Karlheinz Altendorf

Identification of the DCCD-reactive protein of the energy transducing adenosinetriphosphatase complex from Escherichia coli

Convincing evidence exists that the membranebound adenosinetriphosphatase (EC 3.6.1.3.) from Escherichia coli is involved in energy transformation. The energy transducing unit, ATPase complex, consists of two components: One is the ATPase (BF,) itself which can easily be stripped off the membrane; the other component (BF,) is tightly bound to the cytoplasmic membrane. This ATPase complex catalyzes ATP synthesis proper during oxidative phosphorylation and also makes available cytoplasmic ATP for many work functions, including active transport of various nutrients. Consequently, under anaerobic conditions these work functions are strongly inhibited by dicyclohexylcarbodiimide (DCCD) and are deficient in mutants that have a defective ATPase complex (reviews: refs. [ l--.5] ). The observation that DCCD inhibits only the membrane-bound ATPase and not the solubilized one lent support to the notion that the inhibitor exerts its effect on the BF,, component of the ATPase complex [61. There is considerable evidence that the hydrolysis of ATP by the bacterial ATPase complex is coupled to the translocation of protons [7,8]. If that complex itself directly translocates the protons across the cytoplasmic membrane, the BFo component, or at least part of it, should meet the requirement of a channel. Evidence for such a channel, specific for

beta-D-Galactoside transport in Escherichia coli. Reversible inhibition by Aprotic Solvents and its Reconstitution in transport-negative membrane vesicles.

At relatively low concentrations (less than 3M) the aprotic solvents: dimethylsulfoxide, N-methylpyrrolidone, tetramethylurea and hexamethylphosphoric triamide, inhibited beta-D-galactoside transport by whole cells, and the derived membrane vesicles of Escherichia coli. Inhibition was not due to gross leakiness of the membrane and could be largely reversed by a simple washing procedure...

The ATP synthase (F1F0) of Streptomyces lividans: sequencing of the atp operon and phylogenetic considerations with subunit beta

The DNA encoding the subunits of the ATP synthase (F1F0) of Streptomyces lividans 66 strain 1326 was identified using oligodeoxyribonucleotide probes derived from the N-terminal sequence of subunit gamma of the F1 complex. The complete nucleotide sequence of the operon was determined. The atp operon contains nine genes, atpIBEFHAGDC, encoding the eight structural components of the ATP synthase complex and the i protein, a polypeptide of unknown function. The gene order found is identical to that in other non-photosynthetic eubacteria. The determination of the N-terminal amino acid (aa) sequences of the F1 subunits alpha, beta, gamma, delta and epsilon allowed us to identify the translational start points and to define the primary structures of the proteins. The aa sequence deduced for subunit delta revealed an N-terminal extension of about 90 aa, which is not present in any delta subunit or OSCP (oligomycin sensitivity-conferral protein) of other species studied so far. The phylogenetic relationship of eu- and archaebacteria was investigated using sequencing data of the highly conserved beta subunit of different ATP synthases including that of S. lividans. The calculations revealed that S. lividans beta does not form a phylogenetic group together with the Gram+ taxa of low G+C contents, but is more closely related to the beta subunit of Rhodobacteria.

The proton-translocating portion (F0) of the E. coli ATP synthase

Abstract The F 0 part of the ATP synthase complex serves as a proton channel. Recent genetical and biochemical approaches are shedding light on the structure of F 0 and the role of its individual subunits.

[57] Proton-conducting portion (F0) from Escherichia coli ATP synthase: Preparation, dissociation into subunits, and reconstitution of an active complex

Publisher Summary This chapter discusses the preparation, dissociation into subunits, and reconstitution of an active complex for the proton-conducting portion (F 0 ) from Escherichia coli ATP synthase. The F 0 part of the ATP synthase complex (F 1 F 0 ) of E. coli is composed of three different polypeptides and the complex serves as a proton channel. After reconstitution into liposomes a functional F 0 complex can be tested either by measuring passive proton translocation or by adding back F 1 . Passive proton translocation through F 0 is measured using K+-loaded liposomes. ATPase activity of reconstituted F 1 F 0 complexes is measured by determining the liberated orthophosphate. The chapter outlines the procedure for preparation of F 0 by hydrophobic interaction chromatography. Reconstitution of a functional F 0 complex is only achieved by incorporation of all three kinds of subunits into phospholipid vesicles.

Essential Role of Arginine Residues in the Interaction of F0 with F1 in Escherichia Coli ATP Synthase

In a wide variety of organisms the ATP synthase (F1F0; EC 3.6.1.34) plays a key role in energy metabolism. This enzyme has been described for mitochondria, chloroplasts and bacteria (for reviews, see refs. 1–3). Among these the ATP synthase complex of Escherichia coli has been most extensively characterized by both biochemical and genetic studies. As in all other ATP synthase systems, the E. coli complex is composed of two entities: the F1 sector is a peripheral protein and catalyzes the synthesis of ATP, whereas the F0 part is embedded in the cytoplasmic membrane and serves as a proton translocator. The ATP synthase works in a reversible manner, which means that it is also capable of building up an electrochemical proton gradient driven by ATP hydrolysis.