Ulrich Schummer

The influence of calcium on the molecular mobility of fatty acid spin labels in phosphatidylserine and phosphatidylinositol structures.

Abstract The temprature-dependent fluidity of naturally occurirng phosphatidyl-serine and phosphatidylinositol in lamellar structures and in their Ca2+ complexes was investigated within the temperature range from 2 to 60 °C using the stearic acid spin label I(12.3) and I(1.14). Both phospholipids show neither in the presence nor in the absence of Ca2+ thermotropic phase changes. A considerable difference, however, was observed in the temperature-dependent fluidity of the two lipids in the polar and apolar region of the lamellar structure reflected by label I(12.3) and I(1.14).

The effect of calcium on temperature-induced phase changes in liquid-crystalline cardiolipin structure.

Abstract The temperature-dependent fluidity of lamellar and Ca 2+ -precipitated cardiolipin structures was investigated over the temperature range 5–55 °C, using the stearic acid spin labels I(12.3), I(5.10), and I(1.14). In the lamellar phase the I(12.3) label reflects an abrupt thermotropic change of the membrane fluidity at 37 °C. The I(5.10) and I(1.14) labels show two points of phase changes located at 14, 36 °C and 10, 38 °C, respectively. The Ca 2+ -complexed cardiolipin structures provoke a retraction of the hydrocarbon chains, preferentially in the polar region, and at the same time a loss of the phase transitions.

The interaction of a lytic peptide, melittin, with spin-labeled membranes.

Abstract The effect of the amphophilic cationic peptide, melittin, on the biophysical state of erythrocytes, their ghosts, and spherules formed from total erythrocyte lipids was studied using the stearic acid spin labels J(1.14), J(5.10), and J(12.3) incorporated into these membranes. Melittin effects a dose-dependent increase of orientation of the label J(5.10) in the case of ghosts and erythrocyte lipid membranes. The influence on the labels J(1.14) and J(12.3), however, is comparatively small. A distinct dose-dependent difference was found if the labels were incorporated into intact red cells. There is an increase of the molecular motion, particularly of the label J(5.10), in the presence of low melittin concentrations and a decrease at high concentrations. The results suggest that in erythrocyte ghosts and total lipids melittin penetrates into the membrane and its action occurs mainly by retracting the apolar region of the membrane. The effect on intact cells involves an increasing fluidity of the apolar fatty acid chains at low, and a decrease at high melittin concentrations. The influence of the surface active peptide melittin on the physical state of the membranes investigated is not comparable with that of a simple detergent like Triton X-100.

Mycoplasma membrane potentials determined by potential-sensitive fluorescent dyes

The membrane potentials of mycoplasmas were investigated by using potential-sensitive cyanine dyes. The fluorescence response results from a potential-dependent partition of the dyes between the cells and the extracellular medium. Cell hyperpolarization (inside more negative), e.g., by the addition of valinomycin, results in uptake of the dyes into the cells and, by formation of dye aggregates, in quenching of the fluorescence intensity. The magnitude of the fluorescence change upon addition of valinomycin depended on the external K+ concentration. At a defined external K+ concentration, no change in fluorescence occurred. The intracellular K+ concentration was determined by atomic absorption spectroscopy. Mycoplasma membrane potentials were calculated according to the Nernst equation. The membrane potential of bothMycoplasma mycoides subsp.capri andMycoplasma gallisepticum was −48 mV±10%; the membrane potential ofAcholeplasma laidlawii was −28 mV±20%.

Determination of IgG, IgM, and IgA antibodies toMycoplasma pneumoniae by an indirect staphylococcal radioimmunoassay

An indirect staphylococcal radioimmunoassay (SRIA) has been developed for determination ofM. pneumoniae antibodies. This test allows the detection of antibodies in various immunoglobulin (Ig) classes similar to the previously described radioimmunoprecipitation test (RIP). SRIA has two advantages over RIP: first, it uses 100-fold less anti-Ig reagents than RIP; second, bound can be separated from unbound antigen more easily by the relatively heavy staphylococci. SRIA antibodies, belonging to the IgA class of Ig, could be detected in nasal secretions of volunteers infected intranasally with ts H43 ofM. pneumoniae. In sera of patients withM. pneumoniae pneumonia antibodies to the IgG or the IgM class of Igs could be determined separately. This is especially important for an early diagnosis ofM. pneumoniae disease.

A novel method for the determination of electrical potentials across cellular membranes. II. Membrane potentials of acholeplasmas, mycoplasmas, streptococci and erythrocytes☆

The membrane potentials of Acholeplasma laidlawii, Mycoplasma mycoides subsp. capri, Mycoplasma gallisepticum, Streptococcus faecalis and human erythrocytes have been determined by applying a novel technique. The membrane potentials were calculated simply from potassium concentrations determined by atomic absorption spectroscopy, and gravimetry. The versatility of the new technique is demonstrated by comparing our results with data obtained by different techniques.

A staphylococcal radioimmunoassay for detection of antibodies toMycoplasma pneumoniae

A radioimmunoassay (RIA) which depends on the property of protein A ofStaphylococcus aureus to combine with the Fc-fragment of immunoglobulins was developed.This technique was employed to measure antibodies in human and various animal sera. It could be demonstrated that the staphylococcal RIA was at least as sensitive as the previously described radioimmunoprecipitation technique in detecting antibodies toM.pneumoniae in human sera. In addition, antibodies toM.pneumoniae could be demonstrated in sera of hamsters intranasally inoculated with the organisms. Antibodies could also be demonstrated in rabbit sera after immunization withM.pneumoniae. The test proved to be considerably more sensitive than conventional tests for detection of antibodies to the organisms. The test requires only small amounts of reagents and is relatively inexpensive.

A novel method for the determination of electrical potentials across cellular membranes. I. Theoretical considerations and mathematical approach

A new method for the determination of electrical potentials across cellular membranes has been developed. In order to determine the membrane potential, cells were incubated in buffer solutions with increasing concentrations of KCl. Parallel experiments were performed with buffer solutions which additonally contained valinomycin. After sedimentation of the cells, the membrane potential was calculated from data which were obtained by simply measuring the wet mass, the dry mass and the potassium content of cell pellets by atomic absorption spectroscopy.

Investigations of thermotropic phase changes in peripheral nerve of frog and rat. A spin label study.

The temperature-dependent fluidity of myelin of frog and rat peripheral nerve (Nervus ischiadicus) was studied using the spin label technique. In frog nerve a phase change was detected at 38 degree C. In rat nerve no sharp phase change could be established, and the lipid-depleted frog and rat nerve also showed no transition. From the spectral data, it was concluded that in frog and rat nerve the lipid-protein interactions are different, i.e. species dependent. Ca-2+-depletion of frog nerve caused a loss of transition, while rat nerve remained unaffected. Thus it was indicated that, in frog nerve, Ca-2+ is involved in the phase change. In the total lipid extract of frog nerve a phase change centered at 32 degree C occurred, while the total lipid extract of rat nerve again showed no transition. It is suggested that a connexion exists between our results and investigations on the temperature dependence of an axonal conduction block of nerve.

Effect of cell membrane-active antimicrobial agents on membrane potential and viability of mycoplasmas

The membrane potential ofMycoplasma mycoides subsp.capri has been determined to beEM=−48 mV±10%, inside negative. In this study we investigated the influence of cell membrane-active antimicrobial agents, viz., valinomycin, gramicidin, polymyxin, and clotrimazole, on membrane potential and viability ofM. mycoides subsp.capri. Valinomycin, an ionophore with extreme potassium selectivity, induced a membrane hyperpolarization,EM=−110 mV. Valinomycin was not cidal, but static to mycoplasmas. Obviously the potassium drain induced by valinomycin can be compensated for by the organisms. Gramicidin is an antibiotic forming cation conduction channels across membranes. It induced a rapid depolarization,EM=+23 mV, of mycoplasma membranes. At low concentrations, gramicidin had a static effect, whereas at high concentrations it was cidal to mycoplasmas. The rapid permeation of cations through the stationary ion channels formed by gramicidin obviously exerts an inhibitory or even lethal effect on mycoplasma metabolism and growth. Polymyxin B induced a depolarization,EM=−35 mV, of mycoplasma membranes only when the organisms had been pretreated and hyperpolarized with valinomycin. After treatment with both valinomycin and polymyxin B, a slight inhibition of mycoplasma growth was observed. Clotrimazole, a synthetic imidazole antimycotic, hyperpolarized mycoplasma membranes (EM=−80 mV). At high concentrations clotrimazole was cidal, whereas at low concentrations it was static to mycoplasmas.