ABSTRACT
The monomolecular organization of the main tetraether phospholipid from the archaeon Thermoplasma acidophilum was studied by means of a Langmuir film balance integrated into a fluorescence microscope. After transfer to solid surfaces at different pressures the films were further investigated by ellipsometry, small angle X-ray scattering and atomic force microscopy. In order to complete former results about the main tetraether phospholipid of T. acidophilum [Strobl, C., Six, L., Heckmann, K., Henkel, B., Ring, K., 1985. Z. Naturforsch. 40c, 219-222], the thickness and the two-dimensional organization of the monomolecular films were investigated. Two mean heights values were determined, one of 1.5-1.8 nm and another one of 4-5 nm, indicative for two different molecular arrangements. The former one is interpreted as a 'horseshoe' organization with two polar endings in the aqueous subphase, whereas the latter appears to represent the upright population of molecules with one polar end in the subphase and the other one in the air. In freshly spread and compressed films small domains of the upright lipid population are initially observed, which enlarge with increasing pressure. These domains are no longer existent after 12 h of spreading without compression.
Subject(s)
Phospholipid Ethers/chemistry , Thermoplasma/chemistry , Membrane Lipids/chemistry , Microscopy, Atomic Force , Microscopy, Fluorescence , Molecular Conformation , Pressure , Scattering, Radiation , Surface PropertiesABSTRACT
The thermoacidophilic archaeon Sulfolobus acidocaldarius expresses a very unusual quinol oxidase, which contains four heme a redox centers and one copper atom. The enzyme was solubilized with dodecyl maltoside and purified to homogeneity by a combination of hydrophobic interaction and anion exchange chromatography. The oxidase complex consists of four polypeptide subunits with apparent molecular masses of 64, 39, 27, and 14 kDa that are encoded by the soxABCD operon (Lübben, M., Kolmerer, B., and Saraste, M. (1992) EMBO J. 11, 805-812). The optical spectra and redox potentials of the SoxABCD complex have been characterized, and the absorption coefficients of the contributing cytochromes a587 and aa3 were determined. The EPR spectra indicate the presence of three low spin and one high spin heme species, the latter associated with the binuclear heme CuB site. Standard midpoint potentials of the cytochrome a587 heme centers were determined as +210 and +270 mV, respectively. The maximum turnover of the complex (1300 s-1 at 65 degrees C) was found to be about three times greater than that of the previously studied isolated cytochrome aa3 subunit alone (Gleissner, M., Elferink, M. G., Driessen, A. J., Konings, W. N., Anemüller, S., and Schäfer, G. (1994) Eur. J. Biochem. 224, 983-990). With N,N,N',N'-tetramethyl-1,4-phenylenediamine as a reductant, the SoxABCD complex reconstituted into liposomes generates a proton motive force. A new method is described by co-reconstitution of SoxABCD with a Sulfolobus Rieske FeS-protein (SoxL), allowing energization by cytochrome c. It is based on the finding that this Rieske protein can equilibrate electrons between cytochrome c and quinones reversibly (Schmidt, C. L., Anemüller, S., Teixeira, M., and Schäfer, G. (1995) FEBS Lett. 359, 239-243). With this system, generating no scalar protons, the stoichiometry of proton translocation could be determined. A net H+/e- ratio >1 was determined, identifying the SoxABCD complex as a proton-pumping quinol oxidase. According to structural analysis, the cytochrome aa3 moiety of the complex does not contain the signature of a H+ pumping channel as identified in Rhodobacter sphaeroides or Paracoccus denitrificans. Therefore, for H+ translocation, a mechanism different from that in typical heme-copper oxidases of the aa3 or bo3 type is discussed.
Subject(s)
Cytochrome c Group/metabolism , Flavoproteins/metabolism , Iron-Sulfur Proteins/metabolism , Multienzyme Complexes/metabolism , Oxidoreductases/metabolism , Sulfolobus acidocaldarius/enzymology , Bacterial Outer Membrane Proteins/metabolism , Chromatography, High Pressure Liquid , Circular Dichroism , Detergents , Electron Spin Resonance Spectroscopy , Electrophoresis, Polyacrylamide Gel , Heme , Metalloproteins/metabolism , Potentiometry , Protein Conformation , Spectrophotometry, Atomic , Tetramethylphenylenediamine/metabolismABSTRACT
An in vitro method of measuring the bleeding time is described. The test, called the machine bleeding time (MBT), is not affected by heparin and detects the same types of hemostatic abnormalities as the template bleeding time. It is sensitive to thrombocytopenia, aspirin administration, and von Willebrand's disease. It is a reproducible test suitable for replicate and serial determinations and permits in vitro evaluation of the addition of drugs.
