ABSTRACT
The apicoplast is a distinctive organelle associated with apicomplexan parasites, including Plasmodium sp. (which cause malaria) and Toxoplasma gondii (the causative agent of toxoplasmosis). This unusual structure (acquired by the engulfment of an ancestral alga and retention of the algal plastid) is essential for long-term parasite survival. Similar to other endosymbiotic organelles (mitochondria, chloroplasts), the apicoplast contains proteins that are encoded in the nucleus and post-translationally imported. Translocation across the four membranes surrounding the apicoplast is mediated by an N-terminal bipartite targeting sequence. Previous studies have described a recombinant "poison" that blocks plastid segregation during mitosis, producing parasites that lack an apicoplast and siblings containing a gigantic, nonsegregating plastid. To learn more about this remarkable phenomenon, we examined the localization and processing of the protein produced by this construct. Taking advantage of the ability to isolate apicoplast segregation mutants, we also demonstrated that processing of the transit peptide of nuclear-encoded apicoplast proteins requires plastid-associated activity.
Subject(s)
Cell Nucleus/metabolism , Flow Cytometry/methods , Plastids/genetics , Toxoplasma/genetics , Animals , Cell Membrane/metabolism , Cell Separation , Chloramphenicol O-Acetyltransferase/metabolism , Fluorescent Dyes/metabolism , Green Fluorescent Proteins , Luminescent Proteins/metabolism , Plasmids/metabolism , Plastids/metabolism , Recombinant Fusion Proteins/metabolism , Subcellular Fractions , Red Fluorescent ProteinABSTRACT
Apicomplexan parasites--including the causative agents of malaria (Plasmodium sp.) and toxoplasmosis (Toxoplasma gondii)--harbor a secondary endosymbiotic plastid, acquired by lateral genetic transfer from a eukaryotic alga. The apicoplast has attracted considerable attention, both as an evolutionary novelty and as a potential target for chemotherapy. We report a recombinant fusion (between a nuclear-encoded apicoplast protein, the green fluorescent protein and a rhoptry protein) that targets to the apicoplast but grossly alters its morphology, preventing organellar segregation during parasite division. Apicoplast-deficient parasites replicate normally in the first infectious cycle and can be isolated by fluorescence-activated cell sorting, but die in the subsequent host cell, confirming the 'delayed death' phenotype previously described pharmacologically, and validating the apicoplast as essential for parasite viability.
Subject(s)
Plastids/genetics , Toxoplasma/genetics , Animals , Eukaryota/genetics , Genes, Protozoan , Green Fluorescent Proteins , Luminescent Proteins/genetics , Membrane Proteins/genetics , Microscopy, Electron , Mutation , Phenotype , Protozoan Proteins/genetics , Recombinant Fusion Proteins/genetics , Symbiosis/genetics , Toxoplasma/growth & development , Toxoplasma/microbiologyABSTRACT
The role of angiogenesis inhibition in the antitumor activity of recombinant murine interleukin 12 (rmIL-12) was studied in K1735 murine melanomas, the growth of which is rapidly and markedly suppressed by rmIL-12 treatment. On the basis of the prediction that tumor ischemia should result from therapeutic angiogenesis inhibition, tumor cell hypoxia was evaluated as a marker of ischemia using the EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)aceta mide] approach. This method measures intracellular binding of the nitroimidazole EF5, which covalently binds to cellular macromolecules selectively under hypoxic conditions. Whereas 1 week of rmIL-12 treatment effectively inhibited K1735 cell-induced angiogenesis in Matrigel neovascularization assays, 2 weeks of treatment were needed before severe tumor cell hypoxia was detected in K1735 tumors. The hypoxia that developed was regional and localized to tumor areas distant from blood vessels. The great majority of severely hypoxic tumor cells were apoptotic, and in vitro studies indicated that the degree of hypoxia present within treated tumors was sufficient to trigger K1735 apoptosis. Tumor cell apoptosis was also prevalent in the first week of rmIL-12 treatment when few cells were hypoxic. In vitro studies indicated that this non-hypoxia-related apoptosis was induced directly by IFN-gamma produced in response to rmIL-12 administration. These studies reveal that rmIL-12 controls K1735 tumors initially by IFN-gamma-induced apoptosis and later by hypoxia-induced apoptosis. They also establish hypoxia as an expected result of tumor angiogenesis inhibition and a mediator of its therapeutic effect.
Subject(s)
Apoptosis/physiology , Cell Hypoxia/physiology , Interleukin-12/therapeutic use , Melanoma, Experimental/blood supply , Melanoma, Experimental/pathology , Neovascularization, Pathologic/prevention & control , Animals , Apoptosis/drug effects , Cell Hypoxia/drug effects , Collagen , Drug Combinations , Female , Interferon-gamma/pharmacology , Ischemia , Laminin , Melanoma, Experimental/drug therapy , Mice , Mice, Inbred C3H , Proteoglycans , Recombinant Proteins/therapeutic use , Tumor Cells, CulturedABSTRACT
We hypothesized that human hematopoietic cells displaying a CD34+, kit-, rhodamine123(low) phenotype would be highly enriched for cells with stem-like properties. To test this hypothesis, we employed fluorescence activated cell sorting (FACS) to isolate cells with this phenotype from normal light density marrow mononuclear cells (MNC). CD34+, kit+, rhodamine123(low) cells comprised from 0.05-0.01% of the total MNC population. They were small, had scant cytoplasm, and contained nuclei with dense, hyperchromatic chromatin and inconspicuous nucleoli. Additional immunophenotyping revealed that these cells were CD33-, CD38-, CD20-, and glycophorin A-. When plated in semisolid cultures containing optimal concentrations of IL-3, GM-CSF, KL, EPO, IL-6, and IL-1 these cells did not form colonies. However, when cultured over irradiated stromal cells, cobblestone areas were observed to form after 3 weeks, and harvested cells were able to initiate long-term cultures. To further demonstrate that these cells were indeed stem like, we also tested their ability to engraft and mature in immunocompromised (SCID) mice. Irradiated (400 cGy) SCID mice were transplanted with 2 x 10(3) candidate stem cells which were then injected with recombinant human growth factors every other day. Two months post-transplant the animals were sacrificed. PCR and FACS analysis of marrow and spleen cell samples revealed the presence of cells expressing human CD45 consistent with engraftment of human stem cells and the establishment of murine-human chimerism. Moreover, MNC isolated from transplanted mice formed unambiguously human BFU-E, CFU-GM and B cell colonies when stimulated with the appropriate growth factors. Accordingly, we have identified a relatively rapid and simple mechanism for isolating primitive human hematopoietic cells with stem cell-like properties. We anticipate that this strategy will be useful for experimental and therapeutic applications that require human stem cells in quantity.
Subject(s)
Antigens, CD34/analysis , Bone Marrow Cells/chemistry , Cell Separation/methods , Hematopoietic Stem Cells/chemistry , Proto-Oncogene Proteins c-kit/analysis , Rhodamines/metabolism , Animals , Cells, Cultured , Hematopoietic Stem Cell Transplantation , Humans , Mice , Mice, SCID , Phenotype , Rhodamine 123ABSTRACT
Apoptosis is a vital process for organism development and, when disrupted, can lead to abnormalities including cancer and autoimmune diseases. We demonstrate a novel multicolor flow cytometry approach for quantifying apoptosis and cell cycle information of phenotypically distinct populations, using less than 2 x 10(5) cells per sample. We used incorporation of Cy5-dUTP into DNA strand breaks by the terminal dUTP nucleotide end labeling (TUNEL) method to determine apoptosis, while cell cycle information was assessed with an ultraviolet DNA binding dye, DAPI. To simultaneously determine surface phenotype, we used paraformaldehyde fixation and a gentle permeabilization protocol combined with FITC- and PE-labeled surface antibodies. Using these fluorochromes, and three-laser instrumentation, we quantified apoptosis and cell cycle phase in lymphocyte subpopulations from heterogeneous human and murine cell sources, subjected to various culture conditions. Further, we used this method to detect divergent rates of apoptosis in a human, heterogeneous lymphocyte tumor population, demonstrating a potential application for clinical and/or research settings. Thus, we describe a six-parameter, four-color flow cytometry approach for evaluating apoptosis and cell cycle with dual surface labels. This method may also be useful as a generalized scheme to assess simultaneously two intracellular targets in a mixed cell population.
Subject(s)
Apoptosis , B-Lymphocytes/cytology , Cell Cycle , Flow Cytometry/methods , Animals , Antigens, CD19/analysis , Cells, Cultured , Female , Humans , Interleukin-4/pharmacology , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Lipopolysaccharides/pharmacology , Lymphocyte Activation , Mice , Mice, Inbred BALB C , Phenotype , Spleen/immunologyABSTRACT
The importance of glutathione (GSH) in contributing to cancer therapy resistance is well established. Various advantages may accrue from the ability to determine the distribution of GSH content in individual tumor cells disaggregated from solid tumors using flow cytometric techniques compared with biochemical or chemical measurements of the average GSH level in bulk tissue samples. The flow cytometric technique requires a thiol-reactive fluorescent adduct which is stable and which can differentiate cellular GSH from protein thiols. Thiol-reactive compounds specific for GSH require facilitated conjugation by endogenous cellular enzymes, but such compounds have not been found to accurately monitor GSH in human cells. Compounds which react generally with all thiols require a GSH-depleted calibration control to assess GSH vs. non-GSH components of fluorescent-adduct formation. Our report addresses this question, and compares three different thiol assays in several cell lines. We have found a simple way to control for non-GSH adduct formation. This involves a selective permeabilization process to release low molecular weight adducts (dominated by GSH and cysteine). In application to the desired goal of assessing the distribution of GSH in cells disaggregated from tumors, we have identified a problem with cell-line-specific thiol loss during the tumor cell disaggregation process.
Subject(s)
Bridged Bicyclo Compounds , Flow Cytometry , Fluorescent Dyes , Glutathione/analysis , Neoplasms, Experimental/chemistry , Sulfhydryl Reagents , Animals , Chromatography, High Pressure Liquid , Flow Cytometry/methods , Humans , Rats , Tumor Cells, CulturedABSTRACT
Thrombopoietin (TPO) has been reported to stimulate erythropoiesis, but the stimulatory mechanism has not been defined. To address this issue, we performed serum-free cell-culture experiments with recombinant human TPO and purified human progenitor cells. We found that TPO alone was able to stimulate the megakaryocyte colony formation in serum-free cultures, but erythroid colonies were never observed. Only in the presence of EPO (erythropoietin) +IL-3 was TPO able to stimulate a small increase (approximately 25%) in erythroid colony formation. Accordingly, we hypothesized that TPO might have an effect on erythroid progenitor cell viability, rather than a direct stimulatory effect. To test this idea, CD34+ cells were cultured for 7d in serum-free methylcellulose in the presence or absence of TPO, after which time KL+ EPO was added to the cultures. Cells which were pre-cultured for 7 d in the presence of TPO gave rise to approximately 6 times as many burst forming unit-erythroid (BFU-E) colonies as cells which were pre-cultured in the absence of TPO. Further, when primitive CD34+, Kit+ MNC were cultured for 3-7 d under serum-free conditions in the presence or absence of TPO, significantly fewer cells cultured in the presence of TPO displayed apoptotic changes when compared to cells cultured in the absence of TPO. Taken together, these results suggest that TPO has little direct stimulatory effect on erythroid progenitor cells, but might indirectly enhance erythropoiesis by preventing very early erythroid progenitor cells from undergoing apoptotic cell death.
Subject(s)
Apoptosis/physiology , Erythroid Precursor Cells/cytology , Erythropoiesis/physiology , Thrombopoietin/physiology , Cells, Cultured , Culture Media, Serum-Free , Humans , Interleukin-3/physiology , Phenotype , Polymerase Chain ReactionABSTRACT
Basic fibroblast growth factor (FGF-2) and acidic fibroblast growth factor (FGF-1) are mitogens for a variety of cell types. Many reports suggest that haemopoietic cells are among these. Nevertheless, when we examined the effect of recombinant human FGF-1 or 2 on normal human marrow cell proliferation in vitro, only minimal stimulatory activity could be detected. In this regard, the addition of either growth factor to cultures of ancillary cell depleted marrow mononuclear cells (MNC), or to highly enriched CD34+ MNC, failed to enhance haemopoietic colony number and induced only a slight increase in colony size. Perturbation of FGF receptor (FGF-R) expression on CD34+ MNC with antisense (AS) oligodeoxynucleotides (ODN) was also without apparent effect on cell growth. Neither could we demonstrate any effect of FGF-1 or 2 on survival of early progenitor cells in serum-free culture. To explain these findings, we examined progenitor cells for expression of the FGF-R at the mRNA and protein level using RT-PCR and flow cytometry. Primitive CD34+/KIT+ MNC had no detectable FGF-R (FGF-R1, 2, 3 or 4) mRNA or protein expression. In fact, direct immunofluorescence labelling of MNC for CD34 antigen and FGF-R1 demonstrated that expression of these markers was mutually exclusive in the populations examined. FGF-R1 expression was detected on subpopulations of MNC and on cells derived from day-6 CFU-GM and BFU-E colonies. Accordingly, FGF-R1 is either absent, or present at very low levels, on primitive haemopoietic cells. This fact, combined with our in vitro culture data, suggest that receptors are unlikely to play a significant role in the development of these early cells. Nevertheless, the development of mature cells may be influenced by the FGFs since the FGF-Rs are expressed on more mature cells.
Subject(s)
Fibroblast Growth Factor 1/pharmacology , Fibroblast Growth Factor 2/pharmacology , Hematopoietic Stem Cells/cytology , Base Sequence , Cell Division , Cloning, Molecular , Hematopoiesis/physiology , Humans , Molecular Sequence Data , Polymerase Chain ReactionABSTRACT
Murine lymphocytes readily undergo spontaneous and glucocortocoid-induced apoptosis in vitro. It has been previously demonstrated that during apoptosis, many cell types including lymphocytes, enzymatically cleave their DNA, thus demonstrating a sub-G0 DNA peak when stained with propidium iodide and analyzed by flow cytometry. In a mixed population, it is often desirable to phenotypically identify distinct populations or subsets undergoing apoptosis, thus requiring multiparameter analysis of surface phenotype and DNA content. Paraformaldehyde fixation procedures, although common for surface evaluation, have not been extensively used in methods quantifying apoptosis. To measure apoptosis in a mixed lymphocyte population, we evaluated a gentle detergent permeabilization and paraformaldehyde fixation procedure combined with propidium iodide (PI) DNA staining, adapted from existing methods for cell cycle studies. With this method and rigorous gating techniques which we defined, we detected both apoptotic and debris fractions within the sub-G0 cell cycle region of a glucocortocoid-treated murine lymphocyte cell line. Using this cell line, WEHI 231.7, as a lymphocyte model, we developed a logical gating strategy to exclude debris from analysis. We further demonstrated that apoptosis in freshly isolated murine lymphocytes detected with paraformaldehyde fixation and PI staining was quantitatively comparable to PI staining with ethanol fixation, or nick translation labeling of DNA strand breaks (TUNEL). Finally, using fresh murine spleen cells, we demonstrated that paraformaldehyde fixation preserves surface protein staining, allowing multiparameter analysis of immunophenotype and apoptotic or cell cycle status in a mixed lymphocyte population. Thus, this method offers an inexpensive and technically simple alternative for assessing apoptosis and surface phenotype.
Subject(s)
Antigens, Surface/analysis , Apoptosis , Immunophenotyping/methods , Lymphocytes/cytology , Animals , Cell Cycle , Cell Death , Cell Line , Cells, Cultured , Coloring Agents , DNA/analysis , DNA Damage , Electrophoresis, Agar Gel , Fixatives , Flow Cytometry , Formaldehyde/pharmacology , Lymphocytes/drug effects , Mice , Mice, Inbred BALB C , Polymers/pharmacology , Propidium , Spleen/cytologyABSTRACT
To determine the potential role of autocrine growth factor production in regulating primitive human hematopoietic cell development, we examined highly purified CD34+, c-Kit+ marrow mononuclear cells for expression of c-Kit ligand (KL) and stem cell tyrosine kinase 1 (stk1) ligand (STK1-L). Normal marrow mononuclear cells coexpressing CD34 and c-Kit were isolated by a combination of immunomagnetic bead isolation and fluorescence-activated cell sorting. Purified cells were then screened for expression of KL and stk1-L mRNA using a sensitive reverse transcription-polymerase chain reaction method. Using this approach, expression of both cytokine genes at the mRNA level was found in this highly enriched cell population. We then examined the functional significance of these mRNAs by inhibiting their expression with antisense (AS) oligodeoxynucleotides (ODN). In comparison to untreated or control ODN treated cells, inhibition of KL led to a 70% and 89% inhibition in burst-forming unit-erythroid (BFU-E) and colony-forming unit-Mix (CFU-Mix) colonies but had no significant effect on CFU-granulocyte-macrophage (CFU-GM) cloning efficiency. In contrast, inhibition of STK1-L alone had no effect on colony formation. However, when STK1-L AS ODN was combined with KL AS ODN, additive inhibition of CFU-GM and CFU-MIX but not of BFU-E colonies was observed. These findings, along with those of our previous studies showing inhibition of primitive hematopoietic cell growth with antisense ODN directed towards the stk1 receptor, suggest the possibility that both receptor/ligand axes regulate primitive hematopoietic cell growth via an autocrine growth loop.
Subject(s)
Bone Marrow Cells , Hematopoietic Cell Growth Factors/physiology , Hematopoietic Stem Cells/metabolism , Membrane Proteins , Protein-Tyrosine Kinases/physiology , Receptor Protein-Tyrosine Kinases/physiology , Antigens, CD/analysis , Antigens, CD34 , Base Sequence , Cell Division , Cell Separation , Flow Cytometry , Hematopoietic Cell Growth Factors/antagonists & inhibitors , Hematopoietic Cell Growth Factors/biosynthesis , Hematopoietic Cell Growth Factors/genetics , Hematopoietic Stem Cells/cytology , Humans , Immunomagnetic Separation , Molecular Sequence Data , Nucleic Acid Conformation , Oligonucleotides, Antisense/pharmacology , Polymerase Chain Reaction , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/biosynthesis , Protein-Tyrosine Kinases/genetics , Proto-Oncogene Proteins/physiology , Proto-Oncogene Proteins c-kit , RNA, Messenger/antagonists & inhibitors , RNA, Messenger/biosynthesis , RNA, Messenger/chemistry , RNA, Messenger/genetics , Receptor Protein-Tyrosine Kinases/biosynthesis , Receptor Protein-Tyrosine Kinases/genetics , Receptors, Colony-Stimulating Factor/physiology , Stem Cell Factor , fms-Like Tyrosine Kinase 3ABSTRACT
Inhibitory effects of human immunodeficiency virus (HIV) on T lymphocyte function have been linked to perturbation of signaling through the T cell antigen receptor-CD3 complex. Comparative biochemical analyses of signaling responses were performed in T cells that were either uninfected or chronically infected with the HIV-1/IIIB strain. Stimulation with antibodies to CD3 triggered both Ca2+ accumulation and phosphoinositide hydrolysis responses that were equivalent in uninfected and infected cells. Treatment with anti-CD3 or with phorbol diester also stimulated serine phosphorylation of CD4 molecules in uninfected T cells. However, phosphorylation of CD4 was not observed after anti-CD3 treatment in HIV-infected T cells despite normal phosphorylation responses to phorbol diester. Identical results were obtained using a T cell line that was infected with an env (gp160/120-) HIV-1 defective variant. These studies indicate that infection with HIV-1 inhibits the activation of protein kinase associated with the T cell receptor-CD3 complex by a mechanism which is independent of viral env protein components.
Subject(s)
Antigens, Differentiation, T-Lymphocyte/metabolism , CD4 Antigens/metabolism , HIV-1/physiology , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/microbiology , Animals , CD3 Complex , Calcium/metabolism , Cell Line , Cricetinae , Electrophoresis, Polyacrylamide Gel , Enzyme Activation , Flow Cytometry , Gene Products, env/metabolism , HIV Envelope Protein gp120/metabolism , HIV Envelope Protein gp160 , HIV-1/genetics , Humans , Hydrolysis , Phenotype , Phorbol 12,13-Dibutyrate/pharmacology , Phosphatidylinositols/metabolism , Phosphorylation , Precipitin Tests , Protein Kinase C/metabolism , Protein Precursors/metabolism , Signal Transduction , T-Lymphocytes/immunology , T-Lymphocytes/metabolismABSTRACT
Naturally occurring isolates of human immunodeficiency virus (HIV) have been described which are deficient in their ability to fuse with and kill CD4+ target cells. Although the molecular basis for their attenuation has not yet been defined, several lines of evidence point toward the viral envelope gene as a key determinant of viral pathogenicity. In the present article, we report the biological characterization of two highly cytopathic variants derived by repeated cell-free passage of an attenuated isolate of HIV type 2 (HIV-2), termed HIV-2/ST. Unlike the parental virus, the cytopathic variants were found to infect Sup-T1 cells with great efficiency and to induce both cell fusion and profound killing in these cultures. To determine whether changes in the viral envelope gene were responsible for the observed phenotypic differences, we examined the CD4 binding affinity of these viruses using a novel assay designed to quantitate the binding of fluoresceinated CD4 to viral envelope in its native configuration on the cell surface. The results demonstrated that the affinity of parental HIV-2/ST envelope for CD4 was 2 orders of magnitude reduced, while the cytopathic variants exhibited a high CD4 binding affinity, comparable to that of cytopathic HIV-1 and HIV-2 isolates. From these data, we conclude that the cytopathic potential of HIV depends, at least in part, on its receptor-binding affinity. In addition, our study documents strong selection pressures for viruses with increased CD4 affinity during propagation in immortalized T-cell lines, thus emphasizing the need to study HIV envelope biology in natural target cells.
Subject(s)
CD4 Antigens/metabolism , HIV-2/pathogenicity , Cell Fusion , Cytopathogenic Effect, Viral , HIV-1/metabolism , HIV-2/growth & development , HIV-2/metabolism , Humans , In Vitro Techniques , Protein Binding , Virus ReplicationABSTRACT
The amino groups of prothrombin fragment 1 (amino acids 1-156 of prothrombin) were derivatized by acetylation, amidination, and reductive methylation. Conditions that caused complete acetylation of protein amino groups produced a fragment 1 derivative which no longer displayed a metal ion dependent intrinsic fluorescence change and had lost its membrane binding capability as well. However, when derivatized in the presence of calcium ions, extensive acetylation yielded a product that underwent protein fluorescence quenching at metal ion concentrations similar to those observed for the native protein. This derivative bound to membranes in a calcium-dependent manner with only a small reduction in affinity. Several results showed the existence of a partially functional protein that was characterized by a high degree of calcium-dependent protein fluorescence quenching but which had a requirement for 10-fold higher calcium concentration. This derivative was produced by partial acetylation (greater than 3 equiv) of metal-free protein. This partially acetylated protein had greatly diminished membrane binding. The calcium-protected amino group, therefore, was among the most reactive acetylation sites in the metal-free protein. The second site, responsible for abolishing all metal ion induced fluorescence change, was resistant to acetylation and became derivatized at the last stages of amino group acetylation.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Peptide Fragments/metabolism , Protein Precursors/metabolism , Prothrombin/metabolism , Acetylation , Amidines , Animals , Calcium/metabolism , Cattle , Hydrogen-Ion Concentration , Kinetics , Methylation , Oxidation-Reduction , Protein Conformation , Spectrometry, FluorescenceABSTRACT
The molecular interactions between components of the heparin-catalyzed antithrombin III/thrombin reaction were investigated by light scattering. When heparin was added to antithrombin III, the molecular weight increased to a maximum and then decreased to that of a 1:1 (antithrombin III X heparin) complex. The initial molecular weights at low heparin to antithrombin III ratios were consistent with the formation of a 2:1 (antithrombin III X heparin) complex in which only one antithrombin III molecule had undergone the conformational change measured by protein fluorescence enhancement. The peak molecular weight never reached that of a complete 2:1 complex. This behavior was observed for bovine and human antithrombin III in the presence of both unfractionated heparin and high molecular weight-high affinity heparin. Pentosane polysulfate also caused some multiple associations. Bovine antithrombin III and thrombin formed a 1:1 complex that underwent further aggregation within minutes, while the human proteins did not aggregate on this time scale after forming the 1:1 complex. In the presence of stoichiometric amounts of heparin, the bovine proteins formed an initial complex of Mr = 230,000 (corresponding to a dimer of heparin-antithrombin III-thrombin) which underwent further aggregation. The human proteins, however, formed a 1:1 (antithrombin III X thrombin) initial complex in the presence of heparin, followed by aggregation. These interactions of thrombin and antithrombin with heparin suggest complex interactions that could relate to heparin function.
Subject(s)
Antithrombin III/metabolism , Heparin/metabolism , Thrombin/metabolism , Animals , Cattle , Humans , Macromolecular Substances , Molecular Weight , UltracentrifugationABSTRACT
Kinetic characteristics of several heparin preparations and substitute heparins were determined to help understand the bases for activity differences. Several materials were highly active in factor Xa inhibition and the reaction rate at constant factor Xa concentration appeared to be predicted by the extent of intrinsic antithrombin III fluorescence change induced by the polysaccharide. Heparin fractions of different molecular weight and affinity for antithrombin III showed similar kinetic parameters in catalysis of the thrombin-antithrombin III reaction when these parameters were expressed on the basis of antithrombin III-binding heparin. The latter was determined by stoichiometric titration of the antithrombin III fluorescence change by the heparin preparation. However, the various heparin fractions showed very different specific activities per mg of total polysaccharide. This indicated that functional heparin molecules had similar kinetic properties regardless of size or antithrombin III-binding affinity and is possible because the Km for antithrombin III is determined by diffusion rather than by binding affinity. Substitute heparins and depolymerized heparin were poor catalysts for thrombin inhibition, due at least partially to their affinity for thrombin. This latter binary interaction inhibits thrombin reaction in the heparin-catalyzed reaction.
Subject(s)
Antithrombin III/metabolism , Heparin/metabolism , Thrombin/metabolism , Animals , Cattle , Chromatography , Dextran Sulfate , Dextrans/pharmacology , Factor X/metabolism , Factor Xa , Heparin/isolation & purification , Kinetics , Molecular Weight , Pentosan Sulfuric Polyester/pharmacology , Structure-Activity RelationshipABSTRACT
The 43Ca NMR spectra are reported for solutions of prothrombin fragment 1 in the presence and absence of phospholipid. The calcium NMR spectrum permits distinction between three thermodynamic classes of calcium-binding interactions. The calcium ion in the lipid-free solutions was labile, with maximum residence times estimated for the average protein site in the range of 0.5-1 ms. The calcium spectrum was sensitive to the protein association and the addition of phospholipid, which appears to sharpen the calcium specificity for the protein sites. The calcium NMR spectra in the presence of phospholipid are similar to those in lipid-free solutions, which suggests that the calcium ion remains labile in the lipid-protein complex.
Subject(s)
Liposomes , Peptide Fragments/metabolism , Phosphatidylcholines , Phosphatidylserines , Protein Precursors , Prothrombin/metabolism , Animals , Calcium Isotopes , Cattle , Magnetic Resonance SpectroscopyABSTRACT
The kinetics of the heparin-dependent antithrombin/protease reaction were consistent with an ordered sequential two-substrate reaction model under all circumstances tested. In this model, heparin is the catalyst; while antithrombin is the first substrate and the protease is the second substrate. The first step in this reaction, the heparin-antithrombin interaction, has a KD of 25 nM but a diffusionally determined Km of about 150 nM regardless of protease substrate. The second step of the reaction, protease interaction with the heparin-antithrombin complex, was fast with a rate constant of 6.8 X 10(6) M-1.s-1 for Factor Xa and greater than 8 X 10(7) M-1.ls-1 for thrombin. Differences between thrombin and Factor Xa at low (nanomolar) concentrations of heparin were evident in this rate constant and the relative affinities for the heparin-antithrombin complex (Km for Factor Xa = 100 nM; Km for thrombin less than or equal to 2 nM). In agreement with this difference in Km, regardless of protease substrate, active site-blocked thrombin was a potent inhibitor of the antithrombin reaction; while active site-blocked Factor Xa was an ineffective inhibitor. At high heparin concentrations (micromolar), the kinetic parameters for Factor Xa were unchanged but the Km for thrombin increased dramatically to 100 nM. Other kinetic parameters were also estimated. Overall, the two-substrate reaction model provides a versatile approach for studying heparin function.
Subject(s)
Antithrombins/metabolism , Heparin/pharmacology , Protease Inhibitors/metabolism , Animals , Cattle , Factor X/metabolism , Factor Xa , Kinetics , MathematicsABSTRACT
The heparin-catalyzed inactivation of thrombin by antithrombin was examined using saturation kinetics and fractional reaction lifetimes. Based solely on kinetic analysis, the reaction binding sequence was determined to be heparin binding to antithrombin followed by binding of thrombin. Under readily accessible experimental conditions, the rate-determining step was zero order with respect to thrombin and varied from first order to zero order with respect to antithrombin as the concentration of antithrombin was increased. A saturation kinetics model was used to estimate a Km of 12 x 10(-8) M for formation of the kinetically active heparin-antithrombin complex, a kcat of 0.16 s-1 for the rate-limiting step, and an overall rate constant of 13.3 x 10(7) M-1 . s-1 for heparin-antithrombin interaction.
Subject(s)
Antithrombins/metabolism , Heparin/pharmacology , Thrombin/metabolism , Animals , Cattle , Kinetics , Protein BindingABSTRACT
The effects of temperature and pH on the solution conformation of native prothrombin fragment 1 were examined with 1H NMR spectroscopy. A calcium-dependent quenching of the intrinsic protein fluorescence was used to monitor calcium binding to fragment 1 as an indicator of functional protein. The native fragment 1 NMR spectrum contained several features indicative of a folded protein: (a) nonequivalent histidyl C-2 resonances at 7.9 and 8.1 ppm, (b) two resonances of nearly equal intensity at 7.26 and 7.32 ppm, and (c) a resonance at -1.04 ppm. Temperature studies showed that thermal unfolding of fragment 1 (even at 80 degrees C) was reversible; however, there was an irreversible inactivation step which occurred subsequent to the unfolding. The basis for this inactivation appeared to include disulfide exchange reactions. On the basis of NMR spectra, fragment 1 retained its conformation from pH 7.0 to pH 11.5. From pH 7.0 to pH 5.0, the protein showed a reversible conformational change, and below pH 5, the protein self-associated. The pH dependence of the chemical shift of the tyrosyl resonances indicated a pKa of approximately 10 for the tyrosyl residues. These data suggest that the tyrosyl residues are accessible to solvent in the native protein.