ABSTRACT
9-(2-Phosphonylmethoxyethyl)-N6-cyclopropyl-2,6-diaminopurine (cpr-PMEDAP) is an acyclic nucleotide analog of the [9-(2-phosphonylmethoxyethyl)-] (PME) series containing a cyclopropyl substituent on the N6 position of the 2,6-diaminopurine (DAP) base. Growth inhibition assays in a broad range of tumor cell lines demonstrated that this analog had potent antiproliferative activity with IC50 values similar to those of the structurally related guanine analog 9-(2-phosphonylmethoxyethyl)guanine (PMEG). A substantially lower growth inhibitory effect was observed for the 2,6-diaminopurine analog, PMEDAP. To dissect the basis for these varying potencies, the metabolism of the three analogs was examined in a human pancreatic carcinoma cell line, BxPC-3. HPLC analysis of the intracellular metabolites demonstrated that the cpr-PMEDAP was deaminated to PMEG and subsequently phosphorylated to PMEG mono- and diphosphates (PMEGp and PMEGpp). The level of PMEGpp generated from cpr-PMEDAP-treated cells was 50% greater than the level generated from cells incubated with PMEG. The presence of PMEG in the DNA of cells incubated with cpr-PMEDAP confirmed that the cpr-PMEDAP was converted to PMEG. In contrast, PMEDAP was not deaminated to PMEG, but directly phosphorylated to PMEDAPp and PMEDAPpp. The adenylate deaminase inhibitor 2'-deoxycoformycin (dCF) inhibited the conversion of cpr-PMEDAP in a rat liver cytosolic extract and increased the IC50 value for growth inhibition by 40-fold. The antiproliferative activities of PMEG and PMEDAP were unaffected by dCF. Thus, it appears that cpr-PMEDAP, but not PMEDAP, is converted by an adenylate deaminase-like enzyme and functions as a prodrug of PMEG.
Subject(s)
Adenine/analogs & derivatives , Antineoplastic Agents/pharmacology , Guanine/analogs & derivatives , Organophosphorus Compounds/pharmacology , Prodrugs/pharmacology , Adenine/metabolism , Adenine/pharmacology , Animals , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Cell Division/drug effects , Deamination , Dideoxynucleosides/metabolism , Guanine/pharmacology , Humans , Prodrugs/metabolism , Rats , Tumor Cells, CulturedABSTRACT
PMEG (9-(2-phosphonylmethoxyethyl)guanine) is an acyclic nucleotide analog being evaluated for its anti-proliferative activity. We examined the inhibitory effects of PMEG diphosphate (PMEGpp) toward DNA polymerases (pol) delta and epsilon and found it to be a competitive inhibitor of both these enzymes. The apparent Ki values for PMEGpp were 3-4 times lower than the Km values for dGTP. The analog was shown to function as a substrate and to be incorporated into DNA by both enzymes. Examination of the ability of pol delta and pol epsilon to repair the incorporated PMEG revealed that pol epsilon could elongate PMEG-terminated primers in both matched and mismatched positions with an efficiency equal to 27 and 85% that observed for dGMP-terminated control template-primers. Because PMEG acts as an absolute DNA chain terminator, the elongation of PMEG-terminated primers is possible only by cooperation of the 3'-5'-exonuclease and DNA polymerase activities of the enzyme. In contrast to pol epsilon, pol delta exhibited negligible activity on these template-primers, indicating that pol epsilon, but not pol delta, can repair the incorporated analog.
Subject(s)
DNA Polymerase III/metabolism , DNA Polymerase II/metabolism , Guanine/analogs & derivatives , Organophosphorus Compounds/metabolism , Animals , Cattle , DNA Primers/metabolism , Electrophoresis, Polyacrylamide Gel , Guanine/metabolism , Humans , Models, Chemical , Oligonucleotides/metabolism , Templates, GeneticABSTRACT
Previous alanine scanning mutagenesis of thrombin revealed that substitution of residues W50, K52, E229, and R233 (W60d, K60f, E217, and R221 in chymotrypsinogen numbering) with alanine altered the substrate specificity of thrombin to favor the anticoagulant substrate protein C. Saturation mutagenesis, in which residues W50, K52, E229, and R233 were each substituted with all 19 naturally occurring amino acids, resulted in the identification of a single mutation, E229K, that shifted the substrate specificity of thrombin by 130-fold to favor the activation of the anticoagulant substrate protein C over the procoagulant substrate fibrinogen. E229K thrombin was also less effective in activating platelets (18-fold), was resistant to inhibition by antithrombin III (33-fold and 22-fold in the presence and absence of heparin), and displayed a prolonged half-life in plasma in vitro (26-fold). Thus E229K thrombin displayed an optimal phenotype to function as a potent and specific activator of endogenous protein C and as an anticoagulant in vivo. Upon infusion in Cynomolgus monkeys E229K thrombin caused an anticoagulant effect through the activation of endogenous protein C without coincidentally stimulating fibrinogen clotting and platelet activation as observed with wild-type thrombin. In addition, E229K thrombin displayed enhanced potency in vivo relative to the prototype protein C activator E229A thrombin. This enhanced potency may be attributable to decreased clearance by antithrombin III, the principal physiological inhibitor of thrombin.
Subject(s)
Anticoagulants/pharmacology , Protein Engineering , Thrombin/genetics , Thrombin/pharmacology , Animals , Blood Coagulation/drug effects , Drug Evaluation, Preclinical , Fibrinogen/metabolism , Half-Life , Humans , Kinetics , Macaca fascicularis , Models, Molecular , Mutagenesis, Site-Directed , Platelet Activation/drug effects , Protein C/metabolism , Protein Conformation , Substrate Specificity , Thrombin/metabolismABSTRACT
Protein purification has been made significantly easier by the use of affinity tags that can be genetically engineered at either the amino- or carboxyl-terminus of recombinant proteins. One of the most widely used tags is six consecutive histidine residues or 6His tag. These residues bind with high affinity to metal ions immobilized on chelating resins even in the presence of denaturing agents and can be mildly eluted with imidazole. We report the methodology for the immobilization of six histidine-containing proteins onto microtiter plates. A derivative of nitrilotriacetic acid (NTA) was prepared. This derivative, N,N-bis[carboxymethyl]lysine (BCML), was easily coupled to a maleic anhydride-activated polystyrene microtiter plate. The plate was then charged with Ni2+ for the capture of the 6His-tagged proteins. Using two different recombinant proteins with the 6His tag at either the N- or C-terminus, we demonstrated that the binding to the Ni(2+)-NTA plate was specific for six histidine-containing proteins. Proteins lacking the 6His tags did not bind to the plate. The plate was used in a modified enzyme-linked immunoabsorbent assay format to quantitate protein concentrations and determine the affinity of protein-ligand interactions. The technology can also be extended to include high-throughput screening assays for antagonists of protein-protein interactions.
Subject(s)
Chelating Agents , Histidine/analysis , Nickel , Nitrilotriacetic Acid/analogs & derivatives , Organometallic Compounds , Proteins/analysis , Sequence Tagged Sites , Antibodies, Monoclonal , Enzyme-Linked Immunosorbent Assay/methods , Indicators and Reagents , Interleukin-8/analysis , Ligands , Lysine/analogs & derivatives , Lysine/chemical synthesis , Recombinant Proteins/analysis , Reproducibility of Results , Sensitivity and Specificity , Transferrin/analysisABSTRACT
Tissue factor (TF) is a transmembrane protein that functions in the initiation of blood coagulation in vivo. At sites of vascular injury, TF serves as a cell-surface receptor for the serine protease factor VIIa (FVIIa), forming an enzyme--cofactor complex and enhancing the catalytic activity of FVIIa. Tissue factor, along with the receptors for alpha- and gamma-interferons, is a member of the class 2 cytokine receptor superfamily. Crystallographic analysis demonstrated that the extracellular domain of TF consists of two immunoglobulin-like domains joined by a linker region. Each domain is comprised of two antiparallel beta-sheets containing seven conserved beta-strands separated by more variable loop regions. Extensive mutagenesis has been performed in order to map the FVIIa binding site on TF. Results indicated that the discontinuous binding site for FVIIa lies at the domain--domain interface and includes residues from extended loops and beta-strands within both the N- and C-terminal domains. Our previous study provided evidence that three consecutive residues (D44, W45, K46) within the TF loop region between beta-strands C and C' of the N-terminal domain were important for interactions with FVIIa. We have presently extended our alanine-scanning mutagenesis to include the residues within the flanking beta-strands. Thirteen sTF mutants were screened for their ability to enhance FVIIa activity. Three residues within strand C (Y34, Q37, I38) and two residues within C' (K48, Y51) were shown to be important for TF cofactor function.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Factor VIIa/antagonists & inhibitors , Peptide Fragments/chemical synthesis , Peptide Fragments/pharmacology , Thromboplastin/antagonists & inhibitors , Thromboplastin/chemistry , Alanine , Amino Acid Sequence , Binding Sites , Cyclization , Escherichia coli/genetics , Factor VIIa/metabolism , Gene Expression , Molecular Sequence Data , Mutagenesis , Protein Conformation , Protein Structure, Secondary , Recombinant Fusion Proteins , SolubilityABSTRACT
Tissue factor (TF) is a membrane-bound glycoprotein that functions as a cofactor for coagulation factor VIIa (VIIa) and initiates blood coagulation at sites of vascular injury. On the basis of sequence alignments, TF was predicted to be a member of the cytokine receptor superfamily. Utilizing the structural information available for the cytokine receptor superfamily, we have used site-directed mutagenesis to identify the binding site on TF for VIIa. The predicted loop regions in TF were systematically replaced with the homologous loops from the gamma-interferon receptor (gamma-IFN-R), the protein most related to TF in the superfamily of cytokine receptors. Six discontinuous regions (residues 16-20, 40-46, 60-69, 101-111, 129-151, 193-207) were identified that are required for interaction with VIIa and enhancement of activity. Individual substitution of 68 residues within these loops with alanine revealed eight residues (K20, D44, W45, K46, Q110, R135, F140, V207) that are required for cofactor activity. These residues fall into two groups, those that are required only for interactions with VIIa (K46, Q110, R135, F140, V207) and those that are also required to induce the conformational change in VIIa required for enhanced activity (K20, D44, W45). The discontinuous regions of TF required for interactions with VIIa form a single binding surface for VIIa that is analogous to the interface defined by the crystal structure of the complex between growth hormone and its receptor.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Alanine/genetics , Factor VIIa/metabolism , Mutagenesis, Site-Directed , Thromboplastin/chemistry , Amino Acid Sequence , Binding Sites , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Escherichia coli/genetics , Interferon-gamma , Models, Molecular , Molecular Sequence Data , Molecular Structure , Protein Conformation , Receptors, Interferon/chemistry , Receptors, Interferon/genetics , Recombinant Proteins/isolation & purification , Sequence Alignment , Structure-Activity Relationship , Thromboplastin/metabolismABSTRACT
A variety of detergents have been shown to catalyze the dissociation of bound peptides from a soluble from of DRB1*0401. By using a class II molecule lacking the hydrophobic transmembrane region, the need for solubilizing the transmembrane protein was removed and enabled the specific interaction between the class II protein and the amphiphile to be identified. The presence of detergent increased the rate of association of added peptide and the percent occupancy of the receptor, presumably because the dissociation of endogenous peptide was the rate-limiting step in binding. The data help explain the differences reported between peptide binding to class II proteins on the surface of cells and binding to class II proteins solubilized in detergent. The interaction did not correlate with the critical micellar concentration of the detergent nor were all amphiphilic structures equally effective, consistent with a specific interaction between the amphiphile and the MHC class II protein. Of the eight detergents examined, octyl glucoside was the most efficient. These experiments did not distinguish between an allosteric mechanism or direct competition with the peptide for binding.
Subject(s)
Detergents/chemistry , HLA-DR Antigens/chemistry , Histocompatibility Antigens Class II/chemistry , Peptides/immunology , Amino Acid Sequence , Glucosides/chemistry , HLA-DR Antigens/metabolism , HLA-DRB1 Chains , Histocompatibility Antigens Class II/metabolism , Humans , Micelles , Molecular Sequence Data , Phosphatidylinositols/metabolismABSTRACT
Insulin-dependent diabetes mellitus (IDDM) is thought to result from chronic, cell-mediated, autoimmune islet damage. Our aim was to identify the earliest T-cell autoantigen in IDDM, reasoning that this antigen could be causally involved in the initiation of the disease. Identification of the earliest beta-cell-specific autoantigen is extremely important in allowing advances in prevention and treatment of initial events in the development of inflammatory insulitis that precedes beta-cell destruction and overt diabetes. Therefore, we analyzed the proliferative responses of peripheral T-cells from young, female nonobese diabetic (NOD) mice to extracts of pancreatic beta-cell lines. We were able to demonstrate that T-cells responsive to beta-cell antigens exist in the peripheral lymphoid tissue of these mice in the absence of deliberate priming before the manifestation of histologically detectable insulitis. T-cell lines and clones isolated from the peripheral lymphatic tissues of young, unimmunized, female NOD mice were also shown to react with extracts of beta-cells. Fractionation of the beta-cell extracts showed that these T-cell clones recognized multiple beta-cell-specific autoantigens but none of the previously reported putative autoantigens (glutamic acid decarboxylase [GAD]65, GAD67, Hsp65, insulin, ICA 69, carboxypeptidase-H, and peripherin). Thus, we can conclude that these responses are specific for novel beta-cell autoantigens. Finally, NOD T-cell proliferative responses were also seen to an extract of human islets suggesting potential shared antigenic determinants between human and mouse beta-cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Autoantigens/analysis , Diabetes Mellitus, Type 1/immunology , Islets of Langerhans/immunology , Mice, Inbred NOD/immunology , T-Lymphocytes/immunology , Animals , Autoantigens/immunology , Base Sequence , Cell Line , DNA Primers , Female , Glutamate Decarboxylase/analysis , Glutamate Decarboxylase/biosynthesis , Glutamate Decarboxylase/immunology , Heat-Shock Proteins/analysis , Heat-Shock Proteins/biosynthesis , Heat-Shock Proteins/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Molecular Sequence Data , Pancreatic Diseases/immunology , Pancreatic Diseases/pathology , Polymerase Chain ReactionABSTRACT
A new class of thrombin inhibitors based on sequence-specific single-stranded DNA oligonucleotides (thrombin aptamer) has recently been identified. The aptamer-binding site on thrombin was examined by a solid-phase plate binding assay and by chemical modification. Binding assay results demonstrated that the thrombin aptamer bound specifically to alpha-thrombin but not to gamma-thrombin and that hirudin competed with aptamer binding, suggesting that thrombin's anion-binding exosite was important for aptamer-thrombin interactions. To identify lysine residues of thrombin that participated in the binding of the thrombin aptamer, thrombin was modified with fluorescein 5'-isothiocyanate in the presence or absence of the thrombin aptamer, reduced, carboxymethylated, and digested with endoproteinase Arg-C. The digestion products were analyzed by reversed-phase high performance liquid chromatography and the peptide maps compared. Four peptides with significantly decreased modification in the presence of the aptamer were identified and subjected to N-terminal sequence analysis. Results indicated that B chain Lys-21 and Lys-65, both located within the anion-binding exosite, are situated within or in close proximity to the aptamer-binding site of human alpha-thrombin. The thrombin aptamer binds to the anion-binding exosite and inhibits thrombin's function by competing with exosite binding substrates fibrinogen and the platelet thrombin receptor.
Subject(s)
DNA, Single-Stranded/metabolism , Thrombin/metabolism , Amino Acid Sequence , Base Sequence , Binding Sites , DNA, Single-Stranded/chemistry , Fluorescein-5-isothiocyanate , Hirudins/metabolism , Humans , Lysine/analysis , Molecular Sequence DataABSTRACT
The biochemical behavior and peptide binding properties of a soluble form of the human class II DR4Dw4 molecule (PI-DR4Dw4) were compared to DR4Dw4 molecules containing the transmembrane and cytoplasmic domains that were purified both from B and transfected chinese hamster ovary cells. Recombinant and B cell-derived DR4Dw4 molecules bound monoclonal anti-DR4Dw4 antibodies with different affinities and varied in their stability in the presence of sodium dodecyl sulfate. The three forms of DR4Dw4 bound peptides with a similar apparent affinity constant, but soluble class II molecules bound up to ten times more peptide than DR4Dw4 containing a transmembrane region. Peptide binding kinetics for soluble DR4Dw4 molecules were 10-20 times faster than for the other two forms of DR4Dw4 molecules. Finally, soluble PI-DR4Dw4/peptide complexes were shown to stimulate T cell proliferation.
Subject(s)
HLA-DR Antigens/analysis , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Base Sequence , CHO Cells , Carbohydrates/analysis , Cricetinae , Enzyme-Linked Immunosorbent Assay , HLA-DR Antigens/immunology , HLA-DR Antigens/metabolism , Lymphocyte Activation , Molecular Sequence Data , Peptides/metabolism , Protein Conformation , T-Lymphocytes/immunology , TransfectionABSTRACT
An initial event in T cell activation is the specific adherence of T cells via their T cell receptor to the MHC peptide complex. We have studied this adherence by incubating T cells with preformed HLA DR4Dw4 peptide complexes attached to a solid support. Adherence of sodium 51Cr-labeled T cell clones specific for the influenza hemagglutinin peptide, HA 307-319, was maximal after 15 min and was specific for the HLA DR4Dw4-HA 307-319 complex. The binding was temperature dependent and could be blocked with azide or protein kinase C inhibitors, indicating that for adherence the T cells need to be metabolically active and have a functioning protein kinase C pathway. The adherence could be blocked with CD4- or CD3-reactive murine mAb, suggesting that the TCR and CD4 molecules work in concert to induce strong adherence to the HLA DR4Dw4-HA 307-319 complex. A subsequent event in T cell activation is proliferation, which is thought to need additional proteins such as IL-1 or other adhesion molecules. MHC peptide complexes coated on microtiter plates also induced proliferation in the human T cell clones. Removal of any monocytes by treatment of human T cell clones with anti-CD14 in conjunction with C, followed by purification over a nylon wool column, did not abrogate proliferation. After prolonged culture of the T cell clones in plates coated with peptide-pulsed HLA DR4Dw4 in the presence of IL-2, the T cell clones continued to proliferate in response to peptide. These results suggest that human T cell clones do not require a second signal from a monocyte or other APC to proliferate.
Subject(s)
HLA-D Antigens/physiology , HLA-DR Antigens/physiology , Lymphocyte Activation , T-Lymphocytes/immunology , Antibodies, Monoclonal/immunology , Antigen-Presenting Cells/physiology , Antigens, Differentiation, T-Lymphocyte/physiology , CD3 Complex , CD4 Antigens/physiology , Cell Adhesion , Clone Cells , Humans , Receptors, Antigen, T-Cell/physiologyABSTRACT
Full-length tissue factor (263 rTF) and three truncated forms have been expressed in human kidney 293 cells; 1) 243 rTF, which lacks the cytoplasmic tail, is fully functional in the chromogenic assay and has a specific activity comparable with that of the full-length molecule, 263 rTF; 2) 219 rTF, which lacks both the transmembrane and cytoplasmic domains, is not functional; 3) the third variant, referred to as TF-PI, is a fusion protein containing the extracellular domain of TF (amino acids 1-219) fused to the last 37 amino acids of decay-accelerating factor which contain a signal for attachment of a phosphatidylinositol membrane anchor (PI). TF-PI is a membrane-bound protein expressed on the cell surface. The PI anchor restores TF activity lost when the transmembrane domain is deleted from the 219 rTF variant. The ability of the PI anchor to restore activity to 219 rTF clearly demonstrates that while the transmembrane domain is not required for TF activity, lipid association is required.
Subject(s)
Glycolipids/metabolism , Phosphatidylinositols/metabolism , Thromboplastin/genetics , Amino Acid Sequence , Base Sequence , Cell Line , Cell Membrane/metabolism , Chromosome Deletion , Factor VII/metabolism , Fluorescent Antibody Technique , Genetic Vectors , Glycosylphosphatidylinositols , Humans , Molecular Sequence Data , Plasmids , Restriction Mapping , Thromboplastin/analysis , Thromboplastin/metabolism , TransfectionABSTRACT
The possible self-association of tissue factor molecules was investigated by treating cells expressing tissue factor with bifunctional cross-linking agents. The two reagents chosen were 3,3'-dithiobis(sulfosuccinimidylpropionate) and sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate, both of which are membrane-impermeable and thiol-cleavable. A human bladder carcinoma cell line, J82, and a transfected human kidney cell line expressing high amounts of recombinant tissue factor were used in these studies. Exposure of the intact cells to the crosslinking reagents was found to result in the formation of multimeric tissue factor-containing complexes, the extent of which appeared to be dependent upon the amount of tissue factor expressed by the cell. The self-association of tissue factor was prevented in a variant tissue factor molecule harboring a non-homologous transmembrane domain.
Subject(s)
Thromboplastin/chemistry , Amino Acid Sequence , Autoradiography , Blotting, Western , Cells, Cultured , Cross-Linking Reagents , Humans , Kidney/cytology , Molecular Sequence Data , Plasmids , Precipitin Tests , Recombinant Proteins/chemistry , Thromboplastin/genetics , Tumor Cells, Cultured , Urinary Bladder Neoplasms/pathologyABSTRACT
Recombinant human tissue factor (rTF) purified from transfected mammalian cells is a glycoprotein that contains N-linked, but not O-linked oligosaccharides. Two of the three potential N-linked sites in the extracellular portion are fully glycosylated, while one site is approximately 90% utilized. These sites have complex-type oligosaccharides attached. The potential N-linked site in the cytoplasmic domain near the C-terminus is not glycosylated. Characterization of the tryptic map of rTF confirmed most of the proposed amino acid sequence. In addition, the disulfide bonds (between Cys-49 and Cys-57 and between Cys-186 and Cys-209) were demonstrated by FAB-MS analysis of cysteine-containing fragments obtained from the tryptic map.
Subject(s)
Protein Processing, Post-Translational/physiology , Thromboplastin/chemistry , Amino Acid Sequence , Cells, Cultured , Chromatography, High Pressure Liquid , Clone Cells , Disulfides/chemistry , Female , Glycosylation , Humans , Molecular Sequence Data , Placenta/chemistry , Pregnancy , Recombinant Proteins/chemistry , Trypsin , Tunicamycin/pharmacologyABSTRACT
We describe a mammalian cell expression system used to rapidly produce microgram quantities of a membrane protein used as an immunogen. A fusion protein expression vector was constructed which contained the signal sequence and 27 amino acids of the Herpes simplex virus glycoprotein D (gD), followed by a factor VIII (fVIII) thrombin cleavage site and the mature tissue factor (TF) sequence. This fusion protein was transiently expressed and then purified using an antibody to gD. The purified fusion protein, gDTF, was incubated with thrombin to remove the gD-fVIII moiety and the resulting rTF served as antigen for the generation of TF-specific antibodies. The antibodies produced were then used for a comparison of the turnover rates of the constitutively and transiently produced fusion protein. In addition, sensitivity to glycosidases indicated that the transiently and constitutively produced recombinant proteins do not contain identical carbohydrate structures.
Subject(s)
Antigens/genetics , Thromboplastin/genetics , Vaccines, Synthetic , Vaccines , Viral Envelope Proteins/genetics , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Antigens/immunology , Base Sequence , Carbohydrates/analysis , Cells, Cultured , Gene Expression , Glycosylation , Humans , Mice , Molecular Sequence Data , Precipitin Tests , Rabbits , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Simplexvirus/genetics , Simplexvirus/immunology , Thromboplastin/immunology , Viral Envelope Proteins/immunologyABSTRACT
Tissue factor (TF) is a 263 amino acid membrane-bound procoagulant protein that serves as a cofactor for the serine protease factor VII (fVII). Recombinant human TF (rTF) produced in both human kidney 293 cells and Escherichia coli has been immunoaffinity purified by using a TF-specific monoclonal antibody. Recombinant TF produced in 293 cells is glycosylated and migrates on reducing SDS-PAGE with an apparent molecular weight (Mr) of 45K. Some interchain disulfide-bonded rTF dimers are observed under nonreducing conditions. The E. coli produced rTF has a molecular weight of 33K and 35K, with the 33K band missing nine amino acids at the carboxy terminus. Although the E. coli produced rTF does not contain any carbohydrate, it is fully functional in both a chromogenic assay and a one-stage prothrombin time assay. A variant has been constructed wherein the cytoplasmic cysteine (residue 245) has been mutagenized to a serine residue. The amount of disulfide-linked aggregates is dramatically reduced following immunoaffinity purification of this four-cysteine variant (C2455), which is active in the chromogenic and prothrombin time assays.
Subject(s)
Thromboplastin/isolation & purification , Amino Acid Sequence , Base Sequence , Cells, Cultured , Cyanogen Bromide , Cysteine/metabolism , Cytoplasm/metabolism , Escherichia coli/metabolism , Humans , Immunoassay , Kidney/metabolism , Molecular Sequence Data , Plasmids , Prothrombin Time , Recombinant Proteins/isolation & purification , Recombinant Proteins/pharmacology , Thromboplastin/pharmacology , TrypsinABSTRACT
We have studied the binding of radioiodinated human factor VII and its activated form, factor VIIa, to monolayers of a human bladder carcinoma cell line (J82) that expresses functional cell surface tissue factor. The binding of factors VII and VIIa to these cells was found to be time-, temperature-, and calcium-dependent. In addition, the binding of each protein to J82 cells was specific, dose-dependent, and saturable. The binding isotherms for factors VII and VIIa were hyperbolic, and Scatchard plots of the binding data obtained at 37 degrees C indicated a single class of binding sites for each protein with Kd values of 3.20 +/- 0.51 and 3.25 +/- 0.31 nM, respectively. Factors VII and VIIa, respectively, interacted with 256,000 +/- 39,000 and 320,000 +/- 31,000 binding sites/cell. Competition experiments suggested a common receptor for factors VII and VIIa. Binding of factor VIIa to the cells was completely blocked by preincubation of the cells with polyclonal anti-tissue factor IgG, whereas binding of factor VII was inhibited approximately 90%, suggesting the presence of a small number of tissue factor-independent binding sites specific for factor VII on this cell. Functional studies revealed that factor X activation by increasing amounts of cell-bound factor VII or VIIa was hyperbolic in nature. Half-maximal rates of factor Xa formation occurred at factor VII and VIIa concentrations of 3.7 +/- 0.47 and 3.2 +/- 0.31 nM, respectively. No factor VII- or VIIa-mediated activation of factor X was observed when cells were preincubated with anti-tissue factor IgG. Two-chain 125I-factor VIIa recovered from the cells was identical to the offered ligand as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. In contrast, the offered single-chain 125I-factor VII was progressively converted to two-chain 125I-factor VIIa upon binding to the cells. When the J82 cells were pretreated with anti-tissue factor IgG, both factor VII recovered from the cells and factor VII in the supernatant were in the single-chain form, indicating that cell-surface tissue factor was essential for the activation of factor VII on these cells. These data indicate that binding of factor VII to tissue factor appears to be a prerequisite for its conversion to factor VIIa and the initiation of the extrinsic pathway of coagulation on these cells.
Subject(s)
Blood Coagulation , Factor VII/metabolism , Binding, Competitive , Cell Line , Factor VIIa , Factor X/metabolism , Factor Xa , Humans , Kinetics , Protein Binding , Serine Endopeptidases/metabolism , Urinary Bladder Neoplasms/metabolismABSTRACT
We describe the polypeptide structure and some of the catalytic properties of a DNA polymerase alpha.DNA primase complex that can be prepared from KB cells by immunoaffinity purification. The procedure is based on monoclonal antibodies that were raised against a biochemically purified, catalytically active core protomer of the polymerase. In all respects tested, the basic mechanism of substrate recognition and binding by the immunoaffinity-purified polymerase is qualitatively identical to that of the core protomer. The immunoaffinity-purified KB cell polymerase alpha X DNA primase is structurally complex. On the basis of extensive immunochemical analyses with five independent monoclonal antibodies, three of which are potent neutralizers of polymerase alpha activity, peptide mapping studies, and the application of a sensitive immunoassay that permits detection of polymerase alpha antigens in crude cell lysates, we have established that the principal form of catalytically active DNA polymerase alpha in KB cells is a phosphoprotein with a molecular mass of 180 kilodaltons. This protein is stable in vivo, with an estimated half-life of greater than or equal to 15 h. In contrast, the polypeptide is extremely fragile in vitro and generates partial degradation products of p165, p140, and p125 that explain the "microheterogeneity" typically exhibited by polymerase alpha peptides in denaturing polyacrylamide gels. In addition to the catalytically active polymerase alpha polypeptide(s), the immunopurified enzyme fraction typically contains three other proteins, p77, p55, and p49, the functions of which have not yet been established. These proteins do not display polymerase alpha epitopes and have been shown by peptide mapping to be independent species that are unrelated either to the large polymerase peptides or to one another. The polypeptide p77 is also a phosphoprotein, and in both p180 and p77 the phosphorylated amino acids are exclusively serine and threonine.