Evidence against a MgATP-dependent proton pump in rat-liver lysosomes.

1. The effect of MgATP has been studied on the accumulation of the lipid-soluble anion thiocyanate, the accumulation of the lipid-soluble base methylamine, and the fluorescence of bound anilinonaphthalene sulphonate in rat-liver lysosomes. The lysosomes used were isolated from the livers of rats pretreated with Triton WR 1339. 2. The accumulation of thiocyanate is stimulated by the addition of valinomycin in the presence of K+ but not by the addition of MgATP. 3. The fluorescence of anilinonaphthalene sulphonate bound to lysosomes is enhanced by valinomycin in the presence of K+, the extent of the enhancement being dependent on the concentration of K+. In contrast, MgATP has no effect on the fluorescence. 4. The intralysosomal pH, as estimated from the distribution of methylamine, is not affected by the addition of MgATP in media with or without K+, Na+ or HCO3-. 5. These data strongly suggest that there is no MgATP-dependent proton pump in rat-liver lysosomes.     

Nitrate,fumarate, and oxygen as electron acceptors for a late step in microbial heme synthesis

Nitrate can serve as anaerobic electron acceptor for the oxidation of protoporphyrinogen to protoporphyrin in cell-free extracts of Escherichia coli grown anaerobically in the presence of nitrate. Two kinds of experiments indicated this: anaerobic protoporphyrin formation from protoporphyrinogen, followed spectrophotometrically, was markedly stimulated by addition of nitrate; and anaerobic protoheme formation from protoporphyrinogen, determined by extraction procedures, was markedly stimulated by addition of nitrate. In contrast, anaerobic protoheme formation from protoporphyrin was not dependent upon addition of nitrate. This was the first demonstration of the ability of nitrate to serve as electron acceptor for this late step of heme synthesis. Previous studies with mammalian and yeast mitochondria had indicated an obligatory requirement for molecular oxygen at this step.In confirmation of our previous preliminary report, fumarate was also shown to be an electron acceptor for anaerobic protoporphyrinogen oxidation in extracts of E. coli grown anaerobically on fumarate. For the first time, anaerobic protoheme formation from protoporphyrinogen, but not from protoporphyrin, was shown to be dependent upon the addition of fumarate.The importance of these findings is 2-fold. First, they establish that enzymatic protoporphyrinogen oxidation can occur in the absence of molecular oxygen, in contrast to previous observations using mammalian and yeast mitochondria. Secondly, these findings help explain the ability of some facultative and anaerobic bacteria to form very large amounts of heme compounds, such as cytochrome pigments, when grown anaerobically in the presence of nitrate or fumarate. In fact, denitrifying bacteria are known to form more cytochromes when grown anaerobically than during aerobic growth.An unexpected finding was that extracts of another bacterium, Staphylococcus epidermidis, exhibited very little ability to oxidize protoporphyrinogen to protoporphyrin as compared to E. coli extracts. This finding suggests some fundamental differences in these two organisms in this key step in heme synthesis. It is known that these two facultative organisms also differ in that E. coli synthesizes cytochrome during both aerobic and anaerobic growth, while Staphylococcus only synthesizes cytochromes when grown aerobically.     

Inhibition of N-ethylmaleimide of the MgATP-driven proton pump of the chromaffin granules

The thiol reagent N-ethylmaleimide (NEM) completely inhibits the proton pump activity of the H+-ATPase in chromaffin granule 'ghosts' at concentrations which only partly (approximately 20%) inhibit the Mg2+-dependent ATP hydrolysis. Half-maximal inhibition was obtained at approximately 13 microM NEM as compared to 18 microM for the classical proton channel inhibitor N,N'-dicyclohexylcarbodiimide (DCCD), and the apparent stoichiometry of the inhibitors at complete inhibition was NEM : DCCD congruent to 1 : 2. HIgh concentrations of NEM (greater than 100 microM) induce a dissipation of the transmembrane potential generated by MgATP. These findings establish NEM as a valuable proton channel inhibitor in chromaffin granules and explain the rather complex effect of NEM previously reported for catecholamine accumulation in this organelle.     

The acidification of rat liver lysosomes in vitro: a role for the membranous ATPase as a proton pump.

Lysosomes were purified from the livers of rats which had been treated with Triton WR-1339. The ATPase activity of these lysosomes was stimulated by preincubation with NaCl or KCl, conditions which diminish the proton gradient due to Donnan equilibrium. Subsequent to this preincubation measurements of methylamine uptake by lysosomes showed an ATP-dependent enhancement. Simultaneous measurements of the internal volumes of lysosomes confirmed that ATP-dependent methylamine uptake is due to acidification of lysosomes by 0.3 to 0.5 pH units. Because the conditions which stimulated ATP-dependent methylamine uptake also stimulated the ATPase activity it is concluded that acidification of lysosomes requires an ATPase which functions as a proton pump.     

Energy coupling for membrane hyperpolarization in Lemna: Evidence against an ATP-fueled electrogenic pump as the exclusive mechanism

The vacuolar electrical potential of Lemna paucicostata 6746 has an active component of about-130 mV. This hyperpolarization above the diffusion potential was maintained when dicyclohexyl carbodiimide (DCCD) or arsenate (0.1 mM or 5 mM final concentrations, respectively) were added in the light or after the plants had been kept in darkness for 1 h. The ATP level was reduced to 11±3% by DCCD and to 56±6% by arsenate under conditions identical to those during the potential measurements. In this report, it is discussed whether these results could be interpreted in terms of a putative electrogenic ATPase in the plasma membrane of Lemna. Rb⁺-influx in illuminated plants was 12.5% or 52% of the control when ATP generation was inhibited by DCCD or arsenate. This finding is regarded as justifying the assumption that the availability of ATP at plasmalemma-located transport sites is drastically decreased by these inhibitors.A passive proton-permeability in the cell membrane was induced with different concentrations of carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The potential decrease, caused by the current through this shunt, was not affected by DCCD. It therefore seems less conceivable that the cell membrane remains hyperpolarized because of an increase of membrane resistance concomitant to the inhibition of the pump.The significance of respiratory processes for membrane hyperpolarization is displayed by the depolarizing action of anoxia or KCN. As ATP was found to be non-limiting under these conditions, the inhibition of the electrogenic pump is regarded as being in discord with the concept of an electrogenic ATPase, which is solely responsible for membrane hyperpolarization.Abbreviations CCCP carbonyl cyanide m-chlorophenyl hydrazone - DCCD N, N-dicyclohexyl carbodiimide - DES diethylstilbestro - DNP 2,4-dinitrophenol - POPOP 1,4-bis (2-(5-phenyloxazolyl))-benzene - PPO 2,5-diphenyloxazole     

Elimination of proton donor strongly affects directionality and efficiency of proton transport in ESR,a light-driven proton pump from Exiguobacterium sibiricum

ESR from Exiguobacterium sibiricum is a retinal protein which functions as a proton pump. Unusual feature of ESR is that a lysine residue is present at a site for the internal proton donor, which in other proton pumps is a carboxylic residue. Replacement of Lys96 with alanine slows reprotonation of the Schiff base by two orders of magnitude, indicating that Lys96 and interacting water molecules function as internal proton donor to the Schiff base. In this work we examined time resolved generation of light-induced electric potential ΔΨ by the K96A mutant reconstituted into proteoliposomes. We found that the ΔΨ component, which accompanied reprotonation of the Schiff base in wild type ESR, was not only slowed but also decreased greatly in the mutant, and negative phase appeared at high pH. This indicates a higher probability of back reactions in ESR than in bacteriorhodopsin since no negative components have been observed in homologous mutants of BR, D96N and D96A. The higher rate of back reactions in ESR is probably caused by different arrangement of the proton acceptor site compared to that in BR and different sequence of proton release and uptake. Addition of sodium azide, which substitutes for the internal proton donor, restores both the rate and amplitude of the ΔΨ components related to the Schiff base reprotonation in the K96A mutant. This indicates that overall proton transport results from competition of forward and reverse reactions, and emphasizes the importance of internal donor for high efficiency and directionality of H⁺ transfer.     

Steady-state measurements of ΔpH and Δψ in Rhodospirillum rubrum chromatophores by two different methods. Comparison with phosphorylation potential

The pH gradient, ΔpH, and the membrane potential, Δψ, formed during light-induced electron transport in Rhodospirillum rubrum chromatophores were measured by two independent methods: (a) using specific electrodes to monitor light-dependent uptake of NH₄Cl and SCN^? at chromatophore concentrations of about 0.1 mg bacteriochlorophyll/ml and (b) using 9-aminoacridine and 8-anilinonaphthalenesulfonic acid as fluorescent probes for ΔpH and Δψ, respectively, at chromatophore concentrations of about 0.01 mg bacteriochlorophyll/ml. The light intensity was measured and set at a level which saturated the highest bacteriochlorophyll concentration used. The steady-state values obtained with each method under phosphorylating conditions were compared with the phosphorylation potential maintained by the chromatophores under identical conditions. The results indicate that under all conditions employed the ratio $\text{H}{}^{\mathrm{+}}\text{ATP}$ is greater than 2, and varies between 2.4 and 3.4 depending on the method used for estimation of the electrochemical proton gradient.