Photochemistry and Photoinduced Proton-Transfer by Pharaonis Phoborhodopsin

Phoborhodopsin (pR or sensory rhodopsin II, sRII) is a photoreceptor of the negative phototaxis of Halobacterium salinarum, and pharaonis phoborhodopsin (ppR or pharaonis sensory rhodopsin II, psRII) is a corresponding protein of Natronobacterium pharaonis. The photocycle of ppR is essentially as follows: ppR(498) ppR_K(540) ppR_KL(512) ppR_L(488) ppR_M(390) ppR_O(560) ppR (numbers in parenthesis denote the maximum absorbance). The photocycle is very similar to that of bacteriorhodopsin, but the rate of initial pigment recovery is about two-orders of magnitude slower. By low-temperature spectroscopy, two K-intermediates were found but the L intermediate was not detected. The lack of L indicates extraordinary stability of K at low temperature. ppR_M is photoactive similar to M of bR. The ground state ppR contains only all-trans retinal whereas ppR_M and ppR_O contain 13-cis and all-trans, respectively. ppR has the ability of lightinduced proton transport from the inside to the outside. Proton uptake occurs at the formation of ppR_O and the release at its decay. ppR associates with its transducer and this complex transmits a signal to the cytoplasm. The proton transport ability is lost when the complex forms, but the proton uptake and release still occur, suggesting that the proton movement is non-electrogenic (release and uptake occur from the same side). The stoichiometry of the complex between ppR and the transducer is 1 : 1. ppR or pR has absorption maximum at 500 nm, which is blue-shifted from those of other archaeal rhodopsins. The molecular mechanism of this color regulation is not yet solved.     

Effects of Delipidation on Proton Translocation and ATPase Activity in Beef Heart Electron Transport Particles

Delipidation of beef heart electron transport particles with phospholipase A₂ has been examined. When the particles were treated with the lipase and subjected to a low bovine serum albumin wash, ATPase activity was unaffected as was the lipid/protein ratio of the particles. However, energisation by ATP/Mg²⁺ was abolished. Furthermore, unsaturated but not saturated fatty acids discharged the steady-state ATP-driven membrane potential of control samples. When the phospholipase A₂ hydrolysis products were removed, inhibition of energy-linked reactions in the lipid-depleted particles was still observed and was interpreted in terms of non-specific leaks in the vesicle membranes, and ‘specific’ leaks through impaired H⁺-ATPase complexes. ATPase activity was less susceptible to delipidation than energisation but was, nevertheless, strongly inhibited at 50 percent lipid depletion.

Spin label studies indicated a decrease in the fluidity of particle membranes accompanying delipidation. Moreover, the discontinuity seen in Arrhenius plots of ATPase activity was shifted from 17°C (control) to 22°C at 50 percent phospholipid depletion. The data are consistent with a release of unsaturated fatty acids by phospholipase A₂ rendering the transport particles both leakier and the membranes less fluid than controls.     

Hybrid MC/QC simulations of water-assisted proton transfer in nucleosides. Guanosine and its analog acyclovir

To provide an in-depth insight into the molecular basis of spontaneous tautomerism in DNA and RNA base pairs, a hybrid Monte Carlo (MC)–quantum chemical (QC) methodology is implemented to map two-dimensional potential energy surfaces along the reaction coordinates of solvent-assisted proton transfer processes in guanosine and its analog acyclovir in aqueous solution. The solvent effects were simulated by explicit inclusion of water molecules that model the relevant part of the first hydration shell around the solute. The position of these water molecules was estimated by carrying out a classical Metropolis Monte Carlo simulation of dilute water solutions of the guanosine (Gs) and acyclovir (ACV) and subsequently analyzing solute–solvent intermolecular interactions in the statistically-independent MC-generated configurations. The solvent-assisted proton transfer processes were further investigated using two different ab initio MP2 quantum chemical approaches. In the first one, potential energy surfaces of the ‘bare’ finite solute–solvent clusters containing Gs/ACV and four water molecules (MP2/6-31+G(d,p) level) were explored, while within the second approach, these clusters were embedded in ‘bulk’ solvent treated as polarizable continuum (C-PCM/MP2/6-31+G(d,p) level of theory). It was found that in the gas phase and in water solution, the most stable tautomer for guanosine and acyclovir is the 1H-2-amino-6-oxo form followed by the 2-amino-6-(sZ)-hydroxy form. The energy barriers of the water-assisted proton transfer reaction in guanosine and in acyclovir are found to be very similar – 11.74 kcal mol^?1 for guanosine and 11.16 kcal mol^?1 for acyclovir, and the respective rate constants (k = 1.5?×?10¹ s^?1, guanosine and k = 4.09?×?10¹ s^?1, acyclovir), are sufficiently large to generate the 2-amino-6-(sZ)-hydroxy tautomer. The analysis of the reaction profiles in both compounds shows that the proton transfer processes occur through the asynchronous concerted mechanism.     

Factors affecting cation-anion uptake balance and iron acquisition in peanut plants grown on calcareous soils

Dicotyledonous plants subjected to Fe-deficiency stress can decrease pH in the rhizosphere by proton excretion and reduce ferric iron by an activated reduction system in the plasma membranes of the root or by reductants released from the roots. The efficiency by which these plants take up Fe may strongly depend on their cation-anion balance. This study presents results of two experiments conducted to evaluate the effect of K, growth stage and cultivar on ionic balance and Fe acquisition of peanut (Arachis hypogaea L.) plants.Potassium applications to the high calcareous soil (30.3% CaCO₃) favoured proton release, but did not ameliorate plant Fe acquisition. At the earliest stages of plant growth, anion uptake exceeded cation uptake due to intensive N uptake. With time, a shift in the ionic balance was observed as a result of predominant cation uptake. It appears that the relationship between H/OH-ion release and Fe nutrition of peanut plants is actually a complex phenomenon under soil conditions and depends on some soil parameters, such as CaCO₃ content. Even by enhanced H-ion release Fe nutrition of plants can be impaired if soil CaCO₃ is too high.     

Importance of macrophage cholesterol content on the flux of cholesterol mass

Net flux of cholesterol represents the difference between efflux and influx and can result in net cell-cholesterol accumulation, net cell-cholesterol depletion, or no change in cellular cholesterol content. We measured radiolabeled cell-cholesterol efflux and cell-cholesterol mass using cholesterol-normal and -enriched J774 and elicited mouse peritoneal macrophage cells. Net cell-cholesterol effluxes were observed when cholesterol-enriched J774 cells were incubated with 3.5% apolipoprotein (apo) B depleted human serum, HDL₃, and apo A-I. Net cell-cholesterol influxes were observed when cholesterol-normal J774 cells were incubated with the same acceptors except apo A-I. When incubated with 2.5% individual sera, cholesterol mass efflux in free cholesterol (FC)-enriched J774 cells correlated with the HDL-cholesterol (HDL-C) concentrations (r² = 0.4; P=0.003), whereas cholesterol mass influx in cholesterol-normal J774 cells correlated with the LDL cholesterol (LDL-C) concentrations (r² = 0.6; P<0.0001) of the individual sera. A positive correlation was observed between measurements of [³H]cholesterol efflux and reductions in cholesterol mass (r² = 0.4; P=0.001) in FC-enriched J774 cells. In conclusion, isotopic efflux measurements from cholesterol-normal or cholesterol-enriched cells provide an accurate measurement of relative ability of an acceptor to remove labeled cholesterol under a specific set of experimental conditions, i.e., efflux potential. Moreover, isotopic efflux measurements can reflect changes in cellular cholesterol mass if the donor cells are enriched with cholesterol.     

Human Brain Capillary Endothelium: Modulation of K+ Efflux and K+, Ca2+ Uptake by Endothelin

This report describes K⁺ efflux, K⁺ and Ca²⁺ uptake responses to endothelins (ET-1 and ET-3) in cultured endothelium derived from capillaries of human brain (HBEC). ET-1 dose dependently increased K⁺ efflux, K⁺ and Ca²⁺ uptake in these cells. ET-1 stimulated K⁺ efflux occurred prior to that of K⁺ uptake. ET-3 was ineffective. The main contributor to the ET-1 induced K⁺ uptake was ouabain but not bumetanide-sensitive (Na⁺-K⁺-ATPase and Na⁺-K⁺-Cl^– cotransport activity, respectively). All tested paradigms of ET-1 effects in HBEC were inhibited by selective antagonist of ET_A but not ET_B receptors and inhibitors of phospholipase C and receptor-operated Ca²⁺ channels. Activation of protein kinase C (PKC) decreased whereas inhibition of PKC increased the ET-1 stimulated K⁺ efflux, K⁺ and Ca²⁺ uptake in HBEC. The results indicate that ET-1 affects the HBEC ionic transport systems through activation of ET_A receptors linked to PLC and modulated by intracellular Ca²⁺ mobilization and PKC.     

Cholesterol trafficking and raft-like membrane domain composition mediate scavenger receptor class B type 1-dependent lipid sensing in intestinal epithelial cells

Scavenger receptor Class B type 1 (SR-B1) is a lipid transporter and sensor. In intestinal epithelial cells, SR-B1-dependent lipid sensing is associated with SR-B1 recruitment in raft-like/ detergent-resistant membrane domains and interaction of its C-terminal transmembrane domain with plasma membrane cholesterol. To clarify the initiating events occurring during lipid sensing by SR-B1, we analyzed cholesterol trafficking and raft-like domain composition in intestinal epithelial cells expressing wild-type SR-B1 or the mutated form SR-B1-Q445A, defective in membrane cholesterol binding and signal initiation. These features of SR-B1 were found to influence both apical cholesterol efflux and intracellular cholesterol trafficking from plasma membrane to lipid droplets, and the lipid composition of raft-like domains. Lipidomic analysis revealed likely participation of d18:0/16:0 sphingomyelin and 16:0/0:0 lysophosphatidylethanolamine in lipid sensing by SR-B1. Proteomic analysis identified proteins, whose abundance changed in raft-like domains during lipid sensing, and these included molecules linked to lipid raft dynamics and signal transduction. These findings provide new insights into the role of SR-B1 in cellular cholesterol homeostasis and suggest molecular links between SR-B1-dependent lipid sensing and cell cholesterol and lipid droplet dynamics.     

Turgor-dependent unloading of assimilates from coats of developing legume seed. Assessment of the significance of the phenomenon in the whole plant

The in vivo significance of turgor-dependent unloading was evaluated by examining assimilate transport to and within intact developing seeds of Phaseolus vulgaris (cv. Redland Pioneer) and Vicia faba (cv. Coles Prolific). The osmotic potentials of the seed apoplast were low. As a result, the osmotic gradients to the seed coat symplast were relatively small (i.e. 0.1 to 0.3 MPa). Sap concentrations of sucrose and potassium in the seed apoplast and coat symplast accounted for some 45 to 60% of the osmotic potentials of these compartments. Estimated turnover times of potassium and sucrose in the seed apoplast of < 1 h were some 5 to 13 times faster than the respective turnover times in the coat symplast pools. The small osmotic gradient between the seed apoplast and coat symplast combined with the relatively rapid turnover of solutes in the apoplast pool, confers the potential for a small change in assimilate uptake by the cotyledons to be rapidly translated into an amplified shift in the cell turgor of the seed coat. Observed adjustments in the osmotic potentials of solutions infused between the coat and cotyledons of intact seed were consistent with the in vivo operation of turgor-dependent unloading of solutes from the coat. Homeostatic regulation of turgor-dependent unloading was indicated by the maintenance of apoplast osmotic potentials of intact seeds when assimilate balance was manipulated by partial defoliation or elevating pod temperature. In contrast, osmotic potentials of the coat symplast adjusted upward to new steady values over a 2 to 4 h period. The resultant downward shift in coat cell turgor could serve to integrate phloem import into the seed coat with the new rates of efflux to the seed apoplast. Circumstantial evidence for this linkage was suggested by the approximate coincidence of the turgor changes with those in stem levels of ³²P used to monitor phloem transport. The results obtained provide qualified support for the in vivo operation of a turgor homeostat mechanism. It is proposed that the homeostat functions to integrate assimilate demand by the cotyledons with efflux from and phloem import into the coats of developing legume seed.