Relocating the active site of activated protein C eliminates the need for its protein S cofactor. A fluorescence resonance energy transfer study.
The effect of replacing the gamma-carboxyglutamic acid domain of activated protein C (APC) with that of prothrombin on the topography of the membrane-bound enzyme was examined using fluorescence resonance energy transfer. The average distance of closest approach (assuming kappa2 = 2/3) between a fluorescein in the active site of the chimera and octadecylrhodamine at the membrane surface was 89 A, compared with 94 A for wild-type APC. The gamma-carboxyglutamic acid domain substitution therefore lowered and/or reoriented the active site, repositioning it close to the 84 A observed for the APC. protein S complex. Protein S enhances wild-type APC cleavage of factor Va at Arg306, but the inactivation rate of factor Va Leiden by the chimera alone is essentially equal to that by wild-type APC plus protein S. These data suggest that the activities of the chimera and of the APC.protein S complex are equivalent because the active site of the chimeric protein is already positioned near the optimal location above the membrane surface to cleave Arg306. Thus, one mechanism by which protein S regulates APC activity is by relocating its active site to the proper position above the membrane surface to optimize factor Va cleavage. (+info)
Activation of the kallikrein-kinin system in hemodialysis: role of membrane electronegativity, blood dilution, and pH.
BACKGROUND: The kallikrein-kinin system activation by contact with a negatively charged surface has been promulgated to be responsible for hypersensitivity reactions. However, to explain the low frequency and heterogeneity of hypersensitivity reactions, we hypothesized that not only the electronegativity of the membrane, but also other physicochemical parameters could influence the activation of the contact phase system of plasma assessed by the measurement of kallikrein activity and bradykinin concentration. METHODS: Plasma kallikrein activity using chromogenic substrate (S2302) and plasma bradykinin concentration (enzyme immuno assay) were measured during the perfusion of human plasma (2.5 ml/min) through minidialyzers mounted with six different membranes [polyacrylonitrile (PAN) from Asahi (PANDX) and from Hospal (AN69), polymethylmethacrylate (PMMA) from Toray, cellulose triacetate (CT) from Baxter, cuprophane (CUP) from Akzo and polysulfone (PS) from Fresenius]. RESULTS: A direct relationship was shown between the electronegativity of the membrane assessed by its zeta potential and the activation of plasma during the first five minutes of plasma circulation. With the AN69 membrane, the detection of a kallikrein activity in diluted plasma but not in undiluted samples confirmed the importance of a protease-antiprotease imbalance leading to bradykinin release during the first five minutes of dialysis. With PAN membranes, the use of citrated versus heparinized plasma and the use of various rinsing solutions clearly show a dramatic effect of pH on the kallikrein activity and the bradykinin concentration measured in plasma. Finally, increasing the zeta potential of the membrane leads to a significant increase of plasma kallikrein activity and bradykinin concentration. CONCLUSIONS: Our in vitro experimental approach evidences the importance of the control of these physicochemical factors to decrease the activation of the contact system. (+info)
Three-independent-compartment chamber to study in vitro commissural synapses.
We describe a novel chamber in which the two intact neonatal rat hippocampi and the commissural fibers are placed in three independent compartments separated by latex membranes and perfused selectively with different solutions. A set of control tests showed that the compartments are well isolated: 1) methylene blue or eosin applied to one compartment did not diffuse to other compartments when verified via the microscope, and spectrophotometry revealed that <1/10.000th of the dye diffuses to other compartments; 2) tetrodotoxin (1 microM) applied to the commissural compartment blocked the synaptic responses evoked contralaterally without affecting those evoked on the ipsilateral side. This chamber enables a wide range of experiments that cannot be performed in conventional chambers, e.g., to study the maturation and plasticity of the commissural connections, bilateral synchronization of the rhythmic activities in the limbic system, commissural propagation of the epileptiform activities, etc. (+info)
Electrostatic interactions during activation of coagulation factor IX via the tissue factor pathway: effect of univalent salts.
Interaction between the Gla-domain of coagulation proteins and negatively charged phospholipid membranes is essential for blood coagulation reactions. The interaction is calcium-dependent and mediated both by electrostatic and hydrophobic forces. This report focuses on the electrostatic component of factor IX activation via the extrinsic pathway. Effective charges during the reaction are measured by ionic titration of activity, according to the Debye-Huckel and Gouy-Chapman models. Rates of activation decrease with ionic strength independently of the type of monovalent salt used to control ionic strength. Moreover, the effect of ionic strength decreases at concentrations of charged phospholipid approaching saturation levels, indicating that membrane charges participate directly in the ionic interaction measured. The effective charge on calcium-bound factor IX during activation on phospholipid membranes is 0.95+/-0.1. Possible sites mediating contacts between the Gla-domain and membranes are selected by geometrical criteria in several metal-bound Gla-domain structures. A pocket with a solvent opening-pore of area 24-38 A2 is found in the Gla-domain of factors IX, VII, and prothrombin. The pocket contains atoms with negative partial charges, including carboxylate oxygens from Gla residues, and has a volume of 57-114 A3, sufficient to accommodate additional calcium atoms. These studies demonstrate that electrostatic forces modify the activity coefficient of factor IX during functional interactions and suggest a conserved pocket motif as the contact site between the calcium-bound Gla-domain and charged membranes. (+info)
Reconstitution of the human endothelial cell protein C receptor with thrombomodulin in phosphatidylcholine vesicles enhances protein C activation.
Blocking protein C binding to the endothelial cell protein C receptor (EPCR) on the endothelium is known to reduce protein C activation rates. Now we isolate human EPCR and thrombomodulin (TM) and reconstitute them into phosphatidylcholine vesicles. The EPCR increases protein C activation rates in a concentration-dependent fashion that does not saturate at 14 EPCR molecules/TM. Without EPCR, the protein C concentration dependence fits a single class of sites (Km = 2.17 +/- 0.13 microM). With EPCR, two classes of sites are apparent (Km = 20 +/- 15 nM and Km = 3.2 +/- 1.7 microM). Increasing the EPCR concentration at a constant TM concentration increases the percentage of high affinity sites. Holding the TM:EPCR ratio constant while decreasing the density of these proteins results in a decrease in the EPCR enhancement of protein C activation, suggesting that there is little affinity of the EPCR for TM. Negatively charged phospholipids also enhance protein C activation. EPCR acceleration of protein C activation is blocked by anti-EPCR antibodies, but not by annexin V, whereas the reverse is true with negatively charged phospholipids. Human umbilical cord endothelium expresses approximately 7 times more EPCR than TM. Anti-EPCR antibody reduces protein C activation rates 7-fold over these cells, whereas annexin V is ineffective, indicating that EPCR rather than negatively charged phospholipid provide the surface for protein C activation. EPCR expression varies dramatically among vascular beds. The present results indicate that the EPCR concentration will determine the effectiveness of the protein C activation complex. (+info)
Phospholipid-subclass-specific partitioning of lipophilic ions in membrane-water systems.
Herein, we systematically investigate phospholipid-subclass-specific alterations in the partitioning of both cationic and anionic amphiphiles to identify the importance of ester, ether and vinyl ether linkages at the sn-1 position of phospholipids in the partitioning of charged amphiphiles. The results demonstrated that the membrane-water partition coefficient of a prototypic cationic amphiphile (i.e. 3,3'-dipropylthiadicarbocyanine iodide) was approximately 2.5 times higher in membranes comprised of plasmenylcholine in comparison with membranes comprised of either phosphatidylcholine or plasmanylcholine. In striking contrast, the membrane-water partition coefficient of a prototypic anionic amphiphile [i.e. bis-(1,3-dibutylbarbituric acid)trimethine oxonol] in membranes comprised of plasmenylcholine was approximately 2.5 times lower than that manifest in membranes comprised of phosphatidylcholine or plasmanylcholine. Utilizing theseexperimentally determined partition coefficients,the relative membrane dipole potential of membranes comprised of plasmenylcholine was calculated and found to be approximately 25 mV lower than in membranes comprised of phosphatidylcholine or plasmanylcholine. This lower membrane dipole potential in membranes comprised of plasmenylcholine is equivalent to the membrane potential induced by incorporation of approximately 25 mol% of anionic phospholipids in membranes comprised of phosphatidylcholine. Collectively, these results demonstrate that phospholipid-subclass-specific differences in the membrane dipole potential contribute to alterations in the partitioning of lipophilic ions in membrane bilayers comprised of distinct phospholipid subclasses. Moreover, they suggest that these physicochemical differences can be exploited to facilitate the targeting of charged lipophilic drugs to specific cells and subcellular membrane compartments. (+info)
Analysis of the membrane-interacting domains of myelin basic protein by hydrophobic photolabeling.
Myelin basic protein is a water soluble membrane protein which interacts with acidic lipids through some type of hydrophobic interaction in addition to electrostatic interactions. Here we show that it can be labeled from within the lipid bilayer when bound to acidic lipids with the hydrophobic photolabel 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine (TID) and by two lipid photolabels. The latter included one with the reactive group near the apolar/polar interface and one with the reactive group linked to an acyl chain to position it deeper in the bilayer. The regions of the protein which interact hydrophobically with lipid to the greatest extent were determined by cleaving the TID-labeled myelin basic protein (MBP) with cathepsin D into peptides 1-43, 44-89, and 90-170. All three peptides from lipid-bound protein were labeled much more than peptides from the protein labeled in solution. However, the peptide labeling pattern was similar for both environments. The two peptides in the N-terminal half were labeled similarly and about twice as much as the C-terminal peptide indicating that the N-terminal half interacts hydrophobically with lipid more than the C-terminal half. MBP can be modified post-translationally in vivo, including by deamidation, which may alter its interactions with lipid. However, deamidation had no effect on the TID labeling of MBP or on the labeling pattern of the cathepsin D peptides. The site of deamidation has been reported to be in the C-terminal half, and its lack of effect on hydrophobic interactions of MBP with lipid are consistent with the conclusion that the N-terminal half interacts hydrophobically more than the C-terminal half. Since other studies of the interaction of isolated N-terminal and C-terminal peptides with lipid also indicate that the N-terminal half interacts hydrophobically with lipid more than the C-terminal half, these results from photolabeling of the intact protein suggest that the N-terminal half of the intact protein interacts with lipid in a similar way as the isolated peptide. The similar behavior of the intact protein to that of its isolated peptides suggests that when the purified protein binds to acidic lipids, it is in a conformation which allows both halves of the protein to interact independently with the lipid bilayer. That is, it does not form a hydrophobic domain made up from different parts of the protein. (+info)
Scanning near-field fluorescence resonance energy transfer microscopy.
A new microscopic technique is demonstrated that combines attributes from both near-field scanning optical microscopy (NSOM) and fluorescence resonance energy transfer (FRET). The method relies on attaching the acceptor dye of a FRET pair to the end of a near-field fiber optic probe. Light exiting the NSOM probe, which is nonresonant with the acceptor dye, excites the donor dye introduced into a sample. As the tip approaches the sample containing the donor dye, energy transfer from the excited donor to the tip-bound acceptor produces a red-shifted fluorescence. By monitoring this red-shifted acceptor emission, a dramatic reduction in the sample volume probed by the uncoated NSOM tip is observed. This technique is demonstrated by imaging the fluorescence from a multilayer film created using the Langmuir-Blodgett (LB) technique. The film consists of L-alpha-dipalmitoylphosphatidylcholine (DPPC) monolayers containing the donor dye, fluorescein, separated by a spacer group of three arachidic acid layers. A DPPC monolayer containing the acceptor dye, rhodamine, was also transferred onto an NSOM tip using the LB technique. Using this modified probe, fluorescence images of the multilayer film reveal distinct differences between images collected monitoring either the donor or acceptor emission. The latter results from energy transfer from the sample to the NSOM probe. This method is shown to provide enhanced depth sensitivity in fluorescence measurements, which may be particularly informative in studies on thick specimens such as cells. The technique also provides a mechanism for obtaining high spatial resolution without the need for a metal coating around the NSOM probe and should work equally well with nonwaveguide probes such as atomic force microscopy tips. This may lead to dramatically improved spatial resolution in fluorescence imaging. (+info)