Structural requirements of quinone coenzymes for endogenous and dye-mediated coupled electron transport in bacterial photosynthesis

Electron transport in continuous light has been investigated in chromatophores ofRhodopseudomonas capsulata, Ala pho⁺, depleted in ubiquinone-10 and subsequently reconstituted with various ubiquinone homologs and analogs. In addition the restoration of electron transport in depleted chromatophores by the artificial redox compoundsN-methylphenazonium methosulfate andN,N,N,N-tetramethyl-p-phenylenediamine was studied. The following pattern of activities was obtained: (1) Reconstitution of cyclic photophosphorylation with ubiquinone-10 was saturated at about 40 ubiquinone molecules per reaction center. (2) Reconstitution by ubiquinone homologs was dependent on the length of the isoprenoid side chain and the amount of residual ubiquinone in the extracted chromatophores. If two or more molecules of ubiquinone-10 per reaction center were retained, all homologs with a side chain longer than two isoprene units were as active as ubiquinone-10 in reconstitution, and the double bonds in the side chain were not required. If less than two molecules per reaction center remained, an unsaturated side chain longer than five units was necessary for full activity. Plastoquinone, -tocopherol, and naphthoquinones of the vitamin K series were relatively inactive in both cases. (3) All ubiquinone homologs, also ubiquinone-1 and -2, could be reduced equally well by the photosynthetic reaction center, as measured by light-induced proton binding in the presence of antimycin A and uncoupler. Plastoquinone was found to be a poor electron acceptor. (4) Photophosphorylation could be reconstituted byN-methylphenazonium methosulfate as well as byN,N,N,N-tetramethyl-p-phenylenediamine in an antimycin-insensitive way, if more than two ubiquinones per reaction center remained. These compounds were active also in more extensively extracted particles reconstituted with ubiquinone-1, which itself was inactive.Abbreviations UQ-n, n = 1–10 ubiquinone with 1 to 10 isoprene units in the side chain - UQ-9 sat UQ-9 with a saturated side chain - PQ plastoquinone A - PMS N-methylphenazonium methosulfate - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - DAD diaminodurene (2,3,5,6-tetramethyl-p-phenylenediamine) - FCCP carbonyl cyanide-p-trifluoromethoxyphenylhydrazone - E _h redox potential - RC photosynthetic reaction center - BChl bacteriochlorophyll - PES N-methylphenazonium ethosulfate     

Physical composition of the semen of Barbus aeneus, the smallmouth yellowfish (Cyprinidae)

1. The physical composition of the semen of Barbus aeneus (smallmouth yellowfish) was determined. 2. The mean values and standard deviations for some of the physical properties were as follows: sperm count = 8.41 x 10(6)/mm3 +/- 2.55; percentage live cells = 92.5% +/- 9.92; spermatocrit = 61.9% +/- 5.74 and the total motility = 65 +/- 8.95. The colour of the sperm samples were white while the viscosity varied between 2 and 3 on the scale 1-3.     

Modulation of proton pumping efficiency in bacterial ATP synthases

The ATP synthase in chromatophores of Rhodobacter caspulatus can effectively generate a transmembrane pH difference coupled to the hydrolysis of ATP. The rate of hydrolysis was rather insensitive to the depletion of ADP in the assay medium by an ATP regenerating system (phospho-enol-pyruvate (PEP) and pyruvate kinase (PK)). The steady state values of DeltapH were however drastically reduced as a consequence of ADP depletion. The clamped concentrations of ADP obtained using different PK activities in the assay medium could be calculated and an apparent Kd approximately 0.5 microM was estimated. The extent of proton uptake was also strongly dependent on the addition of phosphate to the assay medium. The Kd for this effect was about 70 microM. Analogous experiments were performed in membrane fragment from Escherichia coli. In this case, however, the hydrolysis rate was strongly inhibited by Pi, added up to 3 mM. Inhibition by Pi was nearly completely suppressed following depletion of ADP. The Kd's for the ADP and Pi were in the micromolar range and submillimolar range, respectively, and were mutually dependent from the concentration of the other ligand. Contrary to hydrolysis, the pumping of protons was rather insensitive to changes in the concentrations of the two ligands. At intermediate concentrations, proton pumping was actually stimulated, while the hydrolysis was inhibited. It is concluded that, in these two bacterial organisms, ADP and phosphate induce a functional state of the ATP synthase competent for a tightly coupled proton pumping, while the depletion of either one of these two ligands favors an inefficient (slipping) functional state. The switch between these states can probably be related to a structural change in the C-terminal alpha-helical hairpin of the epsilon-subunit, from an extended conformation, in which ATP hydrolysis is tightly coupled to proton pumping, to a retracted one, in which ATP hydrolysis and proton pumping are loosely coupled.     

Delta12-Prostaglandin J2 inhibits the ubiquitin hydrolase UCH-L1 and elicits ubiquitin-protein aggregation without proteasome inhibition

To investigate molecular mechanisms linking inflammation with neurodegeneration, we treated neuronal cultures with prostaglandins (PGs), which are mediators of inflammation. PGA1, D2, J2, and Delta12-PGJ2, but not PGE2, reduced the viability and raised the levels of ubiquitinated proteins in the neuronal cells. PGJ2 and its metabolite, Delta12-PGJ2, were the most potent of the four neurotoxic PGs tested in inducing both effects. To address the mechanism by which these agents lead to the accumulation of ubiquitinated proteins, we tested their effects on neuronal ubiquitin hydrolases UCH-L1 and UCH-L3 as well as on proteasome activity. Notably, Delta12-PGJ2 inhibited the activities of UCH-L1 (K(i) approximately 3.5 microM) and UCH-L3 (K(i) approximately 8.1 microM) without affecting proteasome activity. Intracellular aggregates containing ubiquitinated proteins were detected in Delta12-PGJ2-treated cells, indicating that these aggregates can form independently of proteasome inhibition. In conclusion, impairment of ubiquitin hydrolase activity, such as triggered by Delta12-PGJ2, may be an important contributor to neurodegeneration associated with accumulation of ubiquitinated proteins and inflammation.     

The stimulation of photophosphorylation and ATPase by artificial redox mediators in chromatophores ofRhodopseudomonas capsulata at different redox potentials

(1) Inhibition of cyclic phosphorylation in chromatophores ofRhodopseudomonas capsulata by antimycin A can be fully reversed by artificial redox mediators, provided the ambient redox potential is maintained around 200 mV. The redox mediator need not be a hydrogen carrier in its reduced form, N-methyl-phenazonium methosulfate and N,N,N,N-tetramethyl-p-phenylenediamine being equally effective. However, the mediator needs to be lipophilic. Endogenous cyclic phosphorylation is fastest around 130 mV. A shift to 200 mV can also be observed if high concentrations of artificial redox mediator are present in the absence of antimycin A. (2) ATPase activity ofRhodopseudomonas capsulata, in the light as well as in the dark, activated or not activated by inorganic phosphate, can also be stimulated by N-methylphenazonium methosulfate. This stimulation is highest at redox potentials between 60 to 80 mV and is sensitive to antimycin A. In this case N,N,N,N-tetramethyl-p-phenylenediamine is much less effective.Abbreviations PES N-methyl-phenazonium ethosulfate - PMS N-methyl-phenazonium methosulfate - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - DAD diaminodurene (2,3,5,6-tetramethyl-p-phenylenediamine) - Bchl bacteriochlorophyll - FCCP carbonylcyanide-p-trifluoromethoxy-phenylhydrazone - E _h redox potential - E _m midpoint redox potential