Human lung tissue as a source of colony stimulating activity.

Medium conditioned by human lung tissue was found to contain colony stimulating activity (CSA). This material was tested against mouse and human bone marrow as target system. Colony forming units (CFUc) from both species responded and gave rise to clonal growth in agar cultures. This colony formation was dose dependent and the relationship was a sigmoid one. Experiments to determine the molecular weight of human lung derived colony stimulating Factors brought evidence for four active molecular weight fractions with approximately 79000, 40000, 23000 and 2000 daltons. The 23000 dalton fraction activated human cells only, whereas the other fractions were active on both human and mouse bone marrow cells.     

The isolation and characterization of a colony stimulating factor from human lung.

Serum-free conditioned medium from human lung obtained at autopsy provides a rich source of colony stimulating factor which stimulates granulocytic and macrophagic colony growth in both mouse and human bone marrow. The appearance of the factor is enhanced by endotoxin and inhibited by either puromycin or actinomycin D. Human lung colony stimulating factor is stable at the pH range of 6.5-10 and temperature of 56 degrees C for 30 min. It is resistant to trypsin and neuraminidase but is sensitive to subtilisin, chymotrypsin and periodate. It shows heterogeneity on Sephadex gel filtration with two activity peaks having molecular weight of 200 000 and 40 000, respectively. Upon gel electrophoresis, human lung colony stimulating factor migrates in the alpha-globulin post-albumin region. Using the combination procedures of hydroxyapatite chromatography and preparative polyacrylamide gel electrophoresis a 600-fold purification was achieved with a final specific activity of 6-10(5) units per mg protein. The purified colony stimulating factor is very labile; however, the activity can be stabilized by the addition of gelatin or bovine serum albumin at the concentration of 0.1% and 0.2 mg/ml, respectively.     

Established cell lines of mouse marrow adherent cells producing differentiation factor(s) for the granulocyte-macrophage lineage

Summary Two continuous cell lines derived from long-term cultures of AKR mouse bone marrow adherent cells were isolated. These cell lines release colony stimulating activity (CSA), a factor that induces in vitro differentiation of granulocyte-macrophage progenitor cells. The colony forming cells and cluster forming cells in mouse marrow responsive to CSA from cell line conditioned medium were compared with those responsive to CSA from mouse lung conditioned medium (MLCM). Colony forming cells were characterized by analysis of their density distribution after equilibrium centrifugation in density gradient. Cluster forming cells were characterized by analyzing the progeny of individual clusters after transfer to fresh semisolid culture medium containing MLCM. The results obtained indicate that the CSA from cell line conditioned medium closely compares with the CSA from MLCM in terms of the populations of colony and cluster forming cells stimulated. This work was supported by a research grant from the Institut National de la Santé et de la Recherche Médicale (CRL 802620), Paris, France.     

Purification of the major protein-tyrosine-phosphatases of human placenta

This report describes the purification of the major protein-tyrosine-phosphatases from human placenta. Enzyme activity was followed with a novel artificial substrate, namely reduced, carboxamidomethylated, and maleylated lysozyme, phosphorylated on tyrosine by a partially purified preparation of insulin and epidermal growth factor receptor kinases, also from human placenta. The key step in the purification of the protein-tyrosine-phosphatases was affinity chromatography on a column of thiophosphorylated, reduced, carboxamidomethylated, and maleylated lysozyme-Sepharose. Purification was carried out separately from both the soluble and particulate fractions. Whereas multiple and distinct enzyme forms were obtained from each of these, little difference could be detected between the behavior of the "soluble" enzyme subtypes and their "particulate" counterparts. The major subtypes were purified to apparent homogeneity with an approximately 23,000-fold enrichment and 10% yield from the soluble fraction and a 4,300-fold enrichment and 13% yield from the particulate fraction. Both samples migrated as bands of 35 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had specific activities of approximately 45,000 nmol of Pi released min-1 mg-1, at least 2-3-fold higher than that of the type 1 and 2A serine/threonine phosphatases. The level of protein-tyrosine-phosphatases in the soluble fraction of human placenta (2,000 units/g of protein) was approximately the same as protein-serine/threonine-phosphatases 1 and 2A in skeletal muscle.     

Protamine-agarose non-charged alkyl derivatives of agarose in the purification of rat-liver phosphoprotein phosphatases.

1. Protamine-agarose and hydrophobic interaction chromatography were found to be effective in the purification of phosphoprotein phosphatase(s) (phosphoprotein phosphohydrolase, EC 3.1.3.16) of rat-liver. The phosphoprotein phosphatase of rat-liver cytosol were first resolved into three fractions, termed A, B and C, in order of elution from DEAE-cellulose. Whereas all fractions displayed activity towards [32P]phosphoprotamine, only fractions B and C displayed appreciable activity towards [32P]phosphopyruvate kinase. Since fraction B exhibited the most properties and the highest recovery of enzymatic activity towards [32P]phosphoprotamine and [32P]phosphopyruvate kinase, it was selected for further purification. The method developed involves sequential chromatography of fraction B on Sephadex G-200, protamineagarose, histone-agarose and then again on Sephadex G-200 as a final step. A 400-fold enrichment in the phosphoprotamine phosphatase activity of fraction B was obtained. Purified fraction B also displayed substantial phosphatase activity towards [32P]phosphopyruvate kinase and [32P]phosphohistones. An apparent molecular weight of about 250 000 was estimated for purified fraction B on a calibrated Sephadex G-200 column. The present data indicate that rat-liver cytosol contains multiple forms of phosphoprotein phosphatases and suggest a technique which might be applied for the further purification of at least fraction B. 2. In a separate approach, a combination of pentyl-agarose and protamineagarose chromatography was shown to be a conbenient method for the enrichment (up to 20-fold of phosphoprotein phosphatase activity from crude liver extracts.     

Enhancement of erythroid progenitor cell growth in medium conditioned by a human cancer cell line, KONT

Erythroid-potentiating activity (EPA) was detected in culture medium conditioned by a human cancer cell line (KONT) that produces colony-stimulating activity (CSA), using erythroid colony formation in vitro. EPA in the medium conditioned by the KONT cells (KONT-CM) was markedly heat stable. After treating KONT-CM at 80 degrees C for 30 min, 30% EPA remained, while CSA was completely inactivated. Both EPA and CSA appeared in approximately the same fractions of the gel filtration, indicating a molecular weight of approximately 30,000 daltons. EPA bound partially to Concanavalin-A Sepharose, whereas CSA almost did not bind. Our results indicate that EPA can be separated from CSA based on heat stability and binding to Concanavalin-A Sepharose.     

Separation of growth-promoting activity for human muscle cells from fetuin.

Whether the growth-promoting activity of Pedersen fetuin is due to fetuin itself or to a contaminant(s) has been a long-standing puzzle. The possibility that the growth-promoting activity of Pedersen fetuin for human muscle satellite cells (HMSC) could be caused by some other component of fetal bovine serum (FBS) that remained in the fetuin as a contaminant has been investigated. One liter of FBS was first precipitated with 50% saturated ammonium sulfate, which leaves the serum albumin in solution, and then with 25% polyethylene glycol, which leaves the fetuin in solution, to generate a fraction 50 PEG 2x that was enriched 11-fold in growth-promoting activity for HMSC, with 68% recovery of total activity. Further purification with FPLC anion exchange chromatography achieved 99-fold enrichment of the activity with 30% overall recovery. The activity is heat labile and pH sensitive, suggesting that it is of protein nature, and the size of the activity is above 70 kDa. SDS-PAGE of the most active fractions shows that they are virtually free of fetuin. Thus, although the active fractions are not homogeneous, these studies demonstrate that the growth-promoting activity for HMSC can be fully separated from fetuin.     

Erythropoietic enhancing activity (EEA) secreted by the human cell line,GCT

Medium conditioned by the monocyte-like cell line GCT contains colony-stimulating activity (CSA), a mediator of in vitro granulopoiesis. Also, the conditioned medium (CM) contains erythroid-enhancing activity (EEA), which can be demonstrated in a system utilizing either nonadherent marrow or blood mononuclear cells, erythropoietin (1–2 units/ml), and 20 ml/dl fetal calf serum. Under these conditions, GCT CM enhances the growth of CFU-E and BFU-E. Attempts were made to characterize the molecular features of EEA. Serum-free GCT cell CM was fractionated on Sephacryl S200 and Ultrogel AcA54. EEA and CSA cochromatographed with apparent molecular weights of ～ 40,000 daltons on Sephacryl and ～ 30,000 daltons on Ultrogel. Fractionation on DEAE Sephacel led to an apparent separation of CSA from EEA; however, when diluted, the fractions containing CSA had EEA. Undiluted fractions containing potent CSA inhibited erythropoiesis; however, dilution of these fractions resulted in marked EEA. Diluted crude GCT CM and DEAE Sephacel fractions enriched in EEA were also capable of sustaining BFU-E in liquid culture and mediating erythropoietin-independent colony growth. CSA could not be unequivocally separated from EEA on concanavalin A-Sepharose, since the diluted void volume containing CSA also had EEA. EEA was present in CM boiled for 60 minutes, whereas CSA was markedly reduced but not abolished. The inverse relationship between CSA concentration and EEA mandates dilution of fractions when bioassayed for these two activities. Although CSA and EEA are similar in molecular weight, they appear to be partially separable by ion-exchange chromatography and heat stability.     

An improved procedure for the isolation of lamellar bodies from human lung. Lamellar bodies free of lysosomes contain a spectrum of lysosomal-type hydrolases

We have recently shown that lamellar body fractions purified from human lung contain a distinct acid alpha-glucosidase distinguishable from lysosomal acid alpha-glucosidase in that it does not cross-react with antibodies raised against the lysosomal enzyme and does not bind to concanavalin A (De Vries, A.C.J., Schram, A.W., Tager, J.M., Batenburg, J.J. and Van Golde, L.M.G. (1985) Biochim. Biophys. Acta 837, 230-238). In order to study the relationship between the non-concanavalin A-binding alpha-glucosidase and lamellar bodies more closely a method was developed for the further purification of the organelles. A purified lamellar body preparation isolated from human lung homogenate by discontinuous sucrose density centrifugation was subjected to gel filtration with Sepharose 4B followed by Percoll density gradient centrifugation, which yielded a lamellar body preparation with a phospholipid phosphorus/protein ratio of 12.57 +/- 0.38 (mumol/mg) (n = 3) as compared to a ratio of 3.34 +/- 0.16 (mumol/mg) (n = 3) in the sucrose density gradient preparation. Concomitantly there was a 3.3 +/- 0.1 (n = 3)-fold enrichment in the content of total acid alpha-glucosidase and a 3.2 +/- 0.1 (n = 3) -fold enrichment of non-concanavalin A-binding acid alpha-glucosidase. The new purification method removes adhering proteins without changing the phospholipid composition. During the successive purification steps the concanavalin A-sensitive and -insensitive alpha-glucosidases remained fully lamellar body fraction associated. Differences between a lysosome-enriched fraction and a lamellar body preparation at varying stages of purification with respect to the ratio between soluble acid hydrolases and the membrane-associated lysosomal enzyme glucocerebrosidase indicate that the purified lamellar bodies were not contaminated with lysosomes. The absence of lysosomes in the purified lamellar body fraction was confirmed by experiments with the weak base glycyl-L-phenylalanine-beta-naphthylamide, which is an artificial substrate for the lysosomal enzyme cathepsin C and brings about lysis of lysosomes. Morphological examination by electron microscopy endorses the absence of contaminating vesicles and organelles and showed a structural integrity of the lamellar bodies in the final preparation. The improved isolation procedure strongly suggests that the concanavalin A-insensitive acid alpha-glucosidase is endogenous to lamellar bodies and supports our earlier idea that it can be used as a lamellar body-specific marker enzyme. In addition, the experiments show that lamellar bodies free of lysosomes contain a spectrum of lysosomal-type enzymes.     

Purification and characterization of a colony stimulating factor from human lung.

Conditioned medium prepared from human autopsy lung tissue contains high level activity of colony stimulating factor which stimulates granulocytes and macrophage colony formation in both mouse and human bone marrow. The lung colony stimulating factor has been purified about 2250-fold by methods including hydroxylapatite chromatography, preparative gel electrophoresis, preparative isoelectric focusing, and gel filtration chromatography. The final specific activity was 2.7 X 10(6) units/mg. The purified factor has a molecular weight of 41 000 as determined by gel filtration. It is stable at the pH range of 6.5--10 and 56 degrees C for 30 min but sensitive to protease digestion and periodate oxidation. On polyacrylamide gel electrophoresis, it migrates in the alpha-globulin post-albumin region. Upon isoelectrofocusing lung colony stimulating factor appears heterogeneous with isoelectric points of 3.7--4.3. Treatment with neuraminidase did not affect its activity, but caused a change in electrophoretic mobility and isoelectric point. Antibody produced by immunizing rabbits with partially purified lung colony stimulating factor exerted strong inhibitory activity on the factor from lung as well as on colony stimulating factor from other human sources including serum, urine, and placenta.     

Isolation of Bacterial Strain from Sediment Core of Pulp and Paper Mill Industries for Production and Purification of Lignin Peroxidase (LiP) Enzyme

Five bacterial strains were isolated and purified (CSA101 to CSA105) from the sediment core of the effluent released from the Century Pulp and Paper Mill Ltd., India. These strains were grown in minimal salt medium (MSM) containing pulp (10% as a carbon source). The production of lignin peroxidase, CMCase, Fpase, and xylanase together with protein and reducing sugar by all bacterial strains was observed. All of the bacterial isolates responded differently with respect to growth and ligninocellulolytic enzyme production. The maximum lignin peroxidase (LiP) was obtained from the cell extract of Bacillus sp. (CSA105) strain, which was used for purification, fractionation and characterization. The culture filtrate from Bacillus sp. (CSA105) was purified with ammonium sulfate precipitation. Crude protein was desalted by dialyzing with Tris buffer. The lignolytic enzyme produced in the liquid medium was fractionated by gel filtration on Sephadex G-100. In the present study, 12.4-fold purification of LiP enzyme was obtained and 35.85% yield of lignin peroxidase was achieved in the cell extract of Bacillus sp. (CSA105). Lignin peroxidase enzyme plays an important role in lignin degradation process. The ligninolytic enzymes were produced by all of the bacterial strains but maximum lignin peroxidase activity was found in cell extract of CSA105. On the basis of the results obtained, the bacterial strain (CSA105) was found most suitable for the purification of the LiP enzyme.     

Isolation and characterization of thrombomodulin from human placenta

Protein C, a plasma protein, is activated by thrombin to a protease (protein Ca) that functions as a physiological anticoagulant. We have isolated thrombomodulin, a cofactor required for the rapid activation of protein C, from human placenta. The purification to near homogeneity was achieved using a crude Triton-solubilized protein fraction from a placental particulate fraction as starting material. Chromatography on DEAE-Sepharose removed 95% of the protein and achieved a 3-fold purification. Thrombomodulin was then isolated by affinity chromatography on a column of thrombin-Sepharose wherein the thrombin had been previously inactivated with diisopropyl fluorophosphate. The final preparation was purified 7,900-fold over the membrane extract with a yield of 7%. We obtained 0.88 mg of thrombomodulin from 100 g of membrane extract derived from 5 kg of placenta. The protein was nearly homogeneous as judged by electrophoresis on 10% acrylamide sodium dodecyl sulfate gels in the presence of 2-mercaptoethanol with an apparent Mr = 105,000. Western blot analysis without 2-mercaptoethanol gave an apparent Mr = 75,000. The protein stimulated the rate of protein C activation by thrombin 800-fold to 10 mol of Ca formed/min/mol of thrombin. Thrombin and thrombomodulin appear to form a 1:1 stoichiometric complex as judged from experiments where we measured the effect of varying the concentration of thrombomodulin with respect to thrombin and the converse, on rates of protein C activation. An antibody directed against rabbit lung thrombomodulin inhibited the human placenta protein by 66%, and the amino acid composition of the proteins from the two species was similar indicating that the proteins are closely related. The apparent Michaelis constant of the thrombin-thrombomodulin complex for protein C is 9.8 microM. The protein C activation reaction requires calcium ions and is maximal at 1 mM Ca2+; higher concentrations inhibited the reaction. Coagulation factor Va and factor Va light chain both stimulate the activity of human thrombomodulin 2- to 3-fold.     

Partial purification of human urinary megakaryocyte colony-stimulating factor

Urinary extracts from patients with aplastic anemia are known to promote murine megakaryocytopoiesis. In this report, we show a simple method for the partial purification of megakaryocyte colony-stimulating factor from human urine. A four-step purification procedure, which included ethanol precipitation, CM Affi-Gel Blue chromatography, wheat germ agglutinin-Sepharose chromatography and high-resolution hydroxyapatite chromatography, resulted in an about 430- to 630-fold increase of specific activity. The final fractions were still contaminated with erythropoietin, but the contaminated content of erythropoietin was not enough to stimulate mouse megakaryocytopoiesis in our culture system. We also demonstrate that human urinary extracts stimulated human megakaryocyte colony formation.     

Purification of human lung angiotensin-converting enzyme by high-performance liquid chromatography: properties and N-terminal amino acid sequence

Angiotensin-converting enzyme from the human lung was purified to apparent homogeneity, using high-performance liquid chromatography following trypsin treatment of the detergent-extract. A 1,750-fold purification was achieved with a 26% yield. The specific activity of the enzyme was 105 units per mg protein with the substrate hippuryl-L-histidyl-L-leucine (HHL) at 37 degrees C, and the Km value for HHL was 1.9 mM. The molecular weight was estimated to be 170,000 by sodium dodecyl sulfate gel electrophoresis, and the isoelectric point was about 4.8, by chromatofocusing. The N-terminal amino acid sequence was (NH2)-X-X-Pro-Gly-Leu-Glu-Pro-Gly-X-Phe-Ser-Ala-Arg-Glu-Ala-Gly-Ala. This is highly homologous to the corresponding sequences of the enzymes from bovine and rabbit lung and from pig, bovine, and mouse kidney, but significantly different from that of the human kidney enzyme.     

Purification and characterization of a human plasma cholesteryl ester transfer protein

The cholesteryl ester transfer protein (CETP) binds to plasma lipoproteins and promotes transfer of cholesteryl esters between the lipoproteins. CETP has been purified 55,000-fold, with a 27% recovery of activity, from the d greater than 1.21 g/ml fraction of human plasma. In the final purification step, partially purified CETP is incubated with a synthetic lipid emulsion consisting of phosphatidylcholine, triglyceride, and fatty acid, and the bound activity, which elutes in the void volume, is separated from nonbound proteins by gel filtration on Sepharose 4B. Sodium dodecyl sulfate-gel analysis of fractions containing bound activity shows the presence of a single protein with an apparent Mr of 74,000. Inclusion of fatty acid in this emulsion was required to prevent the binding of a contaminant protein. However, incubation of CEPT with fatty acid emulsions containing lipid peroxides resulted in substantial inactivation and covalent degradation of the 74-kDa protein. This could be prevented by the inclusion of antioxidants during preparation of the emulsion. Solvent extraction of emulsion-bound CEPT gave a delipidated, active preparation. Purified IgG from a rabbit immunized with the 74-kDa protein completely removed activity from partially purified fractions. Amino acid analysis of the purified protein showed it to contain an unusually high content (45%) of nonpolar residues; the calculated hydrophobicity was greater than that of any other plasma apolipoprotein. These results show human CETP to be a unique plasma apolipoprotein with an apparent Mr of 74,000 which is hydrophobic, self-associating, and susceptible to covalent degradation by lipid peroxides.     

Purification of an autocrine growth factor in conditioned medium obtained from primary cultures of scleral fibroblasts of the chick embryo

Scleral fibroblasts of the chick embryo were found to secrete autocrine growth factors. One of the factors was purified from conditioned medium collected from growing-phase cultures of these cells by DEAE-Sepharose column chromatography and following non-denaturing polyacrylamide gel electrophoresis. The specific activity was increased 1100-fold by this purification. The chromatographically purified growth factor was still active after incubation at 95 degrees C, at pH 10 or pH 3, or with glycosidase H, but inactive after incubation with dithiothreitol or trypsin. An active protein having a molecular weight of 32 kDa was found to be the major component of the final preparation.     

Aptamer affinity chromatography:: combinatorial chemistry applied to protein purification

The systematic evolution of ligands by exponential enrichment process is a combinatorial chemistry method that allows the identification of specific oligonucleotide sequences, known as aptamers, that bind to a desired target molecule with high affinity and specificity. Here, a DNA-aptamer specific for human -selectin was immobilized to a chromatography support to create an affinity column. This column was effectively applied as either the first or second step in the purification of a recombinant human -selectin–Ig fusion protein from Chinese hamster ovary cell-conditioned medium. The fusion protein was efficiently bound to the column and efficiently eluted by gentle elution schemes. Application of the aptamer column as the initial purification step resulted in a 1500-fold purification with an 83% single step recovery. These results demonstrate that oligonucleotide aptamers can be effective affinity purification reagents.     

Isolation and reconstitution of the intestinal Na+/glucose cotransporter

The intestinal Na+/glucose cotransporter was isolated from brush border membrane vesicles using a three-step procedure and Na(+)-dependent phlorizin binding as the measure of cotransporter enrichment. The initial step was to treat the Ca2(+)-precipitated brush border membrane vesicles with 0.02% sodium dodecyl sulfate (SDS) followed by sucrose gradient centrifugation which resulted in a 5-fold enrichment of the Na+/glucose cotransporter. The second step was chromatofocusing chromatography over the pH range from pH 7.4 to pH 4.0. This step resulted in an additional 20-fold purification as compared with the SDS-brush border membrane vesicle protein which served as the starting material. The final step was affinity chromatography on con A-Sepharose which resulted in a 5-fold enrichment of the chromatofocused protein. The glycoprotein fraction from the concanavalin A column reconstituted into phosphatidyl choline: cholesterol liposomes demonstrated Na(+)-dependent, phlorizin-sensitive, and osmotic strength-sensitive glucose uptake. This fraction consisted of a single 75-kDa polypeptide on SDS-polyacrylamide gel electrophoresis upon staining with silver. On the basis of these criteria it appears that a protocol for the isolation of the Na+/glucose cotransporter has been developed.