologically and structurally distinct from antithrombin III and heparin cofactor II, two plasma proteinase inhibitors with similar properties.
Project description:The binding of bovine testicular hyaluronidase to AH-Sepharose (1,6-diaminohexane--Sepharose) gels substituted with (1) dermatan sulphate, (2) desulphated dermatan sulphate, (3) heparin and (4) de-N/O-sulphated, re-N-acetylated heparin was investigated. Hyaluronidase was found to bind to (1) and (3), but not (2) and (4). On the basis of these observations a preparative scheme for the purification of testicular hyaluronidase was developed. This consisted of two steps: (i) chromatography on dermatan sulphate-substituted AH-Sepharose 4B; (ii) chromatography on acetylated AH-Sepharose 4B. This procedure gave hyaluronidase with a specific activity of 19.1 units (mumol/min)/mg in high yield. Polyacrylamide-gel electrophoresis at pH 4.3 revealed two components, both possessing hyaluronidase activity. Sodium dodecyl sulphate polyacrylamide-gel electrophoresis likewise revealed two close bands with approximate molecular weights of 61000 and 67200.
Project description:Cathepsin D was purified by two-step affinity chromatography on concanavalin A-- and pepstatin--Sepharose. The main purification was achieved by washing the enzyme bound to the pepstatin--Sepharose column with buffered 6 M-urea. This step separated cathepsin D from all low- and high-molecular-weight impurities. Although the 1700-fold purified acid proteinase was homogeneous on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, it still showed microheterogeneity.
Project description: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.
Project description:Xyloside-initiated 35SO4(2-)-labelled glycosaminoglycans were isolated from the medium of cultured bovine glomeruli and covalently coupled to Sepharose 4B to construct a solid-phase substrate suitable for the detection of endoglycosidases. The substrate is rendered specific for heparitinase by prior digestion with chondroitin sulphate ABC lyase and is insensitive to proteinase, neuraminidase and hyaluronidase. Normal human mononuclear cells are shown to contain a heparitinase. This enzyme appears to be cell-associated and can be partially purified from human spleen by heparin affinity chromatography.
Project description:The heparin-binding affinity of the tetrameric extracellular superoxide dismutase (EC-SOD) is a result of the cooperative effect of the heparin-binding domains of the subunits, located in the hydrophilic, strongly positively charged C-terminal ends. EC-SOD C, the high-heparin-affinity type, exposed to immobilized trypsin and plasmin was found to rapidly lose its affinity for heparin, without any loss of enzymic activity or major change in molecular mass as judged by size-exclusion chromatography. Heparin and dextran sulphate 5000 inhibited the proteolysis, suggesting that EC-SOD C sequestered by heparan sulphate proteoglycan in vivo is partially protected against proteolysis. The loss of heparin-affinity occurred with the stepwise formation of intermediates, and the pattern upon chromatography on heparin-Sepharose and subsequent immunoblotting was compatible with the notion that the changes are due to sequential truncations of heparin-binding domains from subunits composing the EC-SOD tetramers. A similar pattern with intermediates and apparent truncations has previously been found with EC-SOD of human plasma. The findings show that the unique design of the heparin-binding domain of EC-SOD allows easy modification of the heparin-affinity by means of limited proteolysis, and suggest that such proteolysis is a major contributor to the heterogeneity in heparin-affinity of EC-SOD in mammalian plasma.
Project description:A protein with pore-forming activity has been isolated from the outer membrane of rat liver mitochondria. The purification involves sucrose gradient centrifugation, differential centrifugation in the presence of Triton X-100, and DEAE-Sepharose and CM-Sepharose chromatography. The yield of the purified protein was approx. 2% of the total outer membrane proteins. The protein, when inserted into soya bean phospholipid vesicles, increases the [3H]sucrose permeability of the vesicles but had no effect on the permeability of high-molecular-weight [14C]dextran (Mr 70 000). The protein is very active, since as little as 3-4 micrograms of protein per mg of phospholipid is required for the complete release of [3H]sucrose from the vesicles. Sucrose diffusion channels could not be reconstituted with other membrane proteins such as rat liver cytochrome oxidase or cytochrome b5. Purified pore protein revealed a single band of apparent Mr 30000 when resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This polypeptide could be further resolved by isoelectric focusing into a major (pI7.9) and two relatively minor (pI7.6 and 7.2) components. Proteolytic mapping with V8 proteinase from Staphylococcus aureus suggests that these probably represent a single component showing charge heterogeneity. The reason for the charge heterogeneity is not known. The amino acid composition of the protein revealed 47.8% polar amino acids with a relatively high lysine content.
Project description:S-protein, the main inhibitor of the assembly of the membrane attack complex of complement, was isolated from human plasma by a simple purification procedure, which includes barium citrate adsorption, ammonium sulphate precipitation, chromatography on DEAE-Sephacel and Blue Sepharose and gel filtration on Sephacryl S-200. The homogeneous protein (sedimentation coefficient 4.6 S) was obtained in approx. 5% yield relative to its concentration in plasma, which was found to be 0.3-0.5 mg/ml. The final product did not cross-react with antisera against complement proteins or other proteinase inhibitors of human plasma. On polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, S-protein migrated as a single-chain band with an apparent Mr of 74000 under non-reducing conditions and as a doublet of Mr 78000 and 65000 upon reduction. In plasma or serum S-protein also existed in two forms of corresponding Mr values, as was evidenced by an immunoblot enzyme-linked immunosorbent assay technique. S-protein was found to be an acidic glycoprotein with 10% (W/W) carbohydrate content and several isoelectric points in the range pH 4.75-5.25, and it contained one free thiol group per molecule of protein. The functional properties of S-protein in the complement system were demonstrated by its ability to inhibit complement-dependent cell lysis in a concentration-dependent manner (Ki 0.6 microM) and by its incorporation into the nascent SC5b-7 complex. A new function for S-protein could be revealed in the blood coagulation system. The slow progressive inhibition of thrombin by antithrombin III was not affected by S-protein, whereas the purified protein interfered with the fast inactivation of thrombin clotting as well as amidolytic activity by antithrombin III-heparin complex. The acceleration of this inhibition reaction by heparin was counteracted by S-protein, indicating the ability of S-protein to neutralize heparin activity.
Project description:Purified m beta-acrosin catalysed amidolysis of several p-nitroanilides with C-terminal arginine residues. Antithrombin III inhibited amidolysis catalysed by the enzyme. This effect of antithrombin III was potentiated by heparin, and to a modest extent by heparan sulphate, cellulose sulphate, dextran sulphate and xylan sulphate. De-N-sulphated heparin, de-N-sulphated N-acetylated heparin, heparin of low relative molecular mass, chondroitin 4-sulphate, chondroitin 6-sulphate, dermatan sulphate and hyaluronic acid were ineffective.
Project description:Subconfluent cultures of human embryonic skin fibroblasts were labelled with [35S]sulphate for 3 days, after which cell-free extracellular matrix was isolated. A chondroitin sulphate proteoglycan (CSPG) and a heparan sulphate proteoglycan (HSPG) were purified from the matrix. Chromatography on Sepharose CL-2B gave peak Kav. values of 0.35 and 0.38 respectively for the CSPG and the HSPG. The polysaccharide chains released from the two PGs were of similar size (Kav. 0.50 on Sepharose CL-4B). Approx. 50% of the CSPG showed affinity for hyaluronic acid (HA). However, it differed immunologically from the HA-aggregating CSPG of human articular cartilage, and had a larger core protein (apparent molecular mass 290 kDa) than had the cartilage PG. Neither metabolically [35S]sulphate-labelled PGs, isolated from the medium of fibroblast cultures, nor chemically 3H-labelled polysaccharides (HA, CS, HS and heparin) were incorporated into the extracellular matrix when added to unlabelled cell cultures. These results indicate that the matrix PGs are not derived from the PGs present in the medium and that an interation between polysaccharide chains and matrix components is not sufficient for incorporation of PGs into the matrix. Incubation of cell-free 35S-labelled matrix with unlabelled polysaccharides did not lead to the release of any 35S-labelled material, supporting this conclusion. Furthermore, so-called 'link proteins' were not present in the fibroblast cultures, indicating that the CSPGs were anchored in the matrix in a manner different from the link-stabilized association of CSPG with HA in chondrocyte matrix. The identification of a proteinase, secreted by fibroblasts in culture, that after activation with heparin has the ability to release 35S-labelled PGs from the matrix may also indicate that the core proteins are important for the association of the PGs to the matrix.
Project description:The low potential cytochrome b (b-245) of the microbicidal oxidase of phagocytic cells has been purified from neutrophils from patients with chronic myeloid leukaemia. Cells were homogenized in the presence of proteinase inhibitors and centrifuged to remove the cytoplasm. The pellets containing membranes, granules and other organelles (15 mg/ml) were then washed with buffered sodium cholate (5 mg/ml). Residual pellets were subsequently solubilized with the non-ionic detergent Triton N 101 (10 mg/ml) which extracted about 60% of the cytochrome b. About 10% of the cytochrome b was of mitochondrial origin which was removed on a column of n-amino-octyl-Sepharose that did not adsorb cytochrome b-245. Cytochrome b-245 was chromatographed on a column of heparin-agarose and eluted with NaCl to give a peak specific content of 11-16 nmol of cytochrome b-245/mg of protein, representing a 140-200-fold purification with a recovery of 15%. This technique results in the purification of approx. 100-150 nmol of highly purified cytochrome b-245 from (3-5) X 10(11) cells within 4 days. The most purified material gave a broad band with an apparent Mr of between 68 000 and 78 000 on sodium dodecyl sulphate/polyacrylamide gel electrophoresis, but gel filtration indicated an aggregated form of the protein in Triton N101 . Purified protein (14 nmol of haem/mg of protein) did not contain FAD or FMN and had no NADPH-dependent O2--generating activity.