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We describe here purification and biochemical characterization of the [F.sub.1] [F.sub.o]-ATP synthase from the thermoalkaliphilic organism Bacillus sp. strain TA2.A1. The purified enzyme produced the typical subunit pattern of an [F.sub.1][F.sub.o]-ATP synthase on a sodium dodecyl sulfate-polyacrylamide gel, with [F.sub.1] subunits [alpha], [beta], [gamma], [delta], and [epsilon] and [F.sub.o] subunits a, b, and c. The subunits were identified by N-terminal protein sequencing and mass spectroscopy. A notable feature of the ATP synthase from strain TA2.A1 was its specific blockage in ATP hydrolysis activity. ATPase activity was unmasked by using the detergent lauryldimethylamine oxide (LDAO), which activated ATP hydrolysis > 15-fold. This activation was the same for either the [F.sub.1][F.sub.o] holoenzyme or the isolated [F.sub.1] moiety, and therefore latent ATP hydrolysis activity is an intrinsic property of [F.sub.1] After reconstitution into proteoliposomes, the enzyme catalyzed ATP synthesis driven by an artificially induced transmembrane electrical potential([delta][psi]). A transmembrane proton gradient or sodium ion gradient in the absence of [delta][psi] was not sufficient to drive ATP synthesis. ATP synthesis was eliminated by the electrogenic protonophore carbonyl cyanide m-chlorophenylhydrazone, while the electroneutral N[a.sup.+]/[H.sup.+] antiporter monensin had no effect. Neither ATP synthesis nor ATP hydrolysis was stimulated by N[a.sup.+] ions, suggesting that protons are the coupling ions of the ATP synthase from strain TA2.A1, as documented previously for mesophilic alkaliphilic Bacillus species. The ATP synthase was specifically modified at its c subunits by N,N'-dicyclohexylcarbodiimide, and this modification inhibited ATP synthesis.

Melatonin receptors have been studied for several decades. The low expression of the receptors in tissues led the scientific community to find a substitute for the natural hormone melatonin, the agonist 2-[125I]-iodomelatonin. Using the agonist, several hundreds of studies were conducted, including the discovery of agonists and antagonists for the receptors and minute details about their molecular behavior. Recently, we attempted to expand the panel of radioligands available for studying the melatonin receptors by using the newly discovered compounds SD6, DIV880, and S70254. These compounds were characterized for their affinities to the hMT1 and hMT2 recombinant receptors and their functionality in the classical GTP S system. SD6 is a full agonist, equilibrated between the receptor isoforms, whereas S70254 and DIV880 are only partial MT2 agonists, with Ki in the low nanomolar range while they have no affinity to MT1 receptors. These new tools will hopefully allow for additions to the current body of information on the native localization of the receptor isoforms in tissues. [ABSTRACT FROM AUTHOR]

Synthesis of adenosine triphosphate (ATP) by the F1F0 ATP synthase involves a membrane-embedded rotary engine, the F0 domain, which drives the extra-membranous catalytic F1 domain. The F0 domain consists of subunits a1b2 and a cylindrical rotor assembled from 9-14 α-helical hairpin-shaped c-subunits. According to structural analyses, rotors contain 10 c-subunits in yeast and 14 in chloroplast ATP synthases. We determined the rotor stoichiometry of Ilyobacter tartaricus ATP synthase by atomic force microscopy and cryo-electron microscopy, and show the cylindrical sodium-driven rotor to comprise 11 c-subunits. [ABSTRACT FROM AUTHOR]

Escherichia coli strain PEF42 produces a sodium-ion-dependent hybrid F1F0-ATPase consisting of the Propionigenium modestum subunits a, b, c and δ, of a hybrid α subunit and of the E. coli subunits β, γ and ε The gene encoding subunit c of the P. modestum F1F0-ATPase was cloned into the pT7-7 expression vector to yield plasmid pTTc. E. coli PEF42 was transformed with plasmid pTTc together with plasmid pGP1-2, which harbours the gene for the T7 RNA polymerase. The production of the P. modestum subunit c was induced by a temperature shift from 30 °C to 42 °C for 30 min and led to an increased concentration of this protein in the membrane of the host strain. The c subunit produced in E. coli moved as a monomer in dodecylsulfate electrophoresis. The protein was extracted from the cells with chloroform/methanol, purified and incorporated into sodium dodecylsulfate micelles. Circular dichroism of subunit c in sodium dodecylsulfate showed a temperature-stable spectrum (between 20-60°C) with a high proportion of αhelical structure. Upon incubation of subunit c with [14C]dicyclohexylcarbodiimide the protein became labelled in a sodium-ion-dependent manner, similar to the labelling observed if the purified F1F0-ATPase of P. modestum, was treated with the radioactive carbodiimide. The Na+-specific site was therefore retained in the isolated c subunit dissolved in dodecylsulfate. [ABSTRACT FROM AUTHOR]

Presents a study which solubilized and purified the adenosine triphosphatase (ATPase) of Ilyobacter tartaricus. What was revealed by the sodium doidecyl sulfate-polycrylamide gel electrophories of the purified enzyme; Identification of the polypeptides with different molecular masses; Methods used to obtain two overlapping sequences; Inhibition of proton transport.

We have recently isolated a mutant (aK220R, aV264E, aI278N) of the Na -translocating Escherichia coli/Propionigenium modestum ATPase hybrid with a Na-inhibited growth phenotype on succinate. ATP hydrolysis by the reconstituted mutant ATPase was inhibited by external (N side) NaCl but not by internal (P side) NaCl. In contrast, LiCl activated the ATPase from the N side and inhibited it from the P side. A similar pattern of activation and inhibition was observed with NaCl and the ATPase from the parent strain PEF42. We conclude from these results that the binding sites for the coupling ions on the c subunits are freely accessible from the N side. Upon occupation of these sites, the ATPase becomes more active, provided that the ions can be further translocated to the P side through a channel of the a subunit. If by mutation of the a subunit this channel becomes impermeable for Na , N side Na ions specifically inhibit the ATPase activity. These conclusions were corroborated by the observation that proton transport into proteoliposomes containing the mutant ATPase was abolished by N side but not by P side Na+ ions. In contrast, LiCl affected proton translocation from either side, similar to the sidedness effect of Na+ ions on H+ transport by the parent hybrid ATPase. If the ATPase carrying the mutated a subunit was incubated with 22NaCl and ATP, 1 mol 22Na+/mol enzyme was occluded. With the parent hybrid ATPase, 22Na+ occlusion was not observed. The occluded 22Na+ could be removed from its tight binding site by 20 mM LiCl, while incubation with 20 mM NaCl was without effect. Li+ but not Na+ is therefore apparently able to pass through the mutated a subunit and make the entrapped Na+ ions accessible again to the aqueous environment. These results suggest an ion translocation mechanism through F0 that in the ATP hydrolysis mode involves binding of the coupling ions from the cytoplasm to the multiple c subunits, ATP-driven rotation to bring a Na+, Li+, or H+-loaded c subunit into a contact site with the a subunit and release of the coupling ions through the a subunit channel to the periplasmic surface of the membrane. [ABSTRACT FROM AUTHOR]

We describe here purification and biochemical characterization of the F[sub 1]F[sub o]-ATP synthase from the thermoalkaliphilic organism Bacillus sp. strain TA2.A1. The purified enzyme produced the typical subunit pattern of an F[sub 1]F[sub o]-ATP synthase on a sodium dodecyl sulfate-polyacrylamide gel, with F[sub 1] subunits α, β, γ, δ, and ε and F[sub o] subunits a, b, and c. The subunits were identified by N-terminal protein sequencing and mass spectroscopy. A notable feature of the ATP synthase from strain TA2.A1 was its specific blockage in ATP hydrolysis activity. ATPase activity was unmasked by using the detergent lauryldimethylamine oxide (LDAO), which activated ATP hydrolysis > 15-fold. This activation was the same for either the F[sub 1]F[sub o] holoenzyme or the isolated F[sub 1] moiety, and therefore latent ATP hydrolysis activity is an intrinsic property of F[sub 1]. After reconstitution into proteoliposomes, the enzyme catalyzed ATP synthesis driven by an artificially induced transmembrane electrical potential (Δψ). A transmembrane proton gradient or sodium ion gradient in the absence of Δψ was not sufficient to drive ATP synthesis. ATP synthesis was eliminated by the electrogenic protonophore carbonyl cyanide m-chlorophenylhydrazone, while the electroneutral Na[sup +]/H[sup +] antiporter monensin had no effect. Neither ATP synthesis nor ATP hydrolysis was stimulated by Na[sup +] ions, suggesting that protons are the coupling ions of the ATP synthase from strain TA2.A1, as documented previously for mesophilic alkaliphilic Bacillus species. The ATP synthase was specifically modified at its c subunits by N,N'-dicyclohexylcarbodimide, and this modification inhibited ATP synthesis. [ABSTRACT FROM AUTHOR]

The sodium ion-translocating F1F0 ATP synthase from the bacterium Ilyobacter tartaricus contains a remarkably stable rotor ring composed of 11 c subunits. The rotor ring was isolated, crystallised in two dimensions and analysed by electron cryo-microscopy. Here, we present an α-carbon model of the c-subunit ring. Each monomeric c subunit of 89 amino acid residues folds into a helical hairpin consisting of two membrane-spanning helices and a cytoplasmic loop. The 11 N-terminal helices are closely spaced within an inner ring surrounding a cavity of ∼17 A˚ (1.7 nm). The tight helix packing leaves no space for side-chains and is accounted for by a highly conserved motif of four glycine residues in the inner, N-terminal helix. Each inner helix is connected by a clearly visible loop to an outer C-terminal helix. The outer helix has a kink near the position of the ion-binding site residue Glu65 in the centre of the membrane and another kink near the C terminus. Two helices from the outer ring and one from the inner ring form the ion-binding site in the middle of the membrane and a potential access channel from the binding site to the cytoplasmic surface. Three possible inter-subunit ion-bridges are likely to account for the remarkable temperature stability of I. tartaricus c-rings compared to those of other organisms. [Copyright &y& Elsevier]