Biomedical subjects
Publications and source records attributed to U Schummer.
Chloride fluxes across the cytoplasmic membrane of Acholeplasma laidlawii were studied by using the chloride sensitive fluorescent dye, 6-methoxy-N-(sulfopropyl)quinolinium. Chloride was found to penetrate the membrane passively. Chloride flux was dependent upon the transmembrane electric potential.
Anionic charges, cytochemically identified as lipid phosphate groups, cover the outer membrane surface of Spiroplasma floricola. They induce a negative membrane surface potential which affects the distribution of ions, including protons. Accordingly, the pH at the interface differs from the bulk pH. By using the fluorescent lipoid pH indicator 4-heptadecyl-7-hydroxycoumarin, the pH at the membrane surface was determined. From the difference of the bulk and the interfacial pH the membrane surface potential of S. floricola was calculated to be phi = -118 mV.
In Spiroplasma floricola, the transmembrane proton-motive potential delta p was studied. It is composed of a transmembrane electric potential difference, delta psi, and a transmembrane proton gradient, delta pH, according to delta p = delta psi - (Z.delta pH). Using a potential-sensitive carbocyanine dye and 5,5'-dimethyl[2-14C]oxazolidine-2,4-dione as probes, delta psi and delta pH were measured at different [H+] of the medium, and delta p was calculated to be remarkably constant at -123 mV +/- 16% over a wide range of external pH values. Inhibition experiments indicated that it is generated by a membrane-bound, electrogenic, proton-translocating ATPase.
The influence of transmembrane ion fluxes on mycoplasma membrane potentials was studied. Electric membrane potential was calibrated vs fluorescence intensity of a potential-sensitive carbocyanine dye according to delta psi = (RT/F) X log([aIN(1 - IN) - b]/Kint), where IN = I/I0, I0 = maximal fluorescence intensity (obtained for delta psi----infinity), and a and b are constants. Fluorescence intensity was calibrated vs membrane potential by inducing a K+ diffusion potential. The calibration procedure was based on the assumption that in the presence of valinomycin the membrane potential was determined entirely by K+ diffusion. Then the dependence of fluorescence intensity on the external K+ concentration, Kext, could be described by Ival = I0[1 + a/(Kext + b)]-1. For Mycoplasma mycoides subsp. capri and enterococci, the constants were determined from experimental data using nonlinear least-squares computer-assisted methods. The validity of our assumption was proved using the "null-point" method. Here the Ca2+ ionophore A23187 and varying external Ca2+ concentrations were used to change the membrane potential experimentally. K+ and Na+ diffusion potentials significantly contributed to mycoplasma membrane potential whereas Cl- had no influence. Under growth conditions the mycoplasma membrane potential was estimated to be delta psi = -68 mV.
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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.
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.
Surface carbohydrate structures, containing alpha-D-glucosyl or sterically closely related residues, were visualized on mycoplasma membranes by cytochemical staining procedure with concanavalin A and iron-dextran complexes.
An indirect staphylococcal radioimmunoassay (SRIA) has been developed for determination of M. 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 of M. pneumoniae. In sera of patients with M. pneumoniae pneumonia antibodies to the IgG or the IgM class of Igs could be determined separately. This is especially important for an early diagnosis of M. pneumoniae disease.
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A radioimmunoassay (RIA) which depends on the property of protein A of Stapbylococcus aureus to combine with the Fc-fragment of immunoblobulins was developed. This technique was employed to measure antibodies in human and various animal sera. It coulb be demonstrated that the staphylococcal RIA was at least as sensitive as the previously decribed radioimmunoprecipitation technique in detecting antibodies to M.pneumoniae in human sera. In addition, antibodies to M. pneumoniae could be demonstrated in sera of hamsters intranasally inoculated with the organisms. Antibodies could also be demonstrated in rabbit sera after immunization with M.pneumoniae. The test proved to the 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.
Electron spin resonance measurements were performed in order to investigate the influence of anti-cardiolipin antibodies on cardiolipin-containing liposomes. The physical state of the lipid structures and the alterations caused by the interaction with specific antibody were determined by measuring the freedom of motion of spin-labeled stearic acid derivatives incorporated into the lipid structures. The interaction of the cardiolipin-containing liposomes with the anti-cardiolipin antibodies reduced the mobility of the spin-labeled stearic acid probe I (12, 3), whose nitroxide group is assumed to be located near the polar region of the lipid bilayer. The restricted mobility, which qualitatively resembles the interaction of cardiolipin liposomes with calcium ions, is probably the result of a tighter packing of the polar groups in their crystalline array. The binding sites of the cardiolipin structures for anti-cardiolipin antibodies and Ca2 ions seem to be identical. As indicated by the spin-labeled stearic acid probe I (1, 14), the apolar region of the lipid bilayer is not affected by the interaction of the cardiolipin-containing liposomes with the anti-cardiolipin antibodies.
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Explore the source record for details and available documents.
Explore the source record for details and available documents.
The electrochemical proton gradient across mycoplasmal membranes was studied. The transmembrane proton-motive potential, delta p, is composed of two parameters, a transmembrane electric potential difference, delta psi, and a transmembrane proton gradient, delta pH, according to the formula delta p = delta psi -(A x delta pH). Membrane potentials were determined with use of potential-sensitive cyanine dyes. The delta psi for both Mycoplasma mycoides subspecies capri and Mycoplasma gallisepticum was -48 mV +/- 10%, with the inside negative; the delta psi of Acholeplasma laidlawii was -28mV +/- 20%. The delta pH was determined by measuring the distribution of [14C]5,5-dimethyl oxazolidine-2,4-dione between the intracellular space and the medium. The intracellular pH of glycolyzing mycoplasmas was generally more alkaline than the extracellular medium: at an external pH of 7.0, the internal pH was 7.4 and hence delta pH = 0.4, a value corresponding to -24 mV. Thus, the delta p of both M. mycoides subspecies capri and M. gallisepticum was calculated to be -72 mV and that of A. laidlawii, to be -52 mV. The data further indicate that the delta p is generated by a membrane-bound electrogenic, proton-translocating adenosine triphosphatase that operates in the direction of hydrolysis of adenosine triphosphatase, which formed by glycolysis, and leads to proton extrusion.
The influence of transmembrane ion fluxes on mycoplasma membrane potentials was studied. Fluorescence intensity of potential-sensitive carbocyanine dyes was calibrated vs. electric membrane potential. Potassium and sodium ion diffusion potentials significantly contributed to mycoplasma membrane potential. Chloride ions were obviously freely permeable across mycoplasma membranes. Under growth conditions the mycoplasma membrane potential was estimated to be delta psi = - 80 mV.