Edmund Bäuerlein

Identifying subunits of ATP synthase TF0·F1 in contact with phospholipid head groups α-Subunits are labelled selectively by a new photoreactive phospholipid designed for hydrophilic photolabelling

A new phospholipid photolabel was introduced by modifying 1,2‐dipalmitoylphosphatidylethanolamine with 4‐azidosalicylate to 1,2‐dipalmitoyl‐sn‐glycero‐3‐phospho‐N‐(4‐azido‐2‐hydroxybenzoyl)ethanolamine (ASA‐PE), which could be radioiodinated easily to 125 I‐ASA‐PE. The ATP synthase TF0·F1 of the thermophilic bacterium PS3 was reconstituted with soybean phospholipids forming proteoliposomes with high ATP‐32 Pi exchange activity. These proteoliposomes were incubated with 125I‐ASA‐PE to provide its selective incorporation into the outside of the phospholipid bilayer. Upon illumination with ultraviolet light α‐subunits of TF1 were predominantly labelled.

Lipophilic thiourea and thiouracil as inhibitors of oxidative phosphorylation

In various model reactions for oxidative phosphorylation, Pi as well as ADP were activated by the oxidation of thiol groups in waterfree solution [ 11. Sulfenyl phosphates, RSOP03Hz, were supposed to be the reactive intermediates [2]. A quite stable fi-lactoglobulin sulfenyl iodide, RSJ, reacted very rapidly with thiourea and thiouracil, two antithyroid agents, to form the corresponding mixed disulfides [3]. N-Monoalkyl-thioureas (I) and 6-alkyl-2-thiouracils (II) of increasing chain length showed drastic rate enhancement after a rate minimum with R = C4H9 in (I) and with R = CzHs in (II), supporting the idea that the

Subunit β of adenosine triphosphate synthase of Pectinatus frisingensis and Lactobacillus casei

Summary The genes encoding the β-subunit of adenosine triphosphate synthase of two gram-positive bacteria with a low DNA G+C content, Pectinatus frisingensis and Lactobacillus casei, were cloned and sequenced. The predicted amino acid sequences were compared with 29 homologous sequences of (eu)bacteria. Most of the putative functional amino acid residues are conserved in the molecules investigated. A subunit composition resembling that of F1F0 ATPase complexes from respiring bacateria was found in the case of Pectinatus frisingensis. A phylogenetic analysis revealed closer relationship of Lactobacillus casei and bacilli, whereas Pectinatus frisingensis is only moderately related to the former organisms and to mycoplasmas as previously seen by 16S rRNA analysis.

KINETICS OF ATP HYDROLYSIS BY THE F1-ATPASE FROM BACILLUS PS3: A REAPPRAISAL OF THE EFFECTS OF ATP AND MG2+

Abstract ATPase activity of the F 1 -ATPase from the thermophilic Bacillus PS3 (TF 1 ) was measured as a function of ATP concentration at three different magnesium ion concentrations. A high-performance chromatographic method was used to determine directly ADP concentration in the reaction medium and to measure the steady-state rate of its appearance. Multiphasic curves of ATPase activity versus ATP concentration were obtained, with a first saturating rate mode at low ATP concentrations, a higher rate mode which became predominant at ATP concentrations depending on magnesium concentration, and a marked inhibition of ATP hydrolysis at high ATP concentrations. These curves could be simulated with equivalent residual error either by assuming that the ATP-magnesium chelate is the substrate of the enzyme, free magnesium being an inhibitor, or that free ATP is the substrate, free magnesium being an essential activator. In both cases, the observed hydrolysis rate was assumed to be the sum of two independent rates with different kinetic parameters, as would be the case for an enzyme with functionally heterogeneous and independent catalytic sites. Cross-checking of the different series of kinetic parameters with the binding affinity of TF 1 for ATP, measured by a high-performance chromatographic method, is in favour of a model in which the hydrolysis rate is determined by the concentration of free ATP, free magnesium being an essential activator.

The action of lipophilic maleimides in mitochondrial energy transduction

Therefore the above mentioned inhibition can be related to a protonized disulfide, which reacts like an activated thioester of the sulfenic acid (R-S-OH). It may be a functional group for an energy transfer in mitochondria and a mechanistic linkage to a protondriven ATP synthesis [l] . With the formation of a proton-induced sulfenyl group (RS+) a thiol group is liberated (eq. 1). The sulfenyl groups can be specifically and very rapidly trapped by thioureas and thiouracils [3].

Monothiohydrochinonäther als Vermittler einer oxydativen Synthese von Adenosin-di- und-triphosphat (ADP undATP)

Durch Oxydation von S-Methylmonothio-durohydrochinon (1) oder S-Methylmonothio-2,3-dimethyl-1,4-naphthohydrochinon (2) mit Brom in wasserfr. Pyridin entstehen aus den Tetrabutyl-ammoniumsalzen der Phosphorsaure und des Adenosinmonophosphats: Adenosindiphosphat (ADP) und Adenosintriphosphat (ATP), aus Phosphat und Adenosindiphosphat: etwa 25%ATP.

Redox Properties of Iron in the Binding Site(s) of F1ATPase from Mammalian Mitochondria and Thermophilic Bacterium PS3: A Comparative Study

Iron ions in the two iron centers of beef heart mitochondrial F1ATPase, which we have been recently characterized (FEBS Letters 1996, 379, 231-235), exhibit different redox properties. In fact, the ATP-dependent site is able to maintain iron in the redox state of Fe(II) even in the absence of reducing agents, whereas in the nucleotide-independent site iron is oxidized to Fe(III) upon removal of the reductant. Fe(III) ions in the two sites display different reactivity towards H2O2, because only Fe(III) bound in the nucleotide-independent site rapidly reacts with H2O2 thus mediating a 30% enzyme inactivation. Thermophilic bacterium PS3 bears one Fe(III) binding site, which takes up Fe(III) either in the absence or presence of nucleotides and is unable to maintain iron in the redox state of Fe(II) in the absence of ascorbate. Fe(III) bound in thermophilic F1ATPase in a molar ratio 1:1 rapidly reacts with H2O2 mediating a 30% enzyme inactivation. These results support the presence in mitochondrial and thermophilic F1ATPase of a conserved site involved in iron binding and in oxidative inactivation, in which iron exhibits similar redox properties. On the other hand, at variance with thermophilic F1ATPase, the mitochondrial enzyme has the possibility of maintaining one equivalent of Fe(II) in its peculiar ATP-dependent site, besides one equivalent of Fe(III) in the conserved nucleotide-independent site. In this case mitochondrial F1ATPase undergoes a higher inactivation (75%) upon exposure to H2O2. Under all conditions the inactivation is significantly prevented by PBN and DMSO but not by Cu, Zn superoxide dismutase, thus suggesting the formation of OH radicals as mediators of the oxidative damage. No dityrosines, carbonyls or oxidized thiols are formed. In addition, in any cases no protein fragmentation or aggregation is observed upon the treatment with H2O2.

The action of lipophilic alkylamines on beef heart mitochondria: Evidence for the inhibition of proton translocation linked to electron transport

In a workmg hypothesis for the mechanism of electron transport phosphorylatlon it was proposed that in a hpophdic environment a proton-coupled ATP synthesis may involve the interaction of protein disulfide groups [l-3] . To trap the products of an assumed phosphorylytlc cleavage of the dlsulfide fi_mctlon m a hydrophobic environment, namely a thiol and a sulfenyl group (RS> [ 1,2] , lipophilic maleinimides [4] , for the thlol group, and N-monoalkyl-thioureas [S] ,6-alkyl-2-thiouraclls [5] , 1 -alkyl2-ttioimidazoles [ 61 and 1 -alkyl-2-thiobenumldazoles [6] , for the sulfenyl group, of mcreasmg alkyl chamlengths were synthesized. In tightly-coupled beef heart mitochondrla couphng was itiblted by each of the above tiol and sulfenyl reagents if their alkyl chamlengths were at least 9 carbon atoms long and if glutamate t malate were the substrates; these inhibitions could not be released by various uncouplers [2,7-g]. However, when succmate or ascorbate t TMPD was the substrate each of these lipophlllc substances stimulated state 4 to state 3 respiration. Even though the sulfur-free compounds of 6-nonyl2-tiouracll and N-monononyl-tiourea were without effects m the above-described systems, as expected

The isolation of a low molecular weight protein involved in the energy transduction from complex 1 to the ATP synthase

or /I-hydroxybutyrate were the substrates, whereas with succinate State 4 was stimulated to 90% of State 3 respiration [I] . Concomitantly with the inhibition uncoupling by DNP or CCCP was prevented. For the incubation of well-coupled mitochondria with 6.9 nmol NSPM/mg protein for 5 min caused complete inhibition, the same concentration was used for the labelling experiments with [14C]NSPM. After the extraction with ethanol, removal of the lipids and purification on Sephadex LH-20 a water soluble, [ 14C] NSPM-binding protein of low mol. wt (< 13 000) was isolated which is very probably responsible for the specific inhibitors of Site I coupling.

THE SYNTHESIS OF DISODIUM O-n-ALKYLTHIOPHOSPHATES

Abstract The disodium O-alkylthiophosphates were synthesized by reaction of PSCl3 with the corresponding alcohols to the O-alkylphosphorodichloridothioates (1), which were hydrolyzed in aqueous triethylamin–sodium acetate solution.1 The O-alkylthiophosphates (2) were isolated as barium salts and converted to the disodium salts by Na2SO4. Because the barium salts of O-ethyl- and O-n-propyl-thiophosphate were soluble in water, the corresponding dichlorido compounds were hydrolyzed directly to the disodium salts in aqueous sodium hydroxide plus dioxane according to Gray and Hamer.2 Further purification was achieved by column chromatography on Sephadex LH20 using water as an eluant. Pure products, which were not contaminated by either inorganic thiophosphate or O-alkylphosphates, were thus obtained.