In-gel ligand blotting with 125I-heparin for detection of heparin-binding growth factors

125I-labeled heparin was used to detect basic fibroblast growth factor (bFGF) in crude tumor cell extracts after separation by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. 125I-labeled heparin bound avidly to native recombinant bFGF immobilized on nitrocellulose and eluted with 1.5-2.0 M NaCl. However, Western transfer of bFGF to nitrocellulose after SDS-polyacrylamide gel electrophoresis destroyed heparin-binding ability. In contrast, bFGF was detected by incubation of the polyacrylamide gels directly with 125I-labeled heparin in a gel overly technique. Heparin affinity and NaCl elution pattern from bFGF in gel were similar to those observed for native bFGF spotted on nitrocellulose. This procedure permitted detection of bFGF in crude extracts of a human astrocytoma cell line. In view of the rapid growth of the heparin-binding fibroblast growth factor gene family, this technique should prove useful for the rapid and sensitive detection of other heparin-binding growth factors.     

Separation of mouse crushed muscle extract into distinct mitogenic activities by heparin affinity chromatography

Extracts from gently crushed adult mouse skeletal muscles (CMEs) contain potent myoblast mitogens, and may be used as a model system to investigate myotrophic factors released by adult muscles following injury. CME was separated into four peaks of mitogenic activity by heparin affinity chromatography. The fraction of CME that did not bind to heparin contained transferrin (Tf). Three peaks of mitogenic activity were eluted from the heparin-agarose columns at NaCl concentrations of 0.4 M, 0.9 M, and 2.0 M. A 46 kDa protein that shared antigenicity with the BB isoform of platelet-derived growth factor (PDGF-BB) was present in the 0.4 M NaCl eluant. Mitogenic activity in the 2.0 M NaCl peak eluted identically to purified basic fibroblast growth factor (bFGF), did not act additively to saturating amounts of purified bFGF, and was neutralized by anti-bFGF antibodies. The 0.9 M NaCl eluant acted additively to the combination of three known growth factors for myoblasts, bFGF, insulin-like growth factor I, and epidermal growth factor, to stimulate C2 myoblast proliferation, suggesting this fraction contains a mitogenic activity which does not utilize (and hence compete for) receptors for the known mitogens for myoblasts. Additionally, the 0.9 M NaCl eluant did not stimulate proliferation of fibroblast-like cells derived from muscle tissue. The unbound, 0.4 M NaCl, 0.9 M NaCl, and 2.0 M NaCl eluants from the heparin-agarose column acted additively to one another to stimulate myoblast proliferation. Our data suggest that Tf, PDGF-BB-like molecules, bFGF-like activity, and an uncharacterized heparin-binding myoblast mitogen could be released after muscle injury and act to stimulate satellite cell proliferation. © 1994 Wiley-Liss, Inc.     

Lipoprotein lipase-like activity in the liver of mice with Sarcoma 180

The triglyceride lipase (TGL) activity of liver homogenates of mice with Sarcoma 180 was measured. The liver homogenate of normal or tumor-bearing mice was treated with 0.25% Triton X-100 and centrifuged at 100,000 g for 60 min, and the supernatant was applied to a heparin-Sepharose column. In normal mice, most of the TGL activities in the supernatant was eluted with 0.75 M NaCl from the column. In mice with Sarcoma 180, the TGL gave two peaks on heparin-Sepharose column chromatography, which were eluted with 0.75 M and 1.5 M NaCl, respectively. The activity in the first peak (0.75 M NaCl eluate) decreased; that in the second peak (1.5 M NaCl eluate) increased, and the ratio of the second peak to the first peak increased during tumor development. The livers of normal mice and mice on day 10 after tumor inoculation were perfused with heparin. The highest rate of the TGL release occurred within 1 min of heparin perfusion, and the bulk of heparin-releasable activity appeared within 2 min of perfusion in both normal and tumor-bearing mice. The TGL activity in liver perfusate of tumor-bearing mice, as well as that of liver homogenate, was resolved on a heparin-Sepharose column into two peaks, which were eluted with 0.75 M and 1.5 M NaCl, and most of the activity was eluted with 1.5 M NaCl. The nature of the TGL activity eluted from a heparin-Sepharose column was investigated. In both liver homogenates and liver perfusates, the first peak did not require serum for maximal activity and was relatively resistant to a high concentration of NaCl or protamine sulfate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bovine retina contains three growth factor activities with different affinity to heparin: eye derived growth factor I, II, III

Several ocular tissues have been shown to contain growth factor activity designated under a generic name as Eye Derived Growth Factor. Purification from bovine retina was undertaken and a fraction which could induce target cells to proliferate at doses of 5 ng per ml of culture medium was obtained. Using heparin sepharose chromatography we now show that this mitogenic activity can be fractionated into three different activities. Crude extract of bovine retina used as starting material was separated into two major fractions, one with no affinity for heparin and which was named Eye Derived Growth Factor III, and one with a strong affinity for heparin and eluted from the column with 1.4 M NaCl named Eye Derived Growth Factor I. This fraction EDGF I induces cell proliferation at doses of 100 pg/ml of culture medium. A 10(5) fold purification was achieved by this single chromatography step. Cibacron Blue purified EDGF was also further fractionated by heparin sepharose. All biological activity was found to bind to heparin. One fraction eluted at 1 M NaCl named Eye Derived Growth Factor II had a biological activity at doses of 1 ng while the other growth factor was the EDGF I with biological activity at 25 pg. At this step of purification EDGF I runs as a single band on SDS polyacrylamide gel at a molecular weight of 17 000 d. These data strongly suggest that Eye Derived Growth Factors I and II are respectively similar to Brain Fibroblast Growth Factor and to Endothelial Cell Growth Factor from hypothalamus.     

Purification and amino acid analysis of two human monocyte chemoattractants produced by phytohemagglutinin-stimulated human blood mononuclear leukocytes

Physicochemical characteristics of monocyte chemotactic activity in the culture fluid of PHA-stimulated human mononuclear leukocytes (MNL) were investigated. Among several chemotactic activity peaks eluted from a TSK-2000 gel filtration column, one peak, corresponding to a molecular mass of 17 kDa, accounted for about 40% of total chemotactic activity. On a chromatofocusing column, most of the 17-kDa activity eluted in a pH range of 9.4 to 7.9. It could bind to Orange-A Sepharose. These three characteristics--molecular mass, basic isoelectric point, and dye column binding--were similar to those of human glioma-derived monocyte chemotactic factor (GDCF), recently purified in our laboratory. Therefore, the MNL-derived chemoattractant was purified by the same procedures used for purification of GDCF, namely Orange-A Sepharose chromatography, carboxymethyl (CM)-HPLC, and reverse phase (RP) HPLC. About 50% of the culture fluid chemotactic activity bound to Orange-A Sepharose and was eluted in a single peak by a NaCl gradient. The active pool from the Orange-A column was separated into two sharp peaks by CM-HPLC, each of which eluted at identical acetonitrile concentrations from a RP HPLC column. By SDS-PAGE, the peptides had apparent molecular masses of 15 and 13 kDa and appeared homogeneous. Amino acid analysis showed that the composition of the two peptides was almost identical; and the N terminus of each peptide was apparently blocked. Shared characteristics of these peptides and the GDCF peptides include identical elution patterns from CM- and RP HPLC columns, identical SDS-PAGE migration, almost identical amino acid composition, and blocked N terminus. This suggests that the monocyte attractants isolated from culture fluid of PHA-stimulated MNL are identical to those derived from human glioma cells.     

Fractionation of heparin by affinity chromatography on covalently-bound human alpha-thrombin

Commerical heparin, 135 USP units/mg, was fractionated by human α-thrombin-agarose affinity chromatography. Heparin was applied to an α-thrombin-agarose column equilibrated with 0.01 M Tris HCl (pH 7.4). Unbound heparin was washed from the column with the equilibration buffer. Bound heparin could be eluted with buffer containing 0.025 M NaCl. The specific activity of bound heparin was as great as 500 USP units/mg. Gel filtration was used to fractionate the heparin into molecular size classes. Low molecular weight heparin, with an average specific activity of 100 USP units/mg, was applied to the α-thrombin-agarose column. Gel filtration of the unbound heparin indicated that larger heparin molecules been selectively removed by the α-thrombin-agarose column. Bound heparin had a specific activity of 270 units/mg. Kinetic results of $\text{N-α-tosyl-L-glycyl-L-prolyl-L-arginine-}\text{p}\text{-nitroanilide}$ hydrolysis by α-thrombin in the presence of heparin correlated with the anticoagulant activity.     

Estrogen sulfotransferase of mouse placenta: behaviour and characteristics during partial purification

Mouse placental estrogen sulfotransferase (ST) was partially purified by fast protein liquid chromatography (FPLC) gel filtration in combination with FPLC anion exchange. Owing to the highly unstable nature of the enzyme, large increases in specific activity were not obtained. Storage of the ST in the presence of thiol groups at -20 degrees C stabilized the enzyme considerably. Forty-three percent of the cytosolic ST was bound to an Affi-Gel blue column and eluted as a broad peak at approximately 0.8 M NaCl. The use of the latter procedure, in combination with FPLC gel filtration, did not increase the specific activity substantially. Larger increases in specific activity were obtained using agarose-hexane-adenosine-3',5'-diphosphate affinity chromatography. The bound ST activity was eluted under a single peak at 1 mM ADP. Increases in specific activity following use of this column averaged 54-fold but could reach 90-fold. Attempts at further purification of this material resulted in low recovery and decreased specific activity. Velocity versus substrate concentration curves show that estrone and particularly estradiol inhibit the partially purified mouse placental sulfotransferase above 0.1-0.25 microM substrate concentrations.     

Identification of the hepatocyte mitogen in bovine spleen as heparin-binding growth factors.

Growth promoting activity for rat hepatocytes in bovine spleen was identified as three heparin-binding growth factors. All the features tested, such as heparin affinity, molecular mass, cross reactivity with antibody, and partial amino acid sequence, indicated that one of the three factors was identical to FGF-1 (fibroblast growth factor-1, acidic FGF), another one was related to FGF-2 (fibroblast growth factor-2, basic FGF), whereas it was more potent for hepatocytes than the FGF-2 purified from bovine brain. The third one was eluted from heparin-Sepharose column at 0.75M NaCl, of which activity was not abolished by anti-FGF-1 or FGF-2 antibodies. In addition, the mitogenic effect of this factor was synergistic with that of HGF (hepatocyte growth factor), a known potent hepatocyte mitogen, suggesting that it is a novel growth factor for hepatocytes.     

Heparin-derived oligosaccharides: affinity for acidic fibroblast growth factor and effect on its growth-promoting activity for human endothelial cells

The minimal structural requirements for the interaction of heparin with acidic fibroblast growth factor (aFGF) were investigated. Oligosaccharides (tetra- to decasaccharides) obtained by nitrous acid depolymerisation of standard heparin were separated by affinity chromatography on Sepharose-immobilised aFGF. The shortest fragment retained by the affinity column at 0.2 M NaCl and eluted at 1 M NaCl was a "regular" hexasaccharide, a trimer of the most abundant disaccharide sequence in heparin. More complex octa- and decasaccharides were also retained by the column. The oligosaccharides eluted by 1 M NaCl from the affinity column ("high-affinity" oligosaccharides) and those washed from the column at 0.2 M NaCl ("low-affinity" oligosaccharides) were compared for their capacity to protect aFGF from proteolysis and to potentiate its mitogenic activity. At a low ionic strength, all oligosaccharides tested, except the "regular" disaccharide, protected aFGF against trypsin and collagenase digestion. At higher ionic strength (greater than 0.2 M NaCl), only high-affinity oligosaccharides showed a protective effect. The high-affinity oligosaccharides (hexa- to decasaccharides) potentiated the mitogenic activity of aFGF, as measured by [3H]thymidine incorporation into DNA of human fibroblasts. The effect of the oligosaccharides on human endothelial cell proliferation was more complex: inhibition of proliferation was observed in the presence of serum and low concentrations of aFGF (1-5 ng/ml) and potentiation in the presence of higher concentrations of aFGF. The potentiating effect increased as a function of molecular size of the heparin fragments and, for a given size, as a function of the anionic charge of the oligosaccharide. Our results suggest that inhibition of cell proliferation by heparin may result from interference with an autocrine basic FGF-like activity.     

Resolution of the molecular forms of rat liver glucocorticoid receptor by affinity chromatography on immobilized heparin

Rat liver cytosolic glucocorticoid receptor labelled with [³H] dexamethasone and stabilized with molybdate was bound to heparin-ultrogel and eluted with NaCl or heparin as a single peak of radioactivity. After heat exposure of cytosol, two steroid receptor complexes could be separated by NaCl or heparin. Characterization of the two forms was performed by means of affinity towards isolated nuclei, ssucrose gradient centrigugation and gel exclusion high performance liquid chromatography. The results presented here suggest that the two forms eluted from heparin-agarose correspond to the untransformed and transformed states of the glucocorticoid receptor complex. Taken together, these observations argue in favor of heparin-ultrogel as a suitable procedure to study the mechanism of glucocorticoid-receptor transformation.     

The sucrose carrier of the plant plasmalemma. III. Partial purification and reconstitution of active sucrose transport in liposomes.

The proteins from plasma membranes from sugar beet leaves were solubilized by 1% CHAPS and separated by size exclusion chromatography and by ion-exchange chromatography. The fractions enriched in sucrose transporter were monitored in three ways: differential labeling, ELISA, and reconstitution in proteoliposomes. When the plasma membranes were differentially labeled by N-ethylamaleimide in the presence of sucrose, a major peak of differential labeling was found at 120 kDa upon gel filtration. When this peak was recovered, denaturated by sodium dodecyl sulfate and reinjected on the gel filtration column, it yielded a peak of differential labeling at 42 kDa. When unlabeled membranes were used, the fractions eluted from the column were monitored by ELISA for their ability to recognize a serum directed against a 42 kDa previously identified as a putative sucrose carrier. The results paralleled those obtained by differential labeling, i.e. a major ELISA-reactive peak was found at 120 kDa upon gel filtration, and this peak yielded a peak most reactive at 40 kDa after denaturation. The 120 kDa peak prepared from unlabeled membranes was further separated on a Mono-Q column. The fractions were monitored by ELISA as described above, and reconstituted into proteoliposomes using asolectin. Active transport of sucrose, but not of valine could be observed with the reconstituted 120 kDa fraction. When the eluates from the Mono-Q column were reconstituted, the fractions exhibiting highest transport activity were enriched with a 42 kDa band. The data provide the first report concerning reconstitution of sucrose transport activity and confirm the involvement of a 42 kDa polypeptide in sucrose transport.     

Isolation and characterization of a macrophage-derived heparin-binding growth factor.

Human mononuclear cells were plated in culture, and the conditioned media of these cells were analyzed by heparin-Sepharose affinity chromatography. The fractions were tested for growth factor activity as measured by the stimulation of DNA synthesis in BALB/c 3T3 cells. After 2 d in culture, two peaks of heparin-binding growth factor (HBGF) activity were detected, one eluting with 0.5 M NaCl, which could be shown to be platelet-derived growth factor (PDGF)-like, and the other eluting with 1.0 M NaCl. After 7-11 d in culture, when monocytes had clearly differentiated into macrophages, greater than 95% of the HBGF activity in conditioned medium consisted of the 1.0 M NaCl elution peak. This activity, which was designated macrophage-derived HBGF (MD-HBGF), was found to be a cationic heat-resistant polypeptide with a molecular weight in the range of 14-25 kDa. Analysis using Western blots and specific neutralizing antisera, as well as comparative heparin affinity analysis, indicated that MD-HBGF was not identical to other heparin-binding 3T3 cell growth factors known to be produced by macrophages, such as PDGF (AB, AA, and BB forms), acidic fibroblast growth factor, and basic fibroblast growth factor. In addition to stimulating mitogenesis in 3T3 cells, MD-HBGF also stimulated the proliferation of vascular smooth muscle cells, but did not stimulate the proliferation of vascular endothelial cells.     

Expression of cDNA encoding human basic fibroblast growth factor in E. coli

The cDNA encoding human basic fibroblast growth factor was expressed in E. coli under the control of trp promoter. Bacterially synthesized hbFGF was highly purified using a heparin affinity HPLC column. By this chromatography, hbFGF was eluted as four distinct forms, which were indistinguishable by SDS polyacrylamide gel electrophoresis, amino acid composition, and partial terminal sequence analysis. These molecules stimulated the growth of fibroblasts and endothelial cells although their specific activities varied. The angiogenesis activity of these molecules was also confirmed.     

Human placental fibronectin: demonstration of structural differences between the A and B chains in the extra domain-A region

Fibronectins are a class of cell adhesion proteins produced from a single gene. Soluble plasma fibronectin plays a role in wound healing and the insoluble cellular fibronectin form anchors cells to the substrata. The proteins possess multiple macromolecular binding domains including collagen, fibrin, and heparin. Alternative RNA splicing in at least three regions (ED-A, ED-B, and III CS) is responsible for this fibronectin polymorphism. We have been studying this polymorphism at the protein level in placental fibronectin, a poorly soluble form of cellular fibronectin. Cathepsin D-digested placental fibronectin applied to a heparin-agarose column and eluted with a NaCl stepwise gradient (0.1, 0.3, 0.5 M) gave two polypeptides (80-100 and 65 kDa) in the 0.3 M NaCl peak. Immunoblots with monoclonal antibodies IST-2 (specific for the carboxy-terminal heparin-binding domain) and IST-9 (specific for the ED-A portion of fibronectin) suggest that both peptides contain the carboxy-terminal heparin-binding (Hep-2) domain, but that only the larger fragment possesses the ED-A segment. The two peptides were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electrotransferred to Polybrene-coated polyvinyl difluoride membranes, and characterized by microsequence analysis. This analysis confirmed that both fragments start with the same amino acid sequence, 17 amino acids before the start of ED-A. These results demonstrate that placental fibronectin is a heterodimer, structurally distinct from plasma fibronectin due to the presence of a unique domain modification that is not seen in the plasma form.     

Purification of supercoiled plasmid DNA using chromatographic processes

The interest in purifying injectable-grade plasmid DNA has increased with the development of gene therapy and DNA vaccination technologies. In this paper we develop a method for purifying a 4.8 kb plasmid based on chromatographic processes. An NaCl gradient was optimized on a Q Sepharose column and plasmid was eluted at 800-820 mM NaCl in a broad peak. Supercoiled plasmid was isolated after a final Sepharcryl S1000 SF gel filtration step. Final plasmid preparation was depleted of proteins and RNA, as revealed by the BCA assay and 1% agarose gel electrophoresis.     

Identification and Purification of Calcium-Independent Phospholipase A2 from Bovine Brain Cytosol

Substantial amounts of phospholipase A2 activity were detected in bovine brain cytosol. The major phospholipase A2 activity was present in the precipitate at 40% saturation with solid ammonium sulfate. After the desaltate of the precipitate was loaded onto an Ultrogel AcA 54 gel filtration column, almost all the activity eluted in the void volume when chromatographed without 1 M KCl. However, when buffer with 1 M KCl was used as the eluent, two active peaks were obtained. One peak (peak I) eluted in the void volume, and the other (peak II) eluted with an apparent molecular mass of 39 kDa as compared with standards. The former was active with diacylglycero-3-phosphoethanolamine, whereas the latter was active with both diacylglycero-3-phosphoethanolamine and 1-alk-1'-enyl-2-acylglycero-3-phosphoethanolamine (plasmenylethanolamine). The apparent molecular mass of peak I was estimated to be 110 kDa as compared with standards on an Ultrogel AcA 34 gel filtration column. Both peaks were purified further with a hydrophobic chromatography column (AffiGel 10 coupled with plasmenylethanolamine) and then by high-resolution liquid chromatography on an MA7Q column. The phospholipase A2 obtained from peak II migrated as one main band with a 40-kDa molecular mass and two minor bands with 14- and 25-kDa molecular masses. Phospholipase A2 obtained from peak I eluted as a single peak on high-resolution liquid chromatography but contained two bands with apparent molecular masses of 100 and 110 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of an endothelial cell growth factor from serum-free culture medium of human diploid fibroblast cells

Serum-free culture supernatants of human embryo fibroblast cells contain endothelial cell growth factor (f-ECGF) which supports the serial propagation of human umbilical vein endothelial cells in the serum-free culture medium (medium A). This growth-stimulating activity has been partially purified from serum-free culture-conditioned medium. The stability of the activity to acid (pH 4.0-4.5) was utilized for the first step in purification. f-ECGF had a high affinity to heparin-Sepharose CL-6B, and was isolated by the methods of heparin affinity, of ion-exchange and gel filtration chromatography from the serum-free culture-conditioned medium preparation. The purified f-ECGF had an isoelectric point in the pH range 4.5-6, and a molecular weight of approx. 30 kDa determined by either gel filtration or SDS-polyacrylamide gradient gel electrophoresis. The f-ECGF has high affinity for concanavalin A column, and the activity was partially eluted from the column with ethylene glycol and alpha-methylmannose. The results indicate that f-ECGF is an acidic-glyco-protein with heterogeneous sugar chain(s).     

The flowering of interferon

A microsomal vesicle fraction was prepared from rat liver homogenate by centrifugation in gradients of Percoll. The microsomes were subjected to gel filtration on Sephacryl S-1000 Superfine, which resolved the microsomes from Percoll. The elution pattern of the microsomal marker enzyme NADPH-cytochrome c reductase showed that the main part of the enzyme was present in a peak at Kav about 0.1, while Percoll eluted in a broad peak at Kav about 0.7. The total yield of eluted enzyme activity was 85%. The gel filtration had to be carried out in the presence of 10 mM tris or NaCl. At lower ionic strength or in 0.25 M sucrose alone, anomalous behaviour of the Percoll particles and microsomes on the gel was observed. Electron microscopy of samples from the void volume fraction of the Sephacryl S-1000 Superfine column showed an almost complete removal of Percoll from the microsomes. Furthermore, the vesicle preparation was essentially free of membrane fragments.     

Separation of bovine brain mono- and diacylglycerol lipases by heparin Sepharose affinity chromatography

Mono- and diacylglycerol lipases are differentially inhibited by heparin. No other glycosaminoglycan resembles heparin in this respect. Mono- and diacylglycerol lipases can be separated by heparin Sepharose affinity chromatography. Diacylglycerol lipase was completely retained on a heparin--Sepharose column and was eluted with either 0.5 M NaCl or 2–5 mg/ml heparin, whereas monoacylglycerol lipase was recovered in the washings. Adenosine phosphates markedly affected the activity of diacylglycerol lipase in a concentration dependent manner. ATP was the most potent inhibitor followed by ADP. AMP had no effect and cAMP slightly stimulated the diacylglycerol lipase.