Functions of carotenoids in xanthorhodopsin and archaerhodopsin, from action spectra of photoinhibition of cell respiration

The recent discovery of a carotenoid light-harvesting antenna in xanthorhodopsin, a retinal-based proton pump in Salinibacter ruber, made use of photoinhibition of respiration in whole cells to obtain action spectra [Balashov et al. Science 309, (2005) 2061-2064]. Here we provide further details of this phenomenon, and compare action spectra in three different systems where carotenoids have different functions or efficiencies of light-harvesting. The kinetics of light-induced inhibition of respiration in Salinibacter ruber was determined with single short flashes, and the photochemical cross section of the photoreaction was estimated. These measurements confirm that the xanthorhodopsin complex includes no more than a few, and most likely only one, carotenoid molecule, which is far less than the core complex antenna of photosynthetic bacteria. Although the total cross-section of light absorption in the purple bacterium Rhodospirillum rubrum greatly exceeds that in Salinibacter, the cross-sections are roughly equivalent in the shared wavelength range. We show further that despite interaction of bacterioruberin with archaerhodopsin, another retinal-based proton pump, there is no significant energy transfer from this carotenoid. This emphasizes the uniqueness of the salinixanthin-retinal interaction in xanthorhodopsin, and indicates that bacterioruberin in Halorubrum species has a structural or photoprotective rather than energetic role.     

The toxicity of menthol in short-term bioassays

The toxicity of menthol was studied on 4 different in vitro systems covering organ, cellular and subcellular levels. The 50% inhibitory concentration (IC₅₀) for the cellular and subcellular systems ranged from 0.32 mM to 0.76 mM. At a concentration of 0.5 mM menthol the receptor mediated respiratory stimulation of isolated brown adipocytes was markedly inhibited while the intracellular mitochondrial functions were still unaffected. However, using isolated rat liver mitochondria 0.5 mM menthol was found to cause increase in the ‘state 4’ respiratory rate and osmotic swelling, indicating a leakage of the mitochondrial membrane. We therefore suggest that one effect of menthol is a deterioration of biological membranes. For the determination of the cellular toxicity of foreign compounds isolated brown adipocytes represents a convenient and sensitive model, providing the possibility to localize the primary site of action in terms of mitochondrial or extramitochondrial level.     

Energy transduction in photosynthetic bacteria IV. Light-dependent ATPase in photosynthetic membranes from Rhodopseudomonas capsulata

ATPase activity of photosynthetic membrane fragments from the bacterium Rhodopseudomonas capsulata can be stimulated by continuous illumination under conditions of active cyclic electron flow. The activation corresponds to an increase in the maximum velocity of the reaction and does not affect the apparent K_m for ATP (0.11 mM). No stimulation in the light is observed in the presence of classical uncouplers or oxidized 2,6-dichlorophenolindophenol (DCIP), which, per se, stimulate ATPase in the dark. It is demonstrated, however, that oxidized DCIP acts as an uncoupler of bacterial photophosphorylation.

The effect of light is elicited after a few minutes of preillumination, or in a much shorter time if an ADP trapping system is supplied. Activation does not occur if ADP is added during the preillumination (apparent K_m for inhibition by ADP = 1 μM). The effect of ADP is not related to competitive inhibition with ATP, which can be observed at higher concentrations (apparent K_i = 0.26 mM). ADP, however, is not effective if added after some minutes of preillumination.     

Fumarate permeation in rat heart mitochondria

The possible fumarate translocation in rat heart mitochondria is examined. This substrate, which is claimed to be a non permeant ion in rat liver mitochondria appears to cross the mitochondrial membrane in cardiac mitochondria. This conclusion was proposed on the basis of experimental results which show swelling by rat heart mitochondria in ammonium fumarate, uptake by mitochondria of fumarate, Pi efflux from the matrix induced by fumarate and appearance of malate in the reaction mixture which follows the addition of fumarate to the mitochondria and depends on the fumarase activity. The existence of a carrier unknown so far as well as a possible physiological role of this transport is proposed.     

Bacterial contamination as a cause of spurious cyanide tests

Cyanogenic bacteria were common contaminants in cyanide tests of fungi. This observation demonstrated the need to ensure that the origin of hydrogen cyanide in a test is from the sample and is not microbial. This can be accomplished by recording cyanide test results after short incubation periods or by adding antibiotic solutions.     

Growth, nitrate uptake and respiration rate in bean roots under phosphate deficiency

The decrease in inorganic phosphate concentration in bean (Phaseolus vulgaris L. cv. Złota Saxa) roots induced decrease in respiration rate. The decrease observed in ATP pool in phosphate deficient (-P) roots was greater than it would result from the decline in respiration and possible involvement of alternative pathway, suggesting an increased energy utilization for growth and ion uptake. Indeed, relative growth rate was higher in -P plants until 12 d of culture and later dropped to the rate similar to the control. Net nitrate uptake rate was higher in -P plants than in +P plants at the beginning of phosphate starvation, then during the prolonged culture it decreased rapidly in -P plants and after 19 d it was 8 times lower than that in the control. The decline in ATP production during prolonged phosphate starvation influenced NO₃ ^- uptake more than root growth. This revised version was published online in July 2006 with corrections to the Cover Date.     

Uniport,Not Proton-Symport,in a Non-Mammalian SLC23 Transporter

SLC23 family members are transporters of either nucleobases or ascorbate. While the mammalian SLC23 ascorbate transporters are sodium-coupled, the non-mammalian nucleobase transporters have been proposed, but not formally shown, to be proton-coupled symporters. This assignment is exclusively based on in vivo transport assays using protonophores. Here, by establishing the first in vitro transport assay for this protein family, we demonstrate that a representative member of the SLC23 nucleobase transporters operates as a uniporter instead. We explain these conflicting assignments by identifying a critical role of uracil phosphoribosyltransferase, the enzyme converting uracil to UMP, in driving uracil uptake in vivo. Detailed characterization of uracil phosphoribosyltransferase reveals that the sharp reduction of uracil uptake in whole cells in presence of protonophores is caused by acidification-induced enzyme inactivation. The SLC23 family therefore consists of both uniporters and symporters in line with the structurally related SLC4 and SLC26 families that have previously been demonstrated to accommodate both transport modes as well.