Stoichiometry of proton uptake in isolated pea chloroplasts under different light intensities

The number of protons released inside the chloroplast thylakoids per electron which is transferred through the electron transport chain (H⁺/e^– ratio) was measured in isolated pea chloroplasts at pH 6.0 under continuous illumination and with methyl viologen as an electron acceptor. At saturating light intensity (200 W · m^–2) (strong light) the H⁺/e^– ratio was 3. At low intensity (0.9 W · m^–2) (weak light) the H⁺/e^– ratio was 2 with dark-adapted chloroplasts, but it was close to 3 with chloroplasts that were preilluminated with strong light. It is shown that the presence of azide in the reaction mixture leads to errors in the determination of the H⁺/e^– ratio due to underestimation of the initial rate of H⁺ efflux on switching off the light. To explain the above data, we assume that transformation of the electron transport chain occurs during illumination with strong light, namely, the Q cycle becomes operative.     

1H NMR study of the interaction of bacteriophage λ Cro protein with the O R 3 operator

The 17 base pair operator O _R ³ oligonucleotide, which is the preferential binding site for the Cro repressor of phage , was studied by two-dimensional NMR spectroscopy. A sequential assignment procedure based on two-dimensional Nuclear Overhauser Effect (NOESY) and scalar coupling correlated (COSY) NMR spectroscopy, together with the knowledge of the oligodesoxynucleotide sequence, made it possible to assign the non-exhangeable base protons and the H1 and the H2-H2 sugar protons of the O _R ³ operator DNA. The pattern of the observed NOE connectivities is consistent with a right-handed helical DNA structure. The base and sugar proton assignments provide the necessary information for further studies of the O _R ³ operator — Cro repressor interaction.Abbreviations COSY correlated spectroscopy - FID free induction decay - NOE nuclear Overhauser effect - NOESY nuclear Overhauser effect spectroscopy - RD relaxation delay - TSP sodium 3-trimethylsilyl-(2,2,3,3-²H₄)propionate - EDTA sodium ethylendiamine tetraacetate     

Effects of weak acids on proline accumulation in barley leaves: a comparison between abscisic acid and isobutyric acid

Abstract. When isobutyric acid (IBA) or abscisic acid (ABA) are supplied to leaf sections a similar rapid and marked decrease in the intracellular pH is observed. This acidification is accompanied by an increase in proline level which is about the same for both 3 mol m⁻³ IBA and 1 mol m⁻³ ABA treatments.
Fusicoccin (FC), known to act at the proton pump level, almost completely suppresses the ABA-induced acidification of the cell sap, whereas it only partially counteracts the acidifying effect of IBA, in particular during short periods of treatment. This effect of FC is paralleled by a similar inhibition of the induced proline accumulation: in fact, FC completely suppresses the ABA-induced increase in proline during short treatment periods, whereas it is only effective in inhibiting the IBA-induced proline accumulation after long treatment periods.
These data seem to suggest that the ABA- and IBA-induced changes in proline level might be mediated by changes in the intracellular pH.     

Respiration-dependent proton translocation and the mechanism of protonmotive force generation in Nitrobacter winogradskyi

Abstract Proton translocation associated with electron flow to oxygen has been observed with cells of Nitrobacter winogradskyi in the presence of either potassium ferrocyanide or isoascorbate plus N , N , N ', N ' tetramethyl- p -phenylenediamine. The data are consistent with a proton pumping function for the terminal oxidase, cytochrome aa ₃, in this organism as the mechanism for generating a protonmotive force. The failure of previous work with Nitrobacter [4] to detect proton translocation linked to oxidation of nitrite, the physiological substrate, is discussed.     

In vitro metabolism of N-methyl-dibenzo [c,g]carbazole a potent sarcomatogen devoid of hepatotoxic and hepatocarcinogenic properties

The metabolism of N-methyl substituted 7H-dibenzo[c,g]carbazole (N-Me DBC) was investigated in vitro using liver microsomes from 3-methylcholanthrene (MC)-, benzo[c]carbazole (BC) and Arochlor-pretreated mice and rats. N-Me DBC is a potent sarcomatogen devoid of hepatotoxicity and liver carcinogenic activity. The ethyl acetate-extractable metabolites were separated by high performance liquid chromatography (HPLC) and most of them were identified by proton magnetic resonance (PMR), mass spectrometry (MS) and comparison with synthetically prepared specimens. Mouse and rat microsomes gave rise to the same metabolites. The major metabolites were 5-OH-N-Me DBC (50%), N-hydroxymethyl (HMe) DBC (25-30%) and 3-OH-N-Me DBC (10%). Addition of 1,1,1-trichloropropene-2,3-oxide (TCPO) to the standard incubation medium permitted the identification of two dihydrodiols among the minor metabolites. No metabolite of DBC was observed after incubation of N-Me DBC, or its major metabolite N-HMe DBC, with either mouse or rat microsomes, but the possibility of a slight demethylation cannot be totally excluded. The lack of biotransformation at the nitrogen atom site may explain the lack of hepatotoxicity and liver carcinogenic activity of N-Me DBC. The modulation of metabolism by epoxide hydrolase, cytosol and glutathione was also investigated. The results are discussed in the light of data previously obtained with hepatotoxic and hepatocarcinogenic DBC.     

Membrane potential and surface potential in mitochondria: Uptake and binding of lipophilic cations

Summary The uptake and binding of the lipophilic cations ethidium⁺, tetraphenylphosphonium⁺ (TPP⁺), triphenylmethylphosphonium⁺ (TPMP⁺), and tetraphenylarsonium⁺ (TPA⁺) in rat liver mitochondria and submitochondrial particles were investigated. The effects of membrane potential, surface potentials and cation concentration on the uptake and binding were elucidated. The accumulation of these cations by mitochondria is described by an uptake and binding to the matrix face of the inner membrane in addition to the binding to the cytosolic face of the inner membrane. The apparent partition coefficients between the external medium and the cytosolic surface of the inner membrane (K' _o) and the internal matrix volume and matrix face of the inner membrane (K' _i) were determined and were utilized to estimate the membrane potential from the cation accumulation factorR _c according to the relation =RT/ZF ln [(R _cV_o–K'_o)/(V_i+K'_i)] whereV _o andV _i are the volume of the external medium and the mitochondrial matrix, respectively, andR _c is the ratio of the cation content of the mitochondria and the medium. The values of estimated from this equation are in remarkably good agreement with those estimated from the distribution of⁸⁶Rb in the presence of valinomycin. The results are discussed in relation to studies in which the membrane potential in mitochondria and bacterial cells was estimated from the distribution of lipophilic cations.     

Photosynthetic electron transport and establishment of an associated trans-thylakoid proton electrochemical gradient during development of the wheat leaf

Abstract Photosynthetic electron transport activities and the ability to generate and maintain a trans-thylakoid proton electrochemical gradient were examined during chloroplast development in 4-day-old wheat leaves grown under a diurnal light regime. Polarographic and spectropholometric studies on leaf tissue demonstrated that poorly developed chloroplasls at the leaf base could photo-oxidize water and transfer electrons from photosystem 2 to photosystem 1. The capacity for non-cyclic whole-chain electron transport increased during chloroplast development. Thylakoids isolated from the leaf base, although capable of pumping protons into the inlrathylakoid space, could not maintain a trans-membrane proton electrochemical gradient; this ability developed at later stages of chloroplast biogenesis in the leaf. The implications of these results for the energetics of the developing leaf are discussed.     

Models of localized energy coupling

It is proven that any model of localized protonmotive energy coupling that relies upon properties of a homogeneous surface phase must, when operated in the steady state, lead to bulk phase electrochemical potentials for protons that are as large as those required by the delocalized chemiosmotic theory. To obtain models consistent with experiments supporting localized energy coupling requires some kind of surface heterogeneity for the proton conducting pathways. Two general classes of heterogeneous surface models are mentioned. One class involves phase-separated lipid domains. The second class involves hydrogen-bonded chains in proteins that traverse the membrane laterally.     

Regulation of respiration and ATP synthesis in higher organisms: Hypothesis

The present view on the regulation of respiration and ATP synthesis in higher organisms implies only Michaelis-Menten type kinetics and respiratory control as regulatory principles. Recent experimental observations, suggesting further regulatory mechanisms at respiratory chain complexes, are reviewed. A new hypothesis is presented implying regulation of respiration and ATP synthesis in higher organisms mainly via allosteric modification of respiratory chain complexes, in particular of cytochromec oxidase. The allosteric effectors, e.g., metabolites, cofactors, ions, hormones, and the membrane potential are suggested to change the activity and the coupling degree of cytochromec oxidase by binding to specific sites at nuclear coded subunits. Recent results on the structure and activity of cytochromec oxidase, supporting the hypothesis, are reviewed.Dedicated to Professor Dr. Carl Martius on the occasion of his 80th birthday.     

The significance of the aprotic solvent in monomer studies related to the primary subunit environment in the macromolecule

An advantage of aprotic polar solvent systems in the study of monomer interactions relevant to the macromolecular state is demonstrated with the measurement of nucleoside amino proton exchange rates in DMSO/water mixtures. The DMSO/water solvent provides the first unequivocal observation of general acid catalysis of nucleic acid amino proton exchange, which is undetectable in aqueous solution due to the formation of the endocyclic protonated nucleobase. Suppression of nucleobase protonation in the presence of buffer acid is a consequence of anion desolvation in the aprotic solvent. The detected route of general acid catalysis is demonstrated as a consequence of Watson-Crick H-bonding, leading to the implication that amino chemistry is modulated in the helical state to decrease amino proton lifetime in the closed macromolecular context of conformational information obtained by hydrogen exchange methods. This useful property of the aprotic solvent can be extended to monomeric studies pertaining to specific local site interactions affecting the function and conformation of proteins and nucleic acids.