TISSUE FACTOR OF A HUMAN CELL LINE, RET-1: ITSPRODUCTION, PURIFICATION AND PROPERTIES.

1987 ◽  
Author(s):  
Shin NAKAMURA ◽  
Yukio SUZUKI ◽  
Takayuki HARADA ◽  
Shigeru MORIKAWA ◽  
Shunichiro KAWABATA ◽  
...  
Keyword(s):  
Affinity Chromatography ◽  
Cell Line ◽  
Tissue Factor ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Human Cell Line ◽  
Factor Vii ◽  
Con A ◽  
Sepharose Column ◽  
Triton X

A dendritic cell-like cell line, HIN-Ret-1(RET-1), was found to produce the highest level of tissue factor (TF) among several established cell lines from human lymphoma or leukemia. The TF level expressed by this cell line exceeded 5.6 times that expressed by a monocyte-like cell line, U-937. Unlike other TF producing cell line, i.e. RET-2, HL-60, ML-3, and U-937, the TF expression by RET-1 was spontaneous and unaffected with TP A, PHA, LPS, or MAF. The TF activity of RET-1 was markedly inhibited by Con A as well as that produced by LPS-stimulated monkey monocytes, whereas the TF activity of monkey brain and lung was hardly inhibited by the lectin. Hence, the RET-1 cell lysate solubilized in Triton X-100 was subjected to affinity chromatography on a Con A-Sepharose column, and TF-apoprotein (TF-Apo) was completely bound to the column and eluted with TBS containing 0.15 M α(-methylglucoside and 0.1 % Triton X-100. Further purification of this material was performed with combination of FPLC on a DEAE-5PW column and affinity chromatography using a factor VII-Sepharose column. By these methods, TF-Apo preparation with purification-fold of 9,400 and over-all yield of 7 % was obtained. Its apparent molecular weight was estimated to be 120 kDa by gel filtration in TBS containing 0.1 % Triton X-100. SDS-PAGE gave the value of 47 kDa, which was almost compatible with that of TF-Apo from brain or placenta. TF-Apo frcm the monocytes also bound to the lectin column, suggesting that the apoprotein of these macrophage-related cells has an oligosaccharide chain interacting with the lectin. RET-1 TF-Apo was unstable under acidic condition (<pH 4.0) or in organic solvents such as isopropanol (>18 %) and acetonitrile (>8 %).

scholarly journals The hyaluronate synthase from a eukaryotic cell line

1993 ◽  
Vol 290 (3) ◽  
pp. 791-795 ◽  
Author(s):  
L Klewes ◽  
E A Turley ◽  
P Prehm
Keyword(s):  
Monoclonal Antibodies ◽  
Phase Separation ◽  
Affinity Chromatography ◽  
Cell Line ◽  
Aqueous Phase ◽  
Plasma Membranes ◽  
Eukaryotic Cell ◽  
Molecular Masses ◽  
Triton X ◽  
Hyaluronate Synthase

The hyaluronate synthase complex was identified in plasma membranes from B6 cells. It contained two subunits of molecular masses 52 kDa and 60 kDa which bound the precursor UDP-GlcA in digitonin solution and partitioned into the aqueous phase, together with nascent hyaluronate upon Triton X-114 phase separation. The 52 kDa protein cross-reacted with poly- and monoclonal antibodies raised against the streptococcal hyaluronate synthase and the 60 kDa protein was recognized by monoclonal antibodies raised against a hyaluronate receptor. The 52 kDa protein was purified to homogeneity by affinity chromatography with monoclonal anti-hyaluronate synthase.

scholarly journals Mouse Uterine Alkaline Phosphatase: Improved Purification by Affinity Chromatography and Further Characterization of the Enzyme

1980 ◽  
Vol 33 (3) ◽  
pp. 279 ◽  
Author(s):  
RN Murdoch ◽  
Louise E Buxton ◽  
DJ Kay
Keyword(s):  
Alkaline Phosphatase ◽  
Affinity Chromatography ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Apparent Molecular Weight ◽  
Anion Exchange Chromatography ◽  
Pregnant Mouse ◽  
Exchange Chromatography ◽  
Enzyme Preparations

An improved procedure for the purification of alkaline phosphatase from about 10 g of day 7 pregnant mouse uterine tissue is described. Following homogenization, the procedure involved solubilization and extraction with 0�8% (v/v) Triton X-lOO and 20% (v/v) n-butanol, ammonium sulfate precipitation, concanavalin A-Sepharose 4B affinity chromatography, DEAE-cellulose anion-exchange chromatography and Sephacryl S200 gel filtration. On subjecting 2162-fold purified enzyme preparations to polyacrylamide-gel electrophoresis, a single band of protein coincident with the zone of enzyme activity and having an apparent molecular weight of 205 OOO� lOOOO was identified. Affinity chromatography yielded the largest increase in purity of any step in the procedure and established the glycoprotein nature of the uterine enzyme.

Mannose containing glycopeptides of cells derived from human teratocarcinoma (line PA 1)

1980 ◽  
Vol 58 (5) ◽  
pp. 384-393 ◽  
Author(s):  
Maija-Liisa Rasilo ◽  
Jorma Wartiovaara ◽  
Ossi Renkonen
Keyword(s):  
Molecular Weight ◽  
Paper Chromatography ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Con A ◽  
Whole Cell ◽  
Undifferentiated State

Human teratocarcinoma derived cells, line PA 1, maintained in the undifferentiated state, yielded upon exhaustive pronase digestion unusually large glycopeptides (fraction A), which showed on gel filtration an apparent molecular weight larger than 7400. These glycopeptides derived from whole cell proteins carried large-sized oligosaccharides as evidenced by repeated pronase treatments, hydrazinolysis, and β-elimination experiments. The oligosaccharides consisted of mannose, fucose, galactose, and N-acetylglucosamine.The PA 1 cells contained also oligomannosyl type glycans, presumably linked to asparagine (fraction C glycopeptides). These glycopeptides were strongly bound to Con A – Sepharose and their oligosaccharides were released by endo-β-N-acetylglucosaminidase H. The liberated glycans ranged from Man5GlcNAc to Man9GlcNAc as analyzed by paper chromatography."Pulse–chase" experiments suggest that there is a precursor–product relationship between the mannose label in the fraction C (oligomannosyl type) glycopeptides and the fraction A glycopeptides.

Characterization of a High Affinity Folate Binding Protein in Porcine Serum: Ionic Charge, Concentration—Dependent Polymerization and Ligand Binding Mechanism

2003 ◽  
Vol 23 (5-6) ◽  
pp. 339-351 ◽  
Author(s):  
Jan Holm ◽  
Steen Ingemann Hansen
Keyword(s):  
Affinity Chromatography ◽  
Binding Protein ◽  
Gel Filtration ◽  
Competitive Inhibitor ◽  
Exchange Chromatography ◽  
Ionic Charge ◽  
Folate Binding Protein ◽  
Porcine Serum ◽  
Hill Coefficients ◽  
Triton X

The folate binding protein in porcine serum, present at concentrations of 50-100 nM, is cationic at near neutral pH as evidenced by ion exchange chromatography. The gel filtration profile of the protein isolated from porcine serum by methotrexate affinity chromatography exhibited one peak at 48 kDa and an additional peak of 91 kDa at higher protein concentrations. This could suggest the involvement of concentration-dependent polymerization phenomena. Binding of [3H] folate was of a high-af.nity type with upward convex Scatchard plots and Hill coefficients >1.0 indicative of apparent positive cooperativity. However, binding to protein isolated from porcine serum after affinity chromatography was biphasic (high/low-affinity) in the absence of Triton X-100, 1 g/1. These findings which are similar to those reported for purified milk folate binding proteins are consistent with a model predicting association between unliganded and liganded monomers to weak-ligand affinity heterodimers. Amphiphatic substances, e.g. Triton X-100, form micelles which could separate hydrophobic unliganded monomers from hydrophilic liganded monomers (monomers are hydrophilic in the liganded state) thereby preventing heterodimerization. The folate analogue N10 methyl folate was a potent and competitive inhibitor of [3H] folate binding to the folate binding protein, and moreover changed the binding type to apparent negative cooperativity.

Tissue Factor-Like Activity Of The Human Monocytic Tumor Cell Line U937

1981 ◽  
Author(s):  
D Hudig ◽  
S I Rapaport ◽  
S P Bajaj
Keyword(s):  
Cell Line ◽  
Tissue Factor ◽  
Tumor Cell Line ◽  
Procoagulant Activity ◽  
Factor Vii ◽  
U937 Cells ◽  
Factor X ◽  
Blood Monocytes ◽  
Monocytic Cell ◽  
Coagulant Activity

Cells of the human monocytic cell line U937, derived from a patient with histiocytic lymphoma (Sundstrom and Nilsson, Int. J. Cancer 17:565, 1976) have procoagulant activity similar to that of activated peripheral blood monocytes, although about 10-fold more U937 cells than monocytes are required for equivalent activity. Procoagulant activity of the cells is Ca2+ dependent and is not demonstrable in factor VII deficient or factor X deficient plasma. Culture with E. coli 0127:B8 1ipopolysaccharide increases the procoagulant activity of washed U937 cells two-fold. Exposure of U937 cells to lymphokines from normal lymphocytes does not induce further coagulant activity. The slope of the log/log plot of cells vs. clotting time parallels that of human brain thromboplastin. Other cell lines of myeloid or lymphoid origin, e.g., K562 cells, WI-L2 cells, do not have procoagulant activity. Thus, U937 cells have constitutive factor VII-dependent coagulant activity similar to the tissue factor activity induced by activation of normal monocytes.In further experiments, U937 cells were incubated with purified human factor VII in the presence or absence of Ca2+ and then repeatedly washed. When subsamples of the cells were then added to recalcified factor VII deficient plasma in the absence of added tissue factor, the following clotting times were obtained: for cells incubated with factor VII in the presence of Ca2+,45"; for cells incubated with factor VII in the absence of Ca2+, 150". These data suggest that U937 cells can bind factor VII in a reaction requiring Ca2+, which then enables the cells to express their tissue factor-like activity in factor VII deficient plasma.

Purification of Human Brain Tissue Factor

1988 ◽  
Vol 59 (03) ◽  
pp. 400-403 ◽  
Author(s):  
Sungzong Kang ◽  
Julian Niemetz
Keyword(s):  
Human Brain ◽  
Tissue Factor ◽  
Brain Tissue ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Factor Vii ◽  
Intrinsic Pathway ◽  
Human Brain Tissue ◽  
Agarose Beads

SummaryTissue factor (thromboplastin or Factor III), a glycoprotein cofactor, is required for Factor VII to express its catalytic activity, thereby initiating the extrinsic as well as intrinsic pathway of blood coagulation. Human brain tissue factor was purified 2,500-fold to 98% homogeneity from 2% Tiiton X-100 extraction of acetone dried brain powder with an overall yield of 36%. The method was based upon affinity chromatography utilizing the high affinity binding of tissue factor to Factor VII noncovalently complexed to immobilized anti-Factor VII-agarose beads. The apparent molecular weight of the purified tissue factor is 45,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its isoelectric point is 4.8–5.1 by column chromatofocussing and flat bed agarose isoelectric focussing.

Application of factor VII-sepharose affinity chromatography in the purification of human tissue factor apoprotein

1986 ◽  
Vol 42 (5) ◽  
pp. 635-643 ◽  
Author(s):  
Victor J.J. Bom ◽  
Ingrid E. Ram ◽  
Geertruida H.J. Alderkamp ◽  
Hanneke H. Reinalda-Poot ◽  
Rogier M. Bertina
Keyword(s):  
Affinity Chromatography ◽  
Tissue Factor ◽  
Human Tissue ◽  
Factor Vii

scholarly journals Synthesis and secretion of a cobalamin-binding protein by HT 29 cell line

1991 ◽  
Vol 280 (2) ◽  
pp. 427-430 ◽  
Author(s):  
H Schohn ◽  
J L Guéant ◽  
M Girr ◽  
E Nexø ◽  
L Baricault ◽  
...  
Keyword(s):  
Cell Culture ◽  
Western Blot ◽  
Affinity Chromatography ◽  
Molecular Mass ◽  
Cell Line ◽  
Binding Protein ◽  
Gel Filtration ◽  
Secreted Protein ◽  
Sds Page ◽  
Ht 29

An HT 29 cell line derived from human colonic carcinoma was shown to synthesize and release a cobalamin-binding protein. The cobalamin-binding protein was classified as transcobalamin (TC). By gel filtration on Sephacryl S200 HR, we observed that the secreted protein bound to cobalamin had the same size as plasma transcobalamin. Like transcobalamin, the cobalamin-binding protein bound cobalamin but not cobinamide. Purification of the cobalamin-binding protein was performed by heparin-Sepharose affinity chromatography and by Sephacryl S200 gel filtration. The molecular mass of the purified protein was estimated at 44 kDa by SDS/PAGE. The isoelectric point was determined to be 6.4. The purified cobalamin-binding protein reacted with an antiserum produced against human transcobalamin. A 44 kDa band was also identified by SDS/PAGE of an immunoprecipitated homogenate from HT 29 cells labelled with [35S]methionine and in a Western blot of cell homogenates. The secretion of the cobalamin-binding protein was maximal between 10 and 12 days of cell culture and was inhibited by cycloheximide.

Characterization of triton-solubilized TSH receptors from human thyroid plasma membranes

1981 ◽  
Vol 98 (1) ◽  
pp. 50-56 ◽  
Author(s):  
Yasuhiro Iida ◽  
Junji Konishi ◽  
Kanji Kasagi ◽  
Katsuji Ikekubo ◽  
Kanji Kuma ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Binding Capacity ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Cross Reactivity ◽  
Human Thyroid ◽  
Affinity Constant ◽  
Dependent Manner ◽  
Soluble Receptor ◽  
Triton X

Abstract. The TSH receptor from human thyroid plasma membranes has been solubilized in 10 mm Tris/HCl, 50 mm NaCl, pH 7.4 containing 0.5% triton X-100. Binding of [125I]TSH to the soluble receptor showed rapid and reversible kinetics and reached a maximum within 30 min at 37°C, by 1 h at 25°C and by 24 h at 4°C. Optimal pH was 7.4. The soluble receptor retained specificity with cross-reactivity only to crude hCG (0.03%). Scatchard plots were curvilinear, indicating the presence of at least two binding sites. The high affinity site showed an affinity constant of 1.1 × 109 m−1 with binding capacity of 1.3 × 10−10 m/mg protein. TSH-binding inhibitor immunoglobulins from patients with Graves' disease inhibited [125I]TSH binding to the soluble receptor in a dose-dependent manner. NaCl inhibited the TSH binding and this was ascribed to the decrease in the receptor capacity. Trypsin, neuraminidase and phospholipase C treatment of the solubilized receptor had no effect on TSH binding. The apparent molecular weight of the receptor, determined by gel filtration on Sepharose® 6B, was approximately 300 000.

Cloning of Guinea Pig Tissue Factor cDNA: Comparison of Primary Structure among Six Mammalian Species

2000 ◽  
Vol 83 (03) ◽  
pp. 455-461 ◽  
Author(s):  
Rui-Jin Shi ◽  
Wen-Zhou Li ◽  
Victor Marder ◽  
Lee Sporn
Keyword(s):  
Guinea Pig ◽  
Tissue Factor ◽  
Catalytic Domain ◽  
Mammalian Species ◽  
Coagulation Factor ◽  
Apparent Molecular Weight ◽  
Procoagulant Activity ◽  
Factor Vii ◽  
Reading Frame ◽  
Coagulation Factor Vii

SummaryTissue factor (TF) is a transmembrane glycoprotein that serves as an essential cofactor for plasma coagulation factor VII. TF procoagulant activity exhibits varying species specificity. In particular, guinea pig (GP) TF is unable to activate clotting in heterologous plasma systems, but the molecular basis for this phenomenon is not yet understood. The full-length GP TF cDNA was cloned and sequenced. The open reading frame encoded a predicted precursor protein of 289 amino acids (aa) which was expressed in a reticulocyte lysate system as a protein of apparent molecular weight of 34 kD. The identity of the predicted aa sequence of mature GP TF with rabbit, human, bovine, rat and mouse TF was 66.4, 64.4, 60.6, 53.2 and 52.2%, respectively. With a focus on sites of potential functional significance, we compared sequences within the known binding regions. The eleven residues at the interface region between the TF1 and TF2 modules, which bind to the EGF domain of VIIa, were perfectly conserved among the six species, with the exception of an isoleucine replacing a lysine in the guinea pig sequence. However, only four of the eleven binding residues in the TF1 module, known to interact with the catalytic domain of factor VII, and three of the five residues in the TF2 module, involved in binding the factor VII Gla domain, were conserved among species. We hypothesize that divergence at these regions contributes to the specificity and non-reciprocity of TF procoagulant activity between species.

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