Gebhard von Jagow

Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa

A discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) system for the separation of proteins in the range from 1 to 100 kDa is described. Tricine, used as the trailing ion, allows a resolution of small proteins at lower acrylamide concentrations than in glycine-SDS-PAGE systems. A superior resolution of proteins, especially in the range between 5 and 20 kDa, is achieved without the necessity to use urea. Proteins above 30 kDa are already destacked within the sample gel. Thus a smooth passage of these proteins from sample to separating gel is warranted and overloading effects are reduced. This is of special importance when large amounts of protein are to be loaded onto preparative gels. The omission of glycine and urea prevents disturbances which might occur in the course of subsequent amino acid sequencing.

Ubiquinol-cytochrome c reductase (EC 1.10.2.2). Isolation in Triton X-100 by hydroxyapatite and gel chromatography. Structural and functional properties

1. A mehod for the isolation of a monodisperse ubiquinol-cytochrome c reductase (complex III) from beef heart mitochondria has been developed. The procedure consists of an enzyme solubilization in Triton X-100 followed by hydroxyapatite and gel chromatography. 2. The minimum unit of the isolated complex is composed of 9 polypeptide subunits with Mr of 49000, 47000, 30000, 25000, 12000, 11000 and 6000. It contains 8 mumol of cytochrome b, 4 mumol of cytochrome c1, 7-8 mumol of nonhemne iron, corresponding to 3.5-4 mumol of the Rieske iron-sulfur protein, less than 1.0 mumol of ubiquinone and about 60 mumol of phospholipids, per g of protein. The specific detergent binding amounts to 0.2g of Triton X-100 per g protein. 3. Cytochrome b exhibits an alpha-absorbance maximum at 562 nm. In redox titrations it reveals two half-reduction potentials, i.e. -10 and + 100 mV, at pH 7.0. The absorbance maximum of cytochrome c1 lies at 553 nm and its half-reduction potential amounts to +250 mV. 4. The reductase reveals electron-transferring activity with ubiquinol-1, -2, -3, and -9 as donor and cytochrome c as acceptor. The activity with ubiquinol-9 was analyzed according to the surface dilution scheme developed for the action of phospholipases. The molecular activity amounts to 75 mol of cytochrome c reduced per s at 20%C. 5. A dissociation constant Ks of 5.5 mM has been determined for the Tritonsolubilized enzyme: ubiquinol-containing micelle association. In this case the total concentration of ubiquinol plus Triton X-100 has been substituted for the concentration of binding areas on the ubiquinol-containing micelles. This substitution makes the reasonable assumption that the sum of ubiquinol concentration plus Triton X-100 is proportional to the number of available binding areas. 6. A Km value of 0.025 was found for ubiquinol-9. This is an analog to the Michaelis constant and is expressed as mol fraction of ubiquinol in the ubiquinol-Triton micelle.

Zum Prinzip der Photophosphorylierung und oxidativen Phosphorylierung

The energy transformation systems of Halobacterium halobium, Vibrio succinogenes, chloroplasts and mitochondria are described on the basis of the chemiosmotic theory. Nature seems to utilize three quite different mechanisms for electrogenic proton transport. Structure and function of the ATP-synthetase complexes, however, seem to be quite similar in the various energy-conserving systems.

STRUCTURAL AND FUNCTIONAL FEATURES OF THE ELEVEN CONSTITUENT PROTEINS OF THE MAMMALIAN UBIQUINOL: CYTOCHROME c REDUCTASE

SUMMARY Beef heart ubiquinol:cytochrome c reductase (the reductase) consists of 11 subunits, three catalytic and eight non-catalytic ones. The primary structures of all subunits except the two core proteins have been determined. The three catalytic subunits contain the four redox centres, i.e., three hemes and one iron-su1fur cluster, while the function of the remaining eight non-catalytic subunits has not been clearly established so far. The folding patterns derived from the amino acid sequence data indicate that all six small subunits are integral membrane proteins or anchored in the membrane. The reductase seems to be a multi-subunit complex held together predominantly by ionic bonds. The core of the reductase is a four a-helical antiparallel bundle formed by the transmembrane helices II-V of cytochrome b, incorporating the two b-heme cent res in a transmembrane arrangement.