Interactions of free and immobilized myelin basic protein with anionic detergents

The interaction of free and immobilized myelin basic protein (MBP) with sodium deoxycholate (DOC) and sodium dodecyl sulfate (NaDodSO4) was studied under a variety of conditions. Free MBP formed insoluble complexes with both detergents. Analysis of the insoluble complexes revealed that the molar ratio of detergent/MBP in the precipitate increased in a systematic fashion with increasing detergent concentration until the complex became soluble. At pH 4.8, equilibrium dialysis studies indicated that approximately 15 mol of NaDodSO4 could bind to the protein without precipitation occurring. Regardless of the surfactant, however, minimum protein solubility occurred when the net charge on the protein-detergent complex was between +18 and -9. Complete equilibrium binding isotherms of both detergents to the protein were obtained by using MBP immobilized on agarose. The bulk of the binding of both detergents was highly cooperative and occurred at or above the critical micelle concentration. At I = 0.1, saturation levels of 2.09 +/- 0.15 g of NaDodSO4/g of protein and 1.03 /+- 0.40 g of DOC/g of protein were obtained. Below pH 7.0 the NaDodSO4 binding isotherms revealed an additional cooperative transition corresponding to the binding of 15-20 mol of NaDodSO4/mol of protein. Affinity chromatography studies indicated that, in the presence of NaDodSO4 (but not in its absence), [125I]MBP interacted with agarose-immobilized histone, lysozyme, and MBP but did not interact with ovalbumin-agarose. These data support a model in which the detergent cross-links and causes precipitation of MBP-anionic detergent complexes. Cross-linking may occur through hydrophobic interaction between detergents electrostatically bound to different MBP molecules.     

Reaction of lecithin:cholesterol acyltransferase with micellar complexes of apolipoprotein A-I and phosphatidylcholine, containing variable amounts of cholesterol

In a continued investigation of lecithin:cholesterol acyltransferase reaction with micellar, discoidal complexes of phosphatidylcholine (PC) . cholesterol . apolipoprotein A-I (apo-A-I), we prepared well defined complexes with variable free cholesterol contents and examined their reactivity with purified enzyme. The complexes, prepared by the sodium cholate dialysis method, were fractionated into "small" and "large" classes by gel filtration of the reaction mixtures through a Bio-Gel A-5m column. The small complexes had egg-PC/cholesterol/apo-A-I molar ratios from 68:14:1 to 80:1:1, discoidal shapes with diameters around 114 (+/- 13) A and widths of 42 A by electron microscopy, and Stokes radii from 47 to 49 A corresponding to molecular weights near 2 X 10(5). The corresponding properties of the large complexes, isolated from samples with higher cholesterol contents, were egg-PC/cholesterol/apo-A-I molar ratios from 84:26:1 to 96:17:1, diameters of 161 (+/- 20) A, widths of 43 A, Stokes radii around 80 A, and estimated molecular weights in the vicinity of 5 X 10(5). Both types of complexes, when adjusted to equal apo-A-I concentrations, gave essentially identical initial reaction velocities with purified lecithin:cholesterol acyltransferase over a wide range of cholesterol concentrations (from 2 X 10(-7) to 4 X 10(-4) M), PC/cholesterol molar ratios (from 3:1 to 12:1), and quite different lipid fluidity conditions as detected by diphenylhexatriene fluorescence polarization. When complexes were adjusted to a constant cholesterol concentration, the initial velocities of the lecithin:cholesterol acyltransferase reaction followed Michaelis-Menten kinetics relative to the apo-A-I concentrations. Arrhenius plots of initial reaction rates for various complexes with variable cholesterol content and fluidity, measured at constant apo-A-I concentrations, gave identical temperature dependences with an average activation energy of 18.0 kcal/mol. These results strongly suggest that the cholesterol esterification on high density lipoprotein particles does not depend on their unesterified-cholesterol contents, PC/unesterified-cholesterol molar ratios, nor on the fluidity of their lipid domains.     

N-linked oligosaccharides of murine major histocompatibility complex class II molecule. Role in antigenic peptide binding, T cell recognition, and clonal nonresponsiveness.

To evaluate the functional role of the N-linked oligosaccharides of major histocompatibility complex (MHC) class II molecules, affinity-purified murine IAs class II molecules were deglycosylated in the presence of asparagine amidase enzyme. The deglycosylated IAs molecules were characterized by 12% SDS-polyacrylamide gel analysis under reduced and native conditions and the complete enzymatic removal of all three N-linked sugar components from the alpha/beta heterodimer was confirmed by lectin-link Western blot analysis. Like the native IAs molecules, the deglycosylated IAs molecules were fully capable of binding an antigenic peptide from myelin basic protein MBP(89-101). The kinetics of dissociation of preformed complexes of IAs.MBP(89-101) and deglycosylated IAs.MBP(89-101) were compared at 4 and at 37 degrees C. Both complexes were equally stable at 4 degrees C; however, at 37 degrees C the deglycosylated IAs.MBP(89-101) complexes showed an increased rate of dissociation as compared with the native IAs.MBP(89-101) complexes. When tested for their ability to recognize the T cell receptor on T cells, both complexes bound to cloned HS-1 T cells that recognize and respond to IAs.MBP(89-101). Finally, the complexes of deglycosylated IAs.MBP(89-101) were tested for the induction of in vitro nonresponsiveness and compared with native IAs.MBP(89-101) complexes. Both complexes were capable of inducing 95-100% nonresponsiveness in a proliferation assay. These results suggest that the N-linked oligosaccharide of MHC class II molecules may not be essential for either antigenic peptide binding or T cell recognition. In addition results obtained here provide evidence that the carbohydrate moities of MHC class II molecules may not be involved in induction of T cell clonal anergy.     

Improvement of water solubility of non-competitive AMPA receptor antagonists by complexation with beta-cyclodextrin

The (R,S)-2-acetyl-1-(4'-chlorophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline ((R,S)-1) was previously identified as a potent non-competitive AMPA receptor antagonist able to prevent epileptic seizures and reduce AMPA-induced current in electrophysiological experiments. Through the enantiomeric resolution of racemate by chiral HPLC we already demonstrated that the (R)-1 enantiomer was the eutomer. Considering the poor water solubility, these compounds have been complexed with beta-cyclodextrin (beta-CyD). The effect of beta-cyclodextrin on the spectral features of molecules was quantitatively investigated, in fully aqueous medium by phase-solubility study and the obtained diagrams suggested that it forms complexes with a molar ratio 1:1. The binding constant (K((R)-1)=15889M(-1), K((R,S)(-1))=1079 M(-1)) and the complexation efficiency (CE) were calculated. Then the solid complexes in 1:1 molar ratio were prepared by the co-precipitation method and the FTIR-ATR measurements were carried out in order to confirm the host-guest interactions that drive the complexation process, by monitoring the significant differences of the spectra of the complexes with respect to those of the corresponding physical mixtures in the same molar ratio. The experimental data have been compared with molecular modelling studies and we confirmed our hypothesis.     

Radioligand-dependent differences in the molecular properties of the mouse and rat hepatic aryl hydrocarbon receptor complexes

A comparison of the molecular properties of the male Long-Evans rat and male C57BL/6 mouse hepatic cytosolic aryl hydrocarbon (Ah) receptor complex was determined using 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,7,8-[3H]tetrachlorodibenzofuran (TCDF) as radioligands. In low salt buffer, the sedimentation coefficients, Stokes radii, relative molecular masses, frictional ratios, axial ratios and gel permeation chromatographic properties of the rat receptor complexes were ligand independent. In contrast, there were several ligand-dependent differences in the mouse Ah receptor complexes formed after incubation in low salt buffer and these include: sucrose density gradient analysis of the 2,3,7,8-[3H]TCDF receptor complex gave a 9.5 S specifically bound peak and a 2.6 S nonspecifically bound peak whereas the corresponding 2,3,7,8-[3H]TCDD receptor complex gave a single 9.6 S specifically bound peak; sucrose density gradient analysis of the two major peaks eluted from a Sephacryl S-300 column chromatographic separation of the 2,3,7,8-[3H]TCDF receptor complex gave two specifically bound peaks at 9.2 and 5.1 S. The molecular properties of the rat hepatic cytosolic receptor complexes incubated in high salt (0.4 M KCl) buffer were ligand independent with one exception, namely the significant difference in the sedimentation coefficient of the specifically bound disaggregated 2,3,7,8-[3H]TCDD receptor complex (6.8 S) and the corresponding 2,3,7,8-[3H]TCDF receptor complex (5.0 S). The major ligand-dependent differences in the mouse receptor complexes incubated in high salt (0.4 M KCl) were associated with the sedimentation coefficients of the complexes derived after direct incubation and after gel permeation chromatography. For example, both ligands gave two specifically bound complexes after chromatography on Sephacryl S-300 column and centrifugation of these fractions gave both the approximately 9 and approximately 5 S peaks; this suggested that there was some equilibration between the aggregated and disaggregated receptor complexes. The behavior of the 2,3,7,8-[3H]TCDF mouse receptor complex was similar after incubation in low or high salt buffer except that sucrose density gradient analysis of the gel permeation chromatographic fractions gave an additional specifically bound peak which sedimented at 7.2 S. These studies demonstrate that the molecular properties of the Ah receptor were dependent on the source of the cytosolic receptor preparation, the ionic strength of the incubation media, and the structure of the radioligand.     

Preparation and characterization of a colloidal gold-insulin complex with binding and biological activities identical to native insulin

We studied the binding and biological activities of gold-insulin complexes to develop a complex with properties identical to native insulin. Stabilizing amounts of insulin absorbed to 5-, 10-, or 15-nm gold particles resulted in complexes with 40-327 insulin molecules per gold particle and 4-111 times the biological activity of unlabeled insulin, based on the molar concentration of gold complex. These data suggested that these complexes behaved as multivalent ligands. Gold-insulin complexes were prepared with 5% of the stabilizing insulin concentration and were stabilized with bovine serum albumin. This resulted in a complex with 5-7 insulin molecules per 10-nm gold particle, which stimulated glucose oxidation in rat adipocytes and competed with [125I]-insulin for binding to the insulin receptor identically to unlabeled insulin on an equimolar basis. The organization and distribution of insulin receptors occupied by this monovalent-behaving gold-insulin complex were virtually identical to previous observations using monomeric ferritin-insulin. Since multivalent ligands may affect receptor binding, re-distribution, and intracellular processing, the use of electron-dense probes that resemble the unlabeled ligand in biological and binding properties is appropriate when studying receptor dynamics of in vivo or in vitro biological systems. The gold-insulin complex developed in this study should serve this function.     

胰岛素对3T3-L1脂肪细胞中极低密度脂蛋白受体基因表达的影响

体外培养3T3-L1细胞分化模型,研究不同浓度胰岛素及慢性胰岛素刺激对3T3-L1脂肪细胞中极低密度脂蛋白受体（VLDLR）基因表达的影响.在不同浓度胰岛素及胰岛素慢性刺激的干预下,用半定量RT-PCR检测细胞VLDLR mRNA水平的变化.微量化GOD-PAP法检测培养基中残存的葡萄糖.在细胞诱导分化过程中,胰岛素浓度的增高促进VLDLR的表达;胰岛素慢性刺激下,VLDLR表达因浓度差异呈现不同变化.研究结果表明,胰岛素的浓度及慢性刺激对3T3-L1脂肪细胞的成熟和VLDLR基因的表达有显著作用,而胰岛素抵抗明显减低成熟脂肪细胞VLDLR的表达.     

LDL receptor deficiency unmasks altered VLDL triglyceride metabolism in VLDL receptor transgenic and knockout mice

The very low density lipoprotein receptor (VLDLR) has been proposed to play a role in the delivery of fatty acids to peripheral tissues. However, despite reduced adipose tissue mass in VLDLR-deficient (VLDLR(-)(/-)) mice, this has been difficult to substantiate. In the present study, VLDLR-deficient and VLDLR-overexpressing (PVL) mice were cross-bred onto a low density lipoprotein receptor knockout (LDLR(-)(/-)) background to study the VLDLR under conditions of relatively high serum VLDL and triglyceride levels. Absence of the VLDLR resulted in a significant increase in serum triglyceride levels (1.9-fold) when mice were fed a high fat diet. In contrast, overexpression of the VLDLR resulted in a significant decrease in serum triglyceride levels (2.0-fold) under similar conditions. When kept on a chow diet, a period of prolonged fasting revealed a significant increase in serum triglyceride levels in VLDLR(-)(/-); LDLR(-)(/-) mice (2.3-fold) as compared with LDLR(-)(/-) controls. This could not be attributed to altered apolipoprotein B and VLDL triglyceride production rates. Furthermore, no major differences in nascent VLDL triglyceride content were found between VLDLR(-)(/-); LDLR(-)(/-) mice and LDLR(-)(/-) controls. However, the triglyceride content of circulating VLDL of VLDLR(-)(/-); LDLR(-)(/-) mice (63%) was relatively high as compared with LDLR(-)(/-) controls (49%). These observations suggest that the VLDLR affects peripheral uptake of VLDL triglycerides.In conclusion, under conditions of LDLR deficiency in combination with high fat feeding or prolonged fasting, the effect of the VLDLR on VLDL triglyceride metabolism was revealed.     

Envelope glycoprotein incorporation, not shedding of surface envelope glycoprotein (gp120/SU), Is the primary determinant of SU content of purified human immunodeficiency virus type 1 and simian immunodeficiency virus

Human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) particles typically contain small amounts of the surface envelope protein (SU), and this is widely believed to be due to shedding of SU from mature virions. We purified proteins from HIV-1 and SIV isolates using procedures which allow quantitative measurements of viral protein content and determination of the ratios of gag- and env-encoded proteins in virions. All of the HIV-1 and most of the SIV isolates examined contained low levels of envelope proteins, with Gag:Env ratios of approximately 60:1. Based on an estimate of 1,200 to 2,500 Gag molecules per virion, this corresponds to an average of between 21 and 42 SU molecules, or between 7 and 14 trimers, per particle. In contrast, some SIV isolates contained levels of SU at least 10-fold greater than SU from HIV-1 isolates. Quantification of relative amounts of SU and transmembrane envelope protein (TM) provides a means to assess the impact of SU shedding on virion SU content, since such shedding would be expected to result in a molar excess of TM over SU on virions that had shed SU. With one exception, viruses with sufficient SU and TM to allow quantification were found to have approximately equivalent molar amounts of SU and TM. The quantity of SU associated with virions and the SU:TM ratios were not significantly changed during multiple freeze-thaw cycles or purification through sucrose gradients. Exposure of purified HIV-1 and SIV to temperatures of 55 degrees C or greater for 1 h resulted in loss of most of the SU from the virus but retention of TM. Incubation of purified virus with soluble CD4 at 37 degrees C resulted in no appreciable loss of SU from either SIV or HIV-1. These results indicate that the association of SU and TM on the purified virions studied is quite stable. These findings suggest that incorporation of SU-TM complexes into the viral membrane may be the primary factor determining the quantity of SU associated with SIV and HIV-1 virions, rather than shedding of SU from mature virions.     

Physicochemical properties of the lipopolysaccharide unit that activates B lymphocytes

We have examined the physical state of highly purified deep rough chemotype lipopolysaccharide (ReLPS) from Escherichia coli D31m4 as an aqueous suspension and as complexes with bovine serum albumin min (BSA). The ReLPS suspension showed large ellipsoidal particles 12-38 nm wide and 40-100 nm long. The solubility of this form of ReLPS was determined by equilibrium dialysis experiments to be 3.3 x 10(-8) M at 22 degrees C and 2.8 x 10(-8) M at 37 degrees C in 150 mM Tris-KCl, pH 7.5; 3.0 x 10(-8) M at 37 degrees C in 0.75 mM Tris-KCl, pH 7.5. The BSA-ReLPS complexes were fractionated on a Sephacryl S-200 column to yield peaks I and II with apparent masses of about 240 and 70 kDa, respectively. Peak II was a BSA monomer with estimated BSA:ReLPS molar ratios of 1:1-1:7. The ReLPS suspension and the two complexes were compared as antigens in enzyme-linked immunosorbent assays using three select monoclonal antibodies to lipopolysaccharide. The results were consistent with the high state of disaggregation of the ReLPS in both peaks I and II. Since the ReLPS in these complexes were not visible by electron microscopy, they did not contain vesicles or large particles. All forms of ReLPS tested were capable of stimulating 70Z/3, a lipopolysaccharide-responsive murine pre-B cell line. However, peak II was consistently more stimulatory at very low concentrations than the other preparations. The maximally stimulatory concentration of ReLPS for 70Z/3 cells was 40 ng/ml (1.6 x 10(-8) M) for peak II and 70 ng/ml (2.8 x 10(-8) M) for the ReLPS suspension. As expected, the above concentrations were at or below the solubility of the ReLPS. These results suggested that the highly disaggregated form of ReLPS (possibly the monomer) is the active unit that stimulates the cellular response in 70Z/3 cells.     

Increased CD8+ cytotoxic T cell responses to myelin basic protein in multiple sclerosis

Autoreactive T cells of CD4 and CD8 subsets recognizing myelin basic protein (MBP), a candidate myelin autoantigen, are thought to contribute to and play distinct roles in the pathogenesis of multiple sclerosis (MS). In this study we identified four MBP-derived peptides that had high binding affinity to HLA-A2 and HLA-A24 and characterized the CD8(+) T cell responses and their functional properties in patients with MS. There were significantly increased CD8(+) T cell responses to 9-mer MBP peptides, in particular MBP(111-119) and MBP(87-95) peptides that had high binding affinity to HLA-A2, in patients with MS compared with healthy individuals. The resulting CD8(+) T cell lines were of the Th1 phenotype, producing TNF-alpha and IFN-gamma and belonged to a CD45RA(-)/CD45RO(+) memory T cell subset. Further characterization indicated that the CD8(+) T cell lines obtained were stained with MHC class I tetramer (HLA-A2/MBP(111-119)) and exhibited specific cytotoxicity toward autologous target cells pulsed with MBP-derived peptides in the context of MHC class I molecules. These cytotoxic CD8(+) T cell lines derived from MS patients recognized endogenously processed MBP and lysed COS cells transfected with genes encoding MBP and HLA-A2. These findings support the potential role of CD8(+) CTLs recognizing MBP in the injury of oligodendrocytes expressing both MHC class I molecules and MBP.     

Very low density lipoprotein receptor, a negative regulator of the wnt signaling pathway and choroidal neovascularization

Choroidal neovascularization (CNV) in age-related macular degeneration is a leading cause of blindness. Very low density lipoprotein receptor gene knock-out (Vldlr(-/-)) mice have been shown to develop subretinal neovascularization (NV) with an unknown mechanism. The present study showed that in Vldlr(-/-) mice, NV initiated in the choroid and progressed to penetrate the retinal pigment epithelium layer, proliferating in the subretinal space. This phenotype recapitulated what is seen in wet age-related macular degeneration, suggesting that this is a CNV model. The CNV correlated with overexpression of vascular endothelial growth factor in Vldlr(-/-) eyecups and was blocked by a neutralizing antibody against vascular endothelial growth factor receptor-2. The wnt co-receptor LRP5/6 expression was significantly up-regulated in Vldlr(-/-) eyecups compared with that in wild-type mice. Significantly, Vldlr(-/-) mice showed impaired phosphorylation of downstream effectors of the wnt signaling pathway, glycogen synthase kinase-3beta (GSK-3beta), and beta-catenin, concomitant with increased levels of free GSK-3beta and beta-catenin, suggesting an increased activity of the wnt pathway. Down-regulation of VLDLR by small interference RNA resulted in up-regulation of LRP5/6 expression and activation of beta-catenin in cultured endothelial cells. Furthermore, Dickkopf-1, a specific inhibitor of the wnt pathway, effectively decreased vascular endothelial growth factor and beta-catenin levels in the retinal pigment epithelium of Vldlr(-/-) mice and in cells transfected with the VLDLR small interference RNA. These results suggest that VLDLR functions as a negative regulator of CNV, and this function is mediated through the wnt pathway.     

Study of the assembly of vesicular stomatitis virus N protein: role of the P protein

To derive structural information about the vesicular stomatitis virus (VSV) nucleocapsid (N) protein, the N protein and the VSV phosphoprotein (P protein) were expressed together in Escherichia coli. The N and P proteins formed soluble protein complexes of various molar ratios when coexpressed. The major N/P protein complex was composed of 10 molecules of the N protein, 5 molecules of the P protein, and an RNA. A soluble N protein-RNA oligomer free of the P protein was isolated from the N/P protein-RNA complex using conditions of lowered pH. The molecular weight of the N protein-RNA oligomer, 513,879, as determined by analytical ultracentrifugation, showed that it was composed of 10 molecules of the N protein and an RNA of approximately 90 nucleotides. The N protein-RNA oligomer had the appearance of a disk with outer diameter, inner diameter, and thickness of 148 +/- 10 A, 78 +/- 9 A, and 83 +/- 8 A, respectively, as determined by electron microscopy. RNA in the complexes was protected from RNase digestion and was stable at pH 11. This verified that N/P protein complexes expressed in E. coli were competent for encapsidation. In addition to coexpression with the full-length P protein, the N protein was expressed with the C-terminal 72 amino acids of the P protein. This portion of the P protein was sufficient for binding to the N protein, maintaining it in a soluble state, and for assembly of N protein-RNA oligomers. With the results provided in this report, we propose a model for the assembly of an N/P protein-RNA oligomer.     

Use of a fluorescence spectroscopy technique to study the adsorption of sodium dodecylsulfonate on liposomes

The fluorescent probe 2-(p-toluidinyl)-naphthalene-6-sodium sulfonate (TNS) was used to study the surface adsorption of sublytic concentrations of the anionic surfactant sodium dodecylsulfonate (C(12)-SO(3)) on phosphatidylcholine (PC) bilayers. The number of adsorbed molecules was quantified by determination of the electrostatic potential (psi(o)) of the bilayers. The abrupt decrease in the fluorescence intensity detected even 10 s after the surfactant addition and the slight fluorescence variations with time indicated that the surfactant adsorption was very fast and almost complete. For a given number of monomers adsorbed a linear dependence between the lipid and C12-SO3 concentrations was obtained, indicating similar adsorption mechanism regardless of the surfactant concentration. Hence, a monomeric adsorption is assumed even in systems with a C12-SO3 concentration above its CMC. In addition, this linear correlation allowed us to determine the surfactant/lipid molar ratios (Re) (inversely related to the C12-SO3 ability to be adsorbed on liposomes) and the bilayer/aqueous phase coefficients (K). The fact that the lowest values for Re were always reached after 10 s of incubation corroborates the rapid kinetics of the process. The decrease in the C12-SO3 partitioning (K) when the number of surfactant molecules exceeded 15000 was possibly due to the electrostatic repulsion between the free and the adsorbed monomers, which could hinder the incorporation of new monomers on the charged surface of liposomes.     

Catalytic reaction pathway for the mitogen-activated protein kinase ERK2

The structural, functional, and regulatory properties of the mitogen-activated protein kinases (MAP kinases) have long attracted considerable attention owing to the critical role that these enzymes play in signal transduction. While several MAP kinase X-ray crystal structures currently exist, there is by comparison little mechanistic information available to correlate the structural data with the known biochemical properties of these molecules. We have employed steady-state kinetic and solvent viscosometric techniques to characterize the catalytic reaction pathway of the MAP kinase ERK2 with respect to the phosphorylation of a protein substrate, myelin basic protein (MBP), and a synthetic peptide substrate, ERKtide. A minor viscosity effect on k(cat) with respect to the phosphorylation of MBP was observed (k(cat) = 10 +/- 2 s(-1), k(cat)(eta) = 0.18 +/- 0.05), indicating that substrate processing occurs via slow phosphoryl group transfer (12 +/- 4 s(-1)) followed by the faster release of products (56 +/- 4 s(-1)). At an MBP concentration extrapolated to infinity, no significant viscosity effect on k(cat)/K(m(ATP)) was observed (k(cat)/K(m(ATP)) = 0.2 +/- 0.1 microM(-1) s(-1), k(cat)/K(m(ATP))(eta) = -0.08 +/- 0.04), consistent with rapid-equilibrium binding of the nucleotide. In contrast, at saturating ATP, a full viscosity effect on k(cat)/K(m) for MBP was apparent (k(cat)/K(m(MBP)) = 2.4 +/- 1 microM(-1) s(-1), k(cat)/K(m(MBP))(eta) = 1.0 +/- 0.1), while no viscosity effect was observed on k(cat)/K(m) for the phosphorylation of ERKtide (k(cat)/K(m(ERKtide)) = (4 +/- 2) x 10(-3) microM(-1) s(-1), k(cat)/K(m(ERKtide))(eta) = -0.02 +/- 0.02). This is consistent with the diffusion-limited binding of MBP, in contrast to the rapid-equilibrium binding of ERKtide, to form the ternary Michaelis complex. Calculated values for binding constants show that the estimated value for K(d(MBP)) (/= 1.5 mM). The dramatically higher catalytic efficiency of MBP in comparison to that of ERKtide ( approximately 600-fold difference) is largely attributable to the slow dissociation rate of MBP (/=56 s(-1)), from the ERK2 active site.     

Kinetics of T-cell receptor binding by bivalent HLA-DR. Peptide complexes that activate antigen-specific human T-cells

Monovalent major histocompatibility complex-peptide complexes dissociate within seconds from the T-cell receptor (TCR), indicating that dimerization/multimerization may be important during early stages of T-cell activation. Soluble bivalent HLA-DR2.myelin basic protein (MBP) peptide complexes were expressed by replacing the F(ab) arms of an IgG2a antibody with HLA-DR2.MBP peptide complexes. The binding of bivalent HLA-DR2.peptide complexes to recombinant TCR was examined by surface plasmon resonance. The bivalent nature greatly enhanced TCR binding and slowed dissociation from the TCR, with a t((1)/(2)) of 2.1 to 4.6 min. Soluble bivalent HLA-DR2.MBP peptide complexes activated antigen-specific T-cells in the absence of antigen presenting cells. In contrast, soluble antibodies to the TCR.CD3 complex were ineffective, indicating that they failed to induce an active TCR dimer. TCR/CD3 antibodies induced T-cell proliferation when bound by antigen presenting cells that expressed Fc receptors. In the presence of dendritic cells, bivalent HLA-DR2. MBP peptide complexes induced T-cell activation at >100-fold lower concentrations than TCR/CD3 antibodies and were also superior to peptide or antigen. These results demonstrate that bivalent HLA-DR. peptide complexes represent effective ligands for activation of the TCR. The data support a role for TCR dimerization in early TCR signaling and kinetic proofreading.     

Monitoring of the formation and dissociation of polyethylenimine/DNA complexes by two photon fluorescence correlation spectroscopy

Polyethylenimines (PEI) constitute efficient nonviral vectors for gene transfer. However, because free PEI shows some cytotoxicity and because intracellular dissociation of PEI/DNA complexes seems to be required for efficient transfection, it is important to monitor the concentrations of free and bound partners in the mixtures of DNA and PEI used for transfection. To reach this objective, we used fluorescence correlation spectroscopy with two-photon excitation to characterize the complexes formed with either rhodamine-labeled 25 kDa PEI or DNA plasmid molecules. At the molar ratios of PEI nitrogen atoms to DNA phosphate usually used for transfection, we found that approximately 86% of the PEI molecules were in a free form. The PEI/DNA complexes are composed on the average by 3.5 (+/-1) DNA plasmids and approximately 30 PEI molecules. From this composition and the pK(a) of PEI, it could be inferred that in contrast to DNA condensation by small multivalent cations, only a limited neutralization of the DNA phosphate groups is required for DNA condensation by PEI. Moreover, DNA appears only poorly compacted in the PEI/DNA complexes. As an application, fluorescence correlation spectroscopy was used to monitor the purification of PEI/DNA complexes by ultrafiltration as well as the heparin-induced dissociation of the complexes.     

Transition of nanostructure in DNA-cationic surfactant complexes with the added salt

Nanostructures of complexes of DNA with single-chain surfactant of octadecyltrimethylammonium (OTA) and double-chain surfactant of didodecyldimethylammonium (DDA) in aqueous NaCl solution at concentration, Cs, from 0 to 500 mM were studied using small-angle-scattering techniques (SAXS). SAXS profiles of the DNA-OTA complex show two SAXS peaks with a spacing ratio of 1:3(1/2) in the solution at Cs below 150 mM and three peaks with a spacing ratio of 1:3(1/2):4(1/2) at Cs above 250 mM. Contents of Na+ and Cl- ions in the complexes evaluated from the atomic absorbance for Na+ and the potentiometry for Cl- revealed charge molar ratios of OTA/DNA = 1 and DDA/DNA = 1.25. Contents of Na+ and Cl- ions per ionic unit of DNA molecule in the DNA-OTA complex equilibrating with the solution at Cs below 100 mM were much less than 0.1, while they increased with NaCl concentration at Cs above 200 mM. The DNA-OTA complex in the solution at Cs above 260 mM exhibited an endothermic peak in the DSC measurements, and the others did not. On the basis of the experimental results, the salt concentration dependent nanostructures are discussed.     

Differences between the complexes formed by monomeric fibrin with fragment D and dimer D

The paper is concerned with studies in formation of monomeric fibrin (fm) complexes with fragment D (D) of fibrinogen and dimer D (DD) of stabilized fibrin. The complexes are shown to be essentially different. The fm-D complexes are unstable, their composition is a function of D concentration in the mixture, the ultimate molar D/fm ratio is equal to 3. The fm-DD complexes are quite stable, their composition is constant: the molar DD/fm ratio is equal to 1. In mixtures containing fm, DD and different amounts of D complexes of different composition are formed but the total number of D-units in them approaches 3. A model is suggested showing interaction of fm molecules in protofibril formation with allowance for the retention of binding centres which provide the lateral link between protofibrils.     

Complexes of smooth muscle tropomyosin with F-actin studied by differential scanning calorimetry.

Differential scanning calorimetry (DSC) and light scattering were used to analyze the interaction of duck gizzard tropomyosin (tropomyosin) with rabbit skeletal-muscle F-actin. In the absence of F-actin, tropomyosin, represented mainly by heterodimers, unfolds at 41 degrees C with a sharp thermal transition. Interaction of tropomyosin heterodimers with F-actin causes a 2-6 degrees C shift in the tropomyosin thermal transition to higher temperature, depending on the tropomyosin/actin molar ratio and protein concentration. A pronounced shift of the tropomyosin thermal transition was observed only for tropomyosin heterodimers, and not for homodimers. The most pronounced effect was observed after complete saturation of F-actin with tropomyosin molecules, at tropomyosin/actin molar ratios > 1 : 7. Under these conditions, two well-separated peaks of tropomyosin were observed on the thermogram besides the peak of F-actin, the peak characteristic of free tropomyosin heterodimer, and the peak with a maximum at 45-47 degrees C corresponding to tropomyosin bound to F-actin. By measuring the temperature-dependence of light scattering, we found that thermal unfolding of tropomyosin is accompanied by its dissociation from F-actin. Thermal unfolding of tropomyosin is almost completely reversible, whereas F-actin denatures irreversibly. The addition of tropomyosin has no effect on thermal unfolding of F-actin, which denatures with a maximum at 64 degrees C in the absence and at 78 degrees C in the presence of a twofold molar excess of phalloidin. After the F-actin-tropomyosin complex had been heated to 90 degrees C and then cooled (i.e. after complete irreversible denaturation of F-actin), only the peak characteristic of free tropomyosin was observed on the thermogram during reheating, whereas the thermal transitions of F-actin and actin-bound tropomyosin completely disappeared. Therefore, the DSC method allows changes in thermal unfolding of tropomyosin resulting from its interaction with F-actin to be probed very precisely.