Biomedical subjects

J Seelig

Publications and source records attributed to J Seelig.

At least 19 recordsLinked to original sources

PubMed1992

Peptide binding to lipid bilayers. Nonclassical hydrophobic effect and membrane-induced pK shifts.

The binding of the cyclic peptide (+)-D-Phe1-Cys2-Phe3-D-Trp4-(+)-Lys5-Thr6- Cys7-Thr(ol)8, a somatostatin analogue (SMS 201-995), and the potential-sensitive dye 2-(p-toluidinyl)naphthalene-6-sulfonate (TNS) to lipid membranes was investigated with high-sensitivity titration calorimetry. The binding enthalpy of the peptide was found to vary dramatically with the vesicle size. For highly curved vesicles with a diameter of d congruent to 30 nm, the binding reaction was enthalpy-driven with delta H congruent to -7.0 +/- 0.3 kcal/mol; for large vesicles with more tightly packed lipids, the binding reaction became endothermic with delta H congruent to +1.0 +/- 0.3 kcal/mol and was entropy-driven. In contrast, the free energy of binding was almost independent of the vesicle size. The thermodynamic analysis suggests that the observed enthalpy-entropy compensation of about 8 kcal/mol can be related to a change in the internal tension of the bilayer and is brought about by an entropy increase of the lipid matrix. The "entropy potential" of the membrane may have its molecular origin in the excitation of the hydrocarbon chains to a more disordered configuration and may play a more important role in membrane partition equilibria than the classical hydrophobic effect. The binding of the peptide to the membrane surface induced a pK shift of the peptide terminal amino group. Neutral membranes were found to destabilize the NH3+ group, leading to a decrease in pK; negatively charged membranes, generated an apparent increase in pK due to the increase in proton concentration near the membrane surface. No pK shifts were seen for TNS. Titration calorimetry combined with the Gouy-Chapman theory can be used to determine both the reaction enthalpy and the binding constant of the membrane-binding equilibrium.

Amino Acid Sequence↗

PubMed1992

Carbon-13 magnetic resonance in biology and medicine.

C-13 magnetic resonance spectroscopy is becoming an important tool for the investigation of metabolic processes either in vitro or in vivo. In this review we discuss some methodological aspects of C-13 magnetic resonance spectroscopy and recent applications of this technique to the study of beta-oxidation of fatty acids in the liver and intermediary metabolism in the brain. Finally, two promising applications in the field of clinical investigation are discussed.

Animals↗

PubMed1992

Influence of stigmastanol and stigmastanyl-phosphorylcholine, two plasma cholesterol lowering substances, on synthetic phospholipid membranes. A 2H- and 31P-NMR study.

Cholesterol, stigmastanol, and stigmastanyl-phosphorylcholine (ST-PC) were incorporated into model membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). POPC and ST-PC were deuterated at the lipid headgroup, DOPC at the cis-double bonds. The influence of the three sterols on the motion and conformation of the lipid headgroups and the hydrocarbon chains was monitored with 2H- and 31P-NMR. All three sterols were freely miscible with the lipid matrix in concentrations of up to 50 mol% without inducing phase separations or nonbilayer structures. However, the molecules exert quite different effects on the phospholipid bilayer. Cholesterol and stigmastanol are largely buried in the hydrocarbon part of the membrane, distinctly restricting the flexing motions of the fatty acyl chains whereas the conformation of the phospholipid headgroups is little affected. In contrast, ST-PC is anchored with its headgroup in the layer of phospholipid dipoles, preventing an extensive penetration of the sterol ring into the hydrocarbon layer. Hence ST-PC has almost no effect on the hydrocarbon chains but induces a characteristic conformational change of the phospholipid headgroups. The 2H- and 31P-NMR spectra of mixed phospholipid/ST-PC membranes further demonstrate that the PC headgroup of ST-PC has a similar orientation as the surrounding phosphatidylcholine headgroups. For both types of molecules the -P-N+ dipole is essentially parallel to the membrane surface. Addition of ST-PC induces a small rotation of the POPC headgroup towards the water phase.

Cholesterol↗

PubMed1992

Relationship between the degree of unsaturation of dietary fatty acids and adipose tissue fatty acids assessed by natural-abundance 13C magnetic resonance spectroscopy in man.

Natural-abundance 13C magnetic resonance spectroscopy was used for determining noninvasively the relative concentration of mono- and polyunsaturated fatty acids of adipose tissue in two groups of volunteers. The first consisted of subjects who had followed a fat-reduced diet for at least half a year before the 13C measurements. The second were control subjects who were on a usual high-fat diet. The ratio of unsaturated to total fatty acids in adipose tissue determined by 13C MRS correlated significantly with the same ratio in fat of the diet composition estimated by a dietician according to food records. The results indicate that 13C MRS is capable of assessing the degree of unsaturation of dietary fatty acids consumed during the preceding months.

Adipose Tissue↗

PubMed1991

Glycogen metabolism as detected by in vivo and in vitro 13C-NMR spectroscopy using [1,2-13C2]glucose as substrate.

The metabolism of glucose to glycogen in the liver of fasted and well-fed rats was investigated with 13C nuclear magnetic resonance spectroscopy using [1,2-(13)C2]glucose as the main substrate. The unique spectroscopic feature of this molecule is the 13C-13C homonuclear coupling leading to characteristic doublets for the C-1 and C-2 resonances of glucose and its breakdown products as long as the two 13C nuclei remain bonded together. The doublet resonances of [1,2-(13)C2]glucose thus provide an ideal marker to follow the fate of this exogenous substrate through the metabolic pathways. [1,2-(13)C2]Glucose was injected intraperitoneally into anesthetized rats and the in vivo 13C-NMR measurements of the intact animals revealed the transformation of the injected glucose into liver glycogen. Glycogen was extracted from the liver and high resolution 13C-NMR spectra were obtained before and after hydrolysis of glycogen. Intact [1,2-13C2]glucose molecules give rise to doublet resonances, natural abundance [13C]glucose molecules produce singlet resonances. From an analysis of the doublet-to-singlet intensities the following conclusions were derived. (i) In fasted rats virtually 100% of the glycosyl units in glycogen were 13C-NMR visible. In contrast, the 13C-NMR visibility of glycogen decreased to 30-40% in well-fed rats. (ii) In fed rats a minimum of 67 +/- 7% of the exogenous [1,2-(13)C2]glucose was incorporated into the liver glycogen via the direct pathway. No contribution of the indirect pathway could be detected. (iii) In fasted rats externally supplied glucose appeared to be consumed in different metabolic processes and less [1,2-(13)C2]glucose was found to be incorporated into glycogen (13 +/- 1%). However, the observation of [5,6-(13)C2]glucose in liver glycogen provided evidence for the operation of the so-called indirect pathway of glycogen synthesis. The activity of the indirect pathway was at least 9% but not more than 30% of the direct pathway. (vi) The pentose phosphate pathway was of little significance for glucose but became detectable upon injection of [1-(13)C]ribose.

Animals↗

PubMed1991

Nonclassical hydrophobic effect in membrane binding equilibria.

The enthalpy of transfer of four different amphiphilic molecules from the aqueous phase to the lipid membrane was determined by titration calorimetry. The four molecules investigated were the potential-sensitive dye 2-(p-toluidinyl)naphthalene-6-sulfonate (TNS), the membrane conductivity inducing anion tetraphenylborate (TPB), the Ca2+ channel blocker amlodipine [Bäuerle, H. D., & Seelig, J. (1991) Biochemistry 30, 7203-7211], and the positively charged local anesthetic dibucaine. All four amphiphiles penetrate into the hydrophobic part of the membrane, and their binding constants, after correcting for electrostatic effects, range between 600 M-1 for dibucaine and 60,000 M-1 for tetraphenylborate. The corresponding changes in free energy were about -6 to -9 kcal/mol. Binding of the amphiphiles to membrane vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine was accompanied by exothermic heats of reaction for all four molecules. For TNS, TPB, and amlodipine, the enthalpies of transfer were almost identical and corresponded to delta H approximately -9 kcal/mol, essentially accounting for the total free energy change. Thus, the binding of these charged amphiphiles to the hydrophobic membrane was driven by enthalpy. This is in contrast to the classical hydrophobic effect, where the transfer is considered to be entropy driven. For dibucaine, the enthalpy of transfer was smaller with delta H approximately -2 kcal/mol but was still about one-third of the total free energy change. All enthalpies of transfer exhibited a distinct temperature dependence with molar heat capacities delta Cp of -30 to -100 cal mol-1K-1 for the transfer from water to the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Amlodipine↗

PubMed1991

Interaction of charged and uncharged calcium channel antagonists with phospholipid membranes. Binding equilibrium, binding enthalpy, and membrane location.

The membrane location and the binding mechanism of two Ca2+ channel antagonists, amlodipine and nimodipine, in pure lipid membranes were investigated with deuterium and phosphorus-31 nuclear magnetic resonance, with thermodynamic methods such as high-sensitivity titration calorimetry, and by measuring the membrane surface charge via the zeta-potential. The two drugs exhibit quite different physical-chemical properties. The noncharged nimodipine is strongly hydrophobic, and selective deuteration of the lipid membrane reveals a homogeneous distribution of nimodipine across the whole hydrocarbon layer, but no interaction at the lipid headgroup level. The membrane behavior of the amiphiphilic amlodipine (electric charge z = +1) is distinctly more complex. Deuterium magnetic resonance demonstrates that amlodipine adopts a well-defined position in the bilayer membrane. In particular, the charged ethanolamine side group of amlodipine is located near the water-lipid interface, interacting with the dipoles of the headgroup region according to a nonspecific, electrostatic mechanism and inducing a reorientation of the phosphocholine dipoles toward the water phase. At the level of the hydrocarbon segment, the nonpolar ring system of amlodipine interacts specifically with the cis double bond of the membrane lipid, forming a weak association complex. With increasing amlodipine concentration the deuterium signal of the cis double bond gradually loses intensity, a phenomenon previously observed only in related studies on protein-lipid interactions. The binding equilibrium of amlodipine to phosphatidylcholine membranes was studied by measuring the electrophoretic mobility of lipid vesicles and with a centrifugation assay. Hydrophobic interactions of the nonpolar ring systems and electrostatic repulsions at the membrane surface contribute to the binding energy.(ABSTRACT TRUNCATED AT 250 WORDS)

Amlodipine↗

PubMed1991

13C NMR for the assessment of human brain glucose metabolism in vivo.

Proton-decoupled 13C NMR spectra of the human head were obtained during hyperglycemic glucose clamping using intravenous infusions of [1-13C]glucose in normal volunteers. In addition to 13C signals of mobile lipids, a variety of new metabolite resonances could be resolved for the first time in the human brain. At an enrichment level of 20% [1-13C]glucose, the signals of alpha- and beta-glucose at 92.7 and 96.6 ppm, respectively, could be detected in the human brain after only an infusion period of 15 min. The spatial localization of the different regions of interest was confirmed by 13C NMR spectroscopic imaging with a time resolution of 9 min. Increasing the enrichment level to 99% [1-13C]glucose not only improved the time resolution but allowed the detection of metabolic breakdown products of [1-13C]glucose. The time course of 13C label incorporation into the C2, C3, and C4 resonances of glutamate/glutamine and into lactate could be recorded in the human brain. These results suggest the possibility of obtaining time-resolved, spatially selective, and chemically specific information on the human body.

Adult↗

PubMed1991

Interaction of electric dipoles with phospholipid head groups. A 2H and 31P NMR study of phloretin and phloretin analogues in phosphatidylcholine membranes.

Phloretin, 4-hydroxyvalerophenone, and 2-hydroxy-omega-phenylpropiophenone are lipophilic dipolar substances that modify ionic conductances of bilayer membranes. The structural changes at the level of the head groups and the hydrocarbon chains as induced by the incorporation of phloretin and its analogues were investigated with deuterium and phosphorus nuclear magnetic resonance. Membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were selectively deuterated at the choline head group and at the hydrocarbon chains, and 2H and 31P NMR spectra were recorded with varying concentrations of dipolar agents. Incorporation of phloretin leaves the bilayer structure intact, induces only a small disordering of the hydrocarbon chains, and has no significant effect on the head-group dynamics. On the other hand, quite distinct structural changes are observed for the phosphocholine head group. While the -P-N+ dipole is oriented approximately parallel to the membrane surface for pure POPC bilayers, addition of phloretin, and to a lesser extent 4-hydroxyvalerophenone and 2-hydroxy-omega-phenylpropiophenone, rotates the N+ end of the -P-N+ dipole closer to the hydrocarbon layer. The resulting normal component of the -P-N+ dipole partly compensates the electric field of the dipolar agents. In addition to this structural change, phloretin also modifies the hydration layer at the lipid-water interface. Much less 2H2O is adsorbed to the membrane surface when the bilayer contains phloretin, 4-hydroxyvalerophenone, or 2-hydroxy-omega-phenylpropiophenone. Moreover, a rather large change in the residual phosphorus chemical shielding anisotropy argues in favor of hydrogen-bond formation between the phosphate segment and the phloretin hydroxyl groups.

Cholesterol↗

PubMed1991

Peptide binding to lipid membranes. Spectroscopic studies on the insertion of a cyclic somatostatin analog into phospholipid bilayers.

The cyclic peptide SMS 201-995 (+)D-Phe1-Cys2-Phe3-D-Trp4-(+)Lys5-Thr6-++ +Cys7-Thr(ol)8 is an analog of somatostatin and binds to lipid membranes by an electrostatic/hydrophobic mechanism. The structural changes accompanying the binding process were investigated with circular dichroism (CD), fluorescence spectroscopy, and phosphorus and deuterium nuclear magnetic resonance. The peptide penetrates into the lipid bilayer and the binding is accompanied by a small change in the CD spectrum suggesting the formation of beta-ordered structures. The fluorescence emission spectrum of the tryptophan side chain exhibits a blue shift and an intensity enhancement of the emission maximum, providing evidence that this residue is located in the inner part of the phospholipid headgroup region with a dielectric constant of epsilon approximately 7. The peptide diffuses rapidly in the plane of the membrane, changing the lipid headgroup conformation. This was demonstrated by selectively deuterating the two choline segments and measuring the deuterium spectra as a function of the bound peptide concentrations. A linear variation of the quadrupole splitting with the mol fraction of bound peptide was observed. The molecular origin of this effect is a distinct change in the orientation of the phosphocholine dipole, moving the N+ end of the dipole away from the membrane surface into the water phase. This type of headgroup rotation appears to be the general response of the zwitterionic phosphocholine headgroup to cationic surface charges. However, peptides appear to be the most efficient modulators of the lipid headgroup structure known to date.

Amino Acid Sequence↗

PubMed1990

Peptide binding to lipid bilayers. Binding isotherms and zeta-potential of a cyclic somatostatin analogue.

The binding of the cyclic somatostatin analogue SMS 201-995, (+)-D-Phe1-Cys2-Phe3-D-Trp4-(+)-Lys5-Thr6- Cys7-Thr(ol)8, to neutral and negatively charged lipids was investigated with a centrifugation assay and with electrophoretic and monolayer methods. Monolayers and bilayers were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), either in pure form or in a 75/25 (mol/mol) mixture. The expansion of monolayer films demonstrated the intercalation of the peptide between the lipid molecules with a surface area requirement of 135 A2 per peptide molecule, indicating a parallel alignment of the peptide long axis with the membrane surface. Above a limiting pressure of 32.5 mN/m for POPC and 38.5 mN/m for POPG, peptide penetration was no longer possible. The peptide binding isotherm could be measured for mixed POPC/POPG bilayers up to a peptide concentration of 0.5 mM. Due to electrostatic attraction, binding between the positively charged peptide and the negatively charged membrane surface was enhanced as compared to the binding to a neutral membrane. After correction for electrostatic effects by means of the Gouy-Chapman theory, the binding isotherm as well as the electrophoretic zeta-potential measurement could be described by the same partition equilibrium with a surface partition constant of Kp = 36 +/- 4 M-1 (at 0.1 M NaCl). About 60-70% of SMS 201-995 is probably embedded in the headgroup region with little penetration into the lipid core. The partition constant increases with increasing salt concentration or with decreasing lipid lateral pressure.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗

PubMed1990

Cerebral metabolism of [1,2-13C2]acetate as detected by in vivo and in vitro 13C NMR.

The metabolism of [1,2-13C2]acetate in rat brain was studied by in vivo and in vitro 13C NMR spectroscopy, in particular by taking advantage of the homonuclear 13C-13C spin coupling patterns. Well nourished rats were infused with [1,2-13C2]acetate or [1-13C]acetate in the jugular vein, and the in situ kinetics of 13C labeling during the infusion period was followed by 13C NMR techniques. The in vivo 13C NMR spectra showed signals from (i) the C-1 carbon of [1,2-13C2] acetate or [1-13C]acetate, (ii) 13CO3H-, and (iii) the natural abundance 13C carbons of sufficiently mobile fatty acids. Methanol/HCl/perchloric acid extracts of the brains were prepared and were further analyzed by high resolution 13C NMR. The homonuclear 13C-13C spin coupling patterns after infusion of [1,2-13C2]acetate showed very different isotopomer populations in glutamate, glutamine, and gamma-aminobutyric acid. Analyzing the relative proportions of these isotopomers revealed (i) two different glutamate compartments in the rat brain characterized by the presence and absence, respectively, of glutamine synthase activity, (ii) two different tricarboxylic acid cycles, one preferentially metabolizing [(1,2-13C2]acetate, the other mainly using unlabeled acetyl-coenzyme A, (iii) a hitherto unknown cerebral pyruvate recycling system associated with the tricarboxylic acid cycle, metabolizing primarily unlabeled acetyl-coenzyme A, and (iv) a predominant production of gamma-aminobutyric acid in the glutamate compartment lacking glutamine synthase.

Acetates↗

PubMed1990

Melittin binding to mixed phosphatidylglycerol/phosphatidylcholine membranes.

The binding of bee venom melittin to negatively charged unilamellar vesicles and planar lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) was studied with circular dichroism and deuterium NMR spectroscopy. The melittin binding isotherm was measured for small unilamellar vesicles containing 10 or 20 mol % POPG. Due to electrostatic attraction, binding of the positively charged melittin was much enhanced as compared to the binding to neutral lipid vesicles. However, after correction for electrostatic effects by means of the Gouy-Chapman theory, all melittin binding isotherms could be described by a partition Kp = (4.5 +/- 0.6) x 10(4) M-1. It was estimated that about 50% of the total melittin surface was embedded in a hydrophobic environment. The melittin partition constant for small unilamellar vesicles was by a factor of 20 larger than that of planar bilayers and attests to the tighter lipid packing in the nonsonicated bilayers. Deuterium NMR studies were performed with coarse lipid dispersions. Binding of melittin to POPC/POPG (80/20 mol/mol) membranes caused systematic changes in the conformation of the phosphocholine and phosphoglycerol head groups which were ascribed to the influence of electrostatic charge on the choline dipole. While the negative charge of phosphatidylglycerol moved the N+ end of the choline -P-N+ dipole toward the bilayer interior, the binding of melittin reversed this effect and rotated the N+ end toward the aqueous phase. No specific melittin-POPG complexes could be detected. The phosphoglycerol head group was less affected by melittin binding than its choline counterpart.

Bee Venoms↗

PubMed1990

Numerical analysis of multislice MR excitation and inversion with multifrequency selective rf pulses.

Multifrequency selective excitation and inversion were recently described and tested for multislice imaging and multivolume selective spectroscopy (Magn. Reson. Med. 6, 364 (1988), J. Magn. Reson. 76, 155 (1988]. The technique is based on assumption that a multifrequency rf pulse, a linear superposition of several selective rf pulses with different frequencies, generates a MR signal which can be separated into the spin responses due to each individual frequency. This assumption is investigated theoretically by analyzing the effect of multifrequency selective rf pulses on the magnetization of a homogeneous phantom as a function of slice separation, pulse shape, and rf amplitude using computer simulations of the Bloch equations. It is found that multifrequency selective excitation with sinc pulses--up to eight slices are investigated--and two-frequency inversion with hyperbolic secant pulses lead to profiles comparable in quality and selectivity to those of conventional single-frequency pulses.

Humans↗

PubMed1990

A homonuclear shift correlated and spatially localized spectroscopy using stimulated echoes.

A new method which combines localized high-resolution proton NMR spectroscopy with two-dimensional correlated spectroscopy using stimulated echoes is presented. Stimulated-echo correlated spectroscopy (STECSY) is a straightforward extension of the STEAM method. Experiments with phantoms illustrate the efficacy of STECSY. An in situ application on rat adipose tissue demonstrates that STECSY is a helpful tool with which to select and assign resonances in complex 1H NMR spectra.

Adipose Tissue↗

PubMed1990

Two-volume acquisition in image-guided proton spectroscopy.

By combining two-frequency excitation with 1H NMR STEAM spectroscopy, it is possible to measure two volumes of interest simultaneously without an increase in measuring time compared to single-volume STEAM. Spatial selectivity and spectral resolution of this approach are demonstrated for test solutions and for human 1H NMR brain spectroscopy. First results with a tumor patient are also presented.

Brain↗

PubMed1990

Analysis of glycogen storage disease by in vivo 13C NMR: comparison of normal volunteers with a patient.

Broadband proton-decoupled natural abundance 13C spectra of the human calf, liver, and head were obtained from normal volunteers and a patient with glycogen type IIIA storage disease. Two concentric and coplanar surface coils of diameters 8.0 cm and 13.0 cm were used for 13C (at 16.0 MHz) and 1H (at 63.6 MHz), respectively. A WALTZ-8 sequence lead to homogeneous decoupling over a large volume. In addition to lipid resonances a variety of other metabolite resonances could be resolved. The glycogen concentration in the muscle and the liver of normal volunteers varied considerably depending on dietary preparation and physical exercise. The glycogen level in the liver and the calf of a patient with glycogen type IIIA storage disease was increased by a factor of 2-3 compared to normal, well-trained volunteers. Proton-decoupled 13C spectra of human head are reported for the first time. The spectra are dominated by lipid resonances but an additional resonance at 54.0 ppm is clearly visible. The proton-decoupled 13C head spectrum of a patient with glycogen type IIIA storage disease revealed additional resonances between 71.0 and 85.0 ppm.

Abdominal Muscles↗

PubMed1990

Interaction of phospholipids with Ca2+ ions. On the role of the phospholipid head groups.

Neutral phospholipids play an important role in Ca2+ binding to biomembranes, in particular if the membrane carries a net negative surface charge due to charged lipids or proteins. The concentration of Ca2+ ions in the plane of the phospholipid head groups can be enhanced by at least two orders of magnitude compared to bulk solution. Ca2+ binding furthermore changes the orientation of the phospholipid head groups which is accompanied by variations of the local membrane dipole potential of the order of 10(5) V/cm. Such high electric fields could entail conformational changes of membrane-bound proteins and the Ca2(+)-induced reorientation of the lipid dipoles could thus play a regulatory role in membrane function.

Calcium↗