Biomedical subjects

J Seelig

Publications and source records attributed to J Seelig.

At least 19 recordsLinked to original sources

PubMed2002

Frontocortical N-acetylaspartate reduction associated with long-term i.v. heroin use.

To examine possible metabolic frontal lobe alterations in i.v. heroin-dependent patients with different histories of concomitant substance use, N-acetylaspartate (NAA), a putative marker of neuronal viability, was measured by (1)H-MRS. Compared with controls, NAA levels in patients were reduced by 7% in gray matter (p = 0.015) but not in white matter. To what extent comorbid conditions or substance use, including alcohol, contributed to these frontocortical metabolic changes remains to be elucidated.

Adult↗

PubMed2001

Detergent-like action of the antibiotic peptide surfactin on lipid membranes.

Surfactin is a bacterial lipopeptide with powerful surfactant-like properties. High-sensitivity isothermal titration calorimetry was used to study the self association and membrane partitioning of surfactin. The critical micellar concentration (CMC), was 7.5 microM, the heat of micellization was endothermic with DeltaH(w-->m)(Su) = +4.0 kcal/mol, and the free energy of micellization DeltaG(O,w-->m)(Su) = -9.3 kcal/mol (25 degrees C; 100 mM NaCl; 10 mM TRIS, 1 mM EDTA; pH 8.5). The specific heat capacity of micellization was deduced from temperature dependence of DeltaH(w-->m)(Su) as DeltaC(w-->m)(P) = -250 +/- 10 cal/(mol.K). The data can be explained by combining the hydrophobicity of the fatty acyl chain with that of the hydrophobic amino acids. The membrane partition equilibrium was studied using small (30 nm) and large (100 nm) unilamellar POPC vesicles. At 25 degrees C, the partition coefficient, K, was (2.2 +/- 0.2) x 10(4) M(-1) for large vesicles leading to a free energy of DeltaG(O, w-->b)(Su) = -8.3 kcal/mol. The partition enthalpy was again endothermic, with DeltaH(w-->b)(Su) = 9 +/- 1 kcal/mol. The strong preference of surfactin for micelle formation over membrane insertion explains the high membrane-destabilizing activity of the peptide. For surfactin and a variety of non-ionic detergents, the surfactant-to-lipid ratio, inducing membrane solubilization, R(sat)(b), can be predicted by the simple relationship R(sat)(b) approximately K. CMC.

Bacterial Proteins↗

PubMed2001

Liposomal formulations of Cyclosporin A: a biophysical approach to pharmacokinetics and pharmacodynamics.

There are about 20 publications about liposomal formulations of Cyclosporin A (CyA) in the pharmaceutical and preclinical literature. Liposomal formulations were developed in order to reduce the nephrotoxicity of CyA and to increase pharmacological effects. However, conflicting results have been published as to the therapeutic properties of these formulations. This is also true for the change in pharmacokinetics and organ distribution of the liposomally encapsulated CyA as compared to conventionally formulated CyA. Using biophysical methods, it could be shown that CyA is not tightly entrapped in liposomal membranes, despite its high lipophilicity. CyA shows retardation only at high lipid concentrations in blood, following a massive injection of liposomes. This effect may diminish nephrotoxicity, as could be demonstrated by in vitro studies using a model tubule system. The results of these studies can be used to predict the formulation behavior in vivo and to optimize liposomal formulations. When applied in an early phase of the drug formulation process, these types of biophysical experiments can also help minimize animal experiments. However, these basic interaction studies cannot cover all physiological, pharmacological, and toxic effects in animals and humans.

Biophysical Phenomena↗

PubMed2000

Interaction of a mitochondrial presequence with lipid membranes: role of helix formation for membrane binding and perturbation.

The binding of a peptide to a biological membrane is often accompanied by a transition from a random coil structure to an amphipathic alpha-helix. Recently, we have presented a new approach which allows the determination of the thermodynamic parameters of membrane-induced helix formation [Wieprecht et al. (1999) J. Mol Biol. 294, 785]. It involves a systematic variation of the helix content of a given peptide by double D-substitution and a correlation of the binding parameters with the helicity. Here we have used this method to study membrane-induced helix formation for the presequence of rat mitochondrial rhodanese (RHD). The thermodynamic parameters of binding of the peptide RHD and of four of its double D-isomers were determined for 30 nm (SUVs) and 100 nm (LUVs) unilamellar vesicles composed of phosphatidylcholine/phosphatidylglycerol (3:1) using circular dichroism spectroscopy, fluorescence spectroscopy, and isothermal titration calorimetry. The incremental changes of the thermodynamic parameters of helix formation were found to be very similar for SUVs and LUVs. Membrane-induced helix formation of RHD entailed a negative enthalpy of Delta H(helix) = -0.5 to -0.6 kcal/mol/residue and was opposed by an entropy of about Delta S(helix) = -1 to -1.4 cal/mol K/residue. The free energy of helix formation, Delta G(helix), was about -0.2 kcal/mol, and helix formation accounted for 50-60% of the total free energy of membrane binding. Dye-release experiments were used to assess the role of helix formation for the membrane perturbation potential of the peptides. While helix formation plays a major role for membrane binding, it appears to have little importance for inducing membrane leakiness.

Animals↗

PubMed2000

Titration calorimetry of surfactant-membrane partitioning and membrane solubilization.

The interaction of surfactants with membranes has been difficult to monitor since most detergents are small organic molecules without spectroscopic markers. The development of high sensitivity isothermal titration calorimetry (ITC) has changed this situation distinctly. The insertion of a detergent into the bilayer membrane is generally accompanied by a consumption or release of heat which can be measured fast and reliably with modern titration calorimeters. It is possible to determine the full set of thermodynamic parameters, i.e., the partitioning enthalpy, the partitioning isotherm, the partition coefficient, the free energy, and the entropy of transfer. The application of ITC to the following problems is described: (i) measurement of the critical micellar concentration (CMC) of pure detergent solutions; (ii) analysis of surfactant-membrane partitioning equilibria, including asymmetric insertion; and (iii) membrane-surfactant phase diagrams. Finally, the thermodynamic parameters derived for non-ionic detergents are discussed and the affinity for micelle formation is compared with membrane incorporation.

Calorimetry↗

PubMed2000

Binding of Nisin Z to bilayer vesicles as determined with isothermal titration calorimetry.

Nisin Z, a 34-residue lantibiotic, is secreted by some lactic acid bacteria and exerts its antibacterial activity against various Gram-positive bacteria by permeabilizing the cell membrane. It is a cationic amphiphilic peptide with several unusual dehydro residues and thioether-bridged lanthionines. Isothermal titration calorimetry was used to provide a quantitative thermodynamic description for nisin Z adsorption to and penetration into negatively charged and neutral lipid bilayers. The binding of the cationic peptide (electric charge z approximately 3.8) to anionic membranes was found to be dominated by electrostatic forces which could be described with the Gouy-Chapman theory. For biologically relevant conditions with a membrane surface potential of -40 mV, the peptide concentration near the membrane surface increases by about 2-3 orders of magnitude compared to the bulk concentration. The binding step proper, i.e., the transition from the lipid-water interface into the membrane, is almost exclusively driven by the high surface concentration. Binding can be described by a partition equilibrium of the form X(b) = KC(M) = KC(p,f) exp(-z(p)psi(0)F(0)/RT), where C(M) is the peptide surface concentration, C(p,f) the bulk concentration, and psi(0) the membrane surface potential. The intrinsic partition coefficient (K = 1.8 M(-)(1)) is remarkably small, indicating a correspondingly small hydrophobic energy contribution to the binding process. The electrostatic model was confirmed with nisin Z mutants in which valine-32 was replaced with either lysine (V32K) or glutamate (V32E), increasing or decreasing the electric charge by 1 unit. The extent of peptide binding increased for V32K and decreased for V32E as predicted by the electrostatic theory. In contrast, electrostatic effects were almost negligible for the binding of nisin Z to neutral membranes. However, the binding isotherms were characterized by a distinctly larger intrinsic binding constant K(0) of approximately 540 M(-)(1) and an enhanced hydrophobic free energy of binding. The binding of nisin Z to sonicated lipid vesicles is exothermic with a DeltaH degrees of ca. -9 and -3.4 kcal/mol for charged and neutral membranes, respectively.

Amino Acid Sequence↗

PubMed2000

Binding of the antibacterial peptide magainin 2 amide to small and large unilamellar vesicles

The thermodynamics of binding of the antibacterial peptide magainin 2 amide (M2a) to negatively charged small (SUVs) and large (LUVs) unilamellar vesicles has been studied with isothermal titration calorimetry (ITC) and CD spectroscopy at 45 degrees C. The binding isotherms as well as the ability of the peptide to permeabilize membranes were found to be qualitatively and quantitatively similar for both model membranes. The binding isotherms could be described with a surface partition equilibrium where the surface concentration of the peptide immediately above the plane of binding was calculated with the Gouy-Chapman theory. The standard free energy of binding was deltaG0 approximately -22 kJ/mol and was almost identical for LUVs and SUVs. However, the standard enthalpy and entropy of binding were distinctly higher for LUVs (deltaH0 = -15.1 kJ/mol, deltaS0 = 24.7 J/molK) than for SUVs (deltaH0 = -38.5 kJ/mol, deltaS0 = -55.3 J/molK). This enthalpy-entropy compensation mechanism is explained by differences in the lipid packing. The cohesive forces between lipid molecules are larger in well-packed LUVs and incorporation of M2a leads to a stronger disruption of cohesive forces and to a larger increase in the lipid flexibility than peptide incorporation into the more disordered SUVs. At 45 degrees C the peptide easily translocates from the outer to the inner monolayer as judged from the simulation of the ITC curves.

Journal Article↗

PubMed2000

Effect of ethanol on BOLD response to acoustic stimulation: implications for neuropharmacological fMRI.

The effects of ethanol on acoustically stimulated blood oxygenation level-dependent (BOLD) signal response in healthy humans was examined with echo planar functional magnetic resonance imaging (fMRI). An acquisition mode minimizing neuronal activation by scanner noise in combination with acoustic excitation by a pulsed 1000-Hz sine tone was used. Paradigms were repeated three times before and after the ingestion of 0.7 g of ethanol/kg(body weight). Linear correlation analyses (r>/=0.40) revealed bilateral BOLD responses in the auditory cortex. Significant voxels covered a cortical volume of approximately 3 ml that was reduced by approximately 40% after ethanol. The BOLD signal change initially reaching approximately 3% was reduced by 12-27%, depending on the definition of the region of interest for signal quantitation. Because ethanol produces vasodilation, the hemodynamic contribution to the BOLD signal change was estimated by modeling the relationship between regional cerebral blood flow (rCBF) and BOLD signal changes. Assuming a baseline flow increase by 10% after ethanol intake, the resulting 'Flow-BOLD-Dependence' (FBD) curve suggested that the ethanol-related BOLD signal reduction was approximately 7-12% greater than the reduction contributed purely by vasodilation. However, simultaneous determination of rCBF and regional cerebral blood volume would be required for an exact quantitation of the neuronally induced BOLD response. Although the FBD model needs empirical validation, its cautious implementation appears to be helpful if fMRI is used in combination with vasoactive drugs.

Acoustic Stimulation↗

PubMed2000

Membrane binding and pore formation of the antibacterial peptide PGLa: thermodynamic and mechanistic aspects.

The antibacterial peptide PGLa exerts its activity by permeabilizing bacterial membranes whereas eukaryotic membranes are not affected. To provide insight into the selectivity and the permeabilization mechanism, the binding of PGLa to neutral and negatively charged model membranes was studied with high-sensitivity isothermal titration calorimetry (ITC), circular dichroism (CD), and solid-state deuterium nuclear magnetic resonance ((2)H NMR). The binding of PGLa to negatively charged phosphatidylcholine (PC)/phosphatidylglycerol (PG) (3:1) vesicles was by a factor of approximately 50 larger than that to neutral PC vesicles. The negatively charged membrane accumulates the cationic peptide at the lipid-water interface, thus facilitating the binding to the membrane. However, if bulk concentrations are replaced by surface concentrations, very similar binding constants are obtained for neutral and charged membranes (K approximately 800-1500 M(-)(1)). Membrane selectivity is thus caused almost exclusively by electrostatic attraction to the membrane surface and not by hydrophobic insertion. Membrane insertion is driven by an exothermic enthalpy (DeltaH approximately -11 to -15 kcal/mol) but opposed by entropy. An important contribution to the binding process is the membrane-induced random coil --> alpha-helix transition of PGLa. The peptide is random coil in solution but adopts an approximately 80% alpha-helical conformation when bound to the membrane. Helix formation is an exothermic process, contributing approximately 70% to the binding enthalpy and approximately 30% to the free energy of binding. The (2)H NMR measurements with selectively deuterated lipids revealed small structural changes in the lipid headgroups and in the hydrocarbon interior upon peptide binding which were continuous over the whole concentration range. In contrast, isothermal titration calorimetry of PGLa solutions with PC/PG(3:1) vesicles gave rise to two processes: (i) an exothermic binding of PGLa to the membrane followed by (ii) a slower endothermic process. The latter is only detected at peptide-to-lipid ratios >17 mmol/mol and is paralleled by the induction of membrane leakiness. Dye efflux measurements are consistent with the critical limit derived from ITC measurements. The endothermic process is assigned to peptide pore formation and/or lipid perturbation. The enthalpy of pore formation is 9.7 kcal/mol of peptide. If the same excess enthalpy is assigned to the lipid phase, the lipid perturbation enthalpy is 180 cal/mol of lipid. The functional synergism between PGLa and magainin 2 amide could also be followed by ITC and dye release experiments and is traced back to an enhanced pore formation activity of a peptide mixture.

Anti-Infective Agents↗

PubMed2000

Dynamic susceptibility contrast MR imaging of plaque development in multiple sclerosis: application of an extended blood-brain barrier leakage correction.

Since the pathogenesis of multiple sclerosis (MS) lesions is not yet fully understood, we investigated the potential of dynamic susceptibility contrast (DSC) magnetic resonance (MR) perfusion imaging for a better characterization of lesion pathology. Twenty-five MS patients were examined on a 1.5 T scanner. A single dose of gadolinium (Gd)-DOTA contrast agent was injected, and echoplanar images were acquired every 0.5 seconds for 1 minute. From the signal intensity-versus-time curves, the relative cerebral blood volume (rCBV) was evaluated for regions in plaques and in gray and white matter. The rCBV calculated for acute, Gd-enhancing plaques was corrected for the effects of blood-brain barrier leakage, using a new correction algorithm. Acute plaques had significantly higher blood volumes than normal-appearing white matter (P < = 0.01). Chronic plaques that appeared hypointense on T(1)-weighted images had lower rCBV than T(1)-isointense plaques (P < = 0.03). Our results indicate that the acute phase in MS is accompanied by vasodilation. In later stages of gliosis, the perfusion decreases with increasing axonal injury. Although the DSC technique is less sensitive than conventional MR imaging, the information provided is essentially different from that obtained with any other MR method.

Adult↗

PubMed2000

Simultaneous in vivo monitoring of hepatic glucose and glucose-6-phosphate by (13)C-NMR spectroscopy.

Hepatic glucose-6-phosphate (G6P) was monitored non-invasively in rat liver by in vivo (13)C NMR spectroscopy after infusion of [1-(13)C] glucose. The phosphorylation of glucose to G6P yields small but characteristic displacements for all of its (13)C-NMR resonances relative to those of glucose. It is demonstrated that in vivo (13)C-NMR spectroscopy at 7 Tesla provides the spectral sensitivity and resolution to detect hepatic G6P present at sub-millimolar concentration as partially resolved low-field shoulders of the glucose C1 resonances at 96.86 ppm (C1beta) and 93. 02 ppm (C1alpha). Upon (13)C-labeling, the intracellular conversion of [1-(13)C] glucose to [1-(13)C] G6P could be monitored, which allowed the hepatic glucose-G6P substrate cycle to be assessed in situ. The close correlation found for the (13)C labeling patterns of glucose and G6P supports the concept of an active substrate cycle whose rate exceeds that of net hepatic glucose metabolism. High-resolution (13)C-NMR spectroscopy and biochemical analyses of tissue biopsies collected at the end of the experiments confirmed qualitatively the findings obtained in vivo.

Animals↗

PubMed2000

Amyloid fibril formation from full-length and fragments of amylin.

Amyloiddeposits of fibrillar human amylin (hA) in the pancreas may be a causative factor in type-2 diabetes. A detailed comparison of in vitro fibril formation by full-length hA(1-37) versus fragments of this peptide-hA(8-37) and hA(20-29)-is presented. Circular dichroism spectroscopy revealed that fibril formation was accompanied by a conformational change: random coil to beta-sheet/alpha-helical structure. Fibril morphologies were visualized by electron microscopy and displayed a remarkable diversity. hA(20-29) formed flat ribbons consisting of numerous 3. 6-nm-wide protofibrils. In contrast, hA(1-37) and hA(8-37) formed polymorphic higher order fibrils by lateral association and/or coiling together of 5.0-nm-wide protofibril subunits. For full-length hA(1-37), the predominant fibril type contained three protofibrils and for hA(8-37), the predominant type contained two protofibrils. Polymerization was also monitored with the thioflavin-T binding assay, which revealed different kinetics of assembly for hA(1-37) and hA(8-37) fibrils. hA(20-29) fibrils did not bind thioflavin-T. Together the results demonstrate that the N-terminal region of the hA peptide influences the relative frequencies of the various higher order fibril types and thereby the overall kinetics of fibril formation. Furthermore, while residues 20-29 contribute to the fibrils' beta-sheet core, the flanking C- and N-terminal regions of the hA peptide determine the interactions involved in the formation of higher order coiled polymorphic superstructures.

Amyloid↗

PubMed2000

Correlation of membrane/water partition coefficients of detergents with the critical micelle concentration.

The membrane/water partition coefficients, K, of 15 electrically neutral (non-charged or zwitterionic) detergents were measured with phospholipid vesicles by using isothermal titration calorimetry, and were compared to the corresponding critical micellar concentrations, cmc. The detergents measured were oligo(ethylene oxide) alkyl ethers (C(m)EO(n) with m = 10/n = 3, 7 and m = 12/n = 3.8); alkylglucosides (octyl, decyl); alkylmaltosides (octyl, decyl, dodecyl); diheptanoylphosphatidylcholine; Tritons (X-100, X-114) and CHAPS. A linear relation between the free energies of partitioning into the membrane and micelle formation was found such that K. CMC approximately 1. The identity K. CMC = 1 was used to classify detergents with respect to their membrane disruption potency. "Strong" detergents are characterized by K. CMC < 1 and solubilize lipid membranes at detergent-to-lipid ratios X(b) < 1 (alkylmaltosides, tritons, heptaethylene glycol alkyl ethers). "Weak" detergents are characterized by K. CMC > 1 and accumulate in the membrane- to detergent-to-lipid ratios X(b) > 1 before the bilayer disintegrates (alkylglucosides, pentaethylene glycol dodecyl ether).

Biophysical Phenomena↗

PubMed2000

Serial proton MR spectroscopy of contrast-enhancing multiple sclerosis plaques: absolute metabolic values over 2 years during a clinical pharmacological study.

BACKGROUND AND PURPOSE: The time courses of total creatine (Cr), N-acetylaspartate (NAA), choline (Cho), and myo-inositol have not previously been investigated in the follow-up of contrast-enhancing multiple sclerosis (MS) plaques. Therefore, over a period of 2 years, we compared the absolute concentrations of these metabolites between patients treated with a placebo or 15 +/- deoxyspergualin (DSG) and between clinical groups with relapsing-remitting or secondary-progressive MS. METHODS: Sixteen patients, recruited from a pharmacological study of DSG, and 11 healthy control subjects were investigated by a stimulated-echo acquisition mode sequence (TR/TE = 3000/20). The selected volume initially contained a contrast-enhancing plaque, which was followed up for a period of 2 years. RESULTS: In contrast-enhancing plaques, Cho was significantly elevated and showed a significant reduction after both 3 and 12 months. The initially normal Cr significantly increased between 3 and 12 months, and was negatively correlated with plaque volume on T1-weighted MR images. NAA initially showed normal values, a significant decrease at 1 month, and a slow recovery over 2 years. Myo-inositol did not show a clear tendency. The placebo group did not differ from the treated group, nor did the relapsing-remitting group differ from the secondary-progressive group. CONCLUSION: The contradictory time courses of Cr and NAA show that an absolute quantification in proton MR spectroscopy in MS is necessary to avoid a false interpretation of reduced NAA/Cr ratios. The increase in Cr is probably due to remyelination. The initial dip and later recovery of NAA seem to be related to diminishing edema and remyelination.

Adult↗

PubMed1999

Thermodynamics of the alpha-helix-coil transition of amphipathic peptides in a membrane environment: implications for the peptide-membrane binding equilibrium.

Amphipathic alpha-helices are the membrane binding motif in many proteins. The corresponding peptides are often random coil in solution but are folded into an alpha-helix upon interaction with the membrane. The energetics of this ubiquitous folding process are still a matter of conjecture. Here, we present a new method to quantitatively analyze the thermodynamics of peptide folding at the membrane interface. We have systematically varied the helix content of a given amphipathic peptide when bound to the membrane and have correlated the thermodynamic binding parameters determined by isothermal titration calorimetry with the alpha-helix content obtained by circular dichroism spectroscopy. The peptides investigated were the antibiotic magainin 2 amide and three analogs in which two adjacent amino acid residues were substituted by their d-enantiomers. The thermodynamic parameters controlling the alpha-helix formation were found to be linearly related to the helicity of the membrane-bound peptides. Helix formation at the membrane surface is characterized by an enthalpy change of DeltaH(helix) approximately -0.7 kcal/mol per residue, an entropy change of DeltaS(helix) approximately -1.9 cal/molK residue and a free energy change of DeltaG(helix)=-0.14 kcal/mol residue. Helix formation is a strong driving force of peptide insertion into the membrane and accounts for about 50 % of the free energy of binding. An increase in temperature entails an unfolding of the membrane-bound helix. The temperature dependence can be described with the Zimm-Bragg theory and the enthalpy of unfolding agrees with that deduced from isothermal titration calorimetry.

Calorimetry↗

PubMed1999

Binding of antibacterial magainin peptides to electrically neutral membranes: thermodynamics and structure.

Magainins are positively charged amphiphatic peptides which permeabilize cell membranes and display antimicrobial activity. They are usually thought to bind specifically to anionic lipids, and binding studies have been performed almost exclusively with negatively charged membranes. Here we demonstrate that binding of magainins to neutral membranes, a reaction which is difficult to assess with spectroscopic means, can be followed with high accuracy using isothermal titration calorimetry. The binding mechanism can be described by a surface partition equilibrium after correcting for electrostatic repulsion by means of the Gouy-Chapman theory. Unusual thermodynamic parameters are observed for the binding process. (i) The three magainin analogues that were investigated bind to neutral membranes with large exothermic reaction enthalpies DeltaH of -15 to -18 kcal/mol (at 30 degrees C). (ii) The reaction enthalpies increase with increasing temperature, leading to a large positive heat capacity DeltaC(p) of approximately 130 cal mol(-)(1) K(-)(1) (at 25 degrees C). (iii) The Gibbs free energies of binding DeltaG are between -6.4 and -8.6 kcal/mol, resulting in a large negative binding entropy DeltaS. The binding of magainin to small unilamellar vesicles is hence an enthalpy-driven reaction. The negative DeltaH and DeltaS and the large positive DeltaC(p) contradict the conventional understanding of the hydrophobic effect. CD experiments reveal that the membrane-bound fraction of magainin is approximately 80% helical at 8 degrees C, decreasing to approximately 60% at 45 degrees C. Since the random coil --> alpha-helix transition in aqueous solution is known to be an exothermic process, the same process occurring at the membrane surface is shown to account for up to 65% of the measured reaction enthalpy. In addition to membrane-facilitated helix formation, the second main driving force for membrane binding is the insertion of the nonpolar amino acid side chains into the lipid bilayer. It also contributes a negative DeltaH and follows the pattern for the nonclassical hydrophobic effect. Addition of cholesterol drastically reduces the extent of peptide binding and reveals an enthalpy-entropy compensation mechanism. Membrane permeability was measured with a dye assay and correlated with the extent of peptide binding. The level of dye efflux is linearly related to the amount of surface-bound peptide and can be traced back to a membrane perturbation effect.

Amino Acid Sequence↗

PubMed1999

Conformation and self-association of human recombinant transforming growth factor-beta3 in aqueous solutions.

The transforming growth factors-beta (TGF-beta) are important regulatory peptides for cell growth and differentiation with therapeutic potential for wound healing. Among the several TGF-beta isoforms TGF-beta3 has a particularly low solubility at physiological pH and easily forms aggregates. A spectroscopic structural analysis of TGF-beta3 in solution has thus been difficult. In this study, circular dichroism spectroscopy was used to determine the secondary structural elements of TGF-beta3. In addition, the aggregation of TGF-beta3 was investigated systematically as a function of pH and salt concentration using a rapid screening method. Sedimentation equilibrium and sedimentation velocity analysis revealed that TGF-beta3 exists predominantly in two major forms: (i) monomers in solution at low pH and (ii) large precipitating aggregates at physiological pH. Under acidic conditions (pH < 3.8) the protein was not aggregated. At pH approximately 3.9, a monomer right arrow over left arrow dimer equilibrium could be detected that transformed into larger aggregates at pH > 4.1. Aggregation was pronounced in the pH range of 4.3 < pH < 9.8 with the aggregation maximum between pH 6.5 and 8. 5. The aggregation process was accompanied by a structural change of the protein. The CD spectra were characterized by an isodichroic point at 209.5 nm indicating a two-state equilibrium between TGF-beta3 dissolved in solution and aggregated TGF-beta3. Aggregated TGF-beta3 showed a higher beta-sheet content and lower beta-turn and random coil contributions compared with monomeric TGF-beta3. Both the solution structure and the aggregate structure of TGF-beta3 were different from the crystal structure. This was in contrast to TGF-beta2, which showed very similar crystal and solution structures. Under alkaline conditions (pH > 9.8) the turbidity disappeared and a further conformational change was induced. The pH dependence of the TGF-beta3 conformation in solution in the range of 2.3 < pH < 11. 0 was reversible. Aggregation of TGF-beta3 was, furthermore, influenced by the presence of salt. For pH > 3.8 the addition of salt greatly enhanced the tendency to aggregate, even in the very basic domain. Under physiological conditions (pH 7.4, cNaCl = 164 mM) TGF-beta3 has almost the highest tendency to aggregate and will remain in solution only at nanomolar concentrations.

Circular Dichroism↗

PubMed1999

New drugs for the Na+/H+ exchanger. Influence of Na+ concentration and determination of inhibition constants with a microphysiometer.

The NHE-1 isoform of the Na+/H+ exchanger is excessively activated in cardiac cells during ischemia. Hence NHE-1 specific inhibitors are being developed since they could be of beneficial influence under conditions of cardiac ischemia and reperfusion. In this study, the Cytosensortrade mark microphysiometer was used to measure the potency of four new drug molecules, i.e., EMD 84021, EMD 94309, EMD 96785 and HOE 642 which are inhibitors of the isoform 1 of the Na+/H+ exchanger. The experiments were performed with Chinese hamster ovary cells (CHO K1) which are enriched in the NHE-1 isoform of the Na+/H+ antiporter. The Na+/H+ exchanger was stimulated with NaCl and the rate of extracellular acidification was quantified with the Cytosensor. The proton exchange rate was measured as a function of the NaCl concentration in the range of 10-138 mm NaCl stimulation. The proton exchange rate followed Michaelis-Menten kinetics with a KM = 30 +/- 4 mm for Na+. Addition of either one of the four inhibitors decreased the acidification rate. The IC50 values of the four compounds could be determined as 23 +/- 7 nm for EMD 84021, 5 +/- 1 nm for EMD 94309, 9 +/- 2 nm for EMD 96785 and 8 +/- 2 nm for HOE 642 at 138 mm NaCl, in good agreement with more elaborate biological assays. The IC50 values increased with the NaCl concentration indicating competitive binding of the inhibitor. The microphysiometer approach is a fast and simple method to measure the activity of the Na+/H+ antiporter and allows a quantitative kinetic analysis of the proton excretion rate.

Animals↗