A covalently bound photoisomerizable agonist. Comparison with reversibly bound agonists at electrophorus electroplaques

After disulphide bonds are reduced with dithiothreitol, trans-3- (α-bromomethyl)-3’-[α- (trimethylammonium)methyl]azobenzene (trans-QBr) alkylates a sulfhydryl group on receptors. The membrane conductance induced by this “tethered agonist” shares many properties with that induced by reversible agonists. Equilibrium conductance increases as the membrane potential is made more negative; the voltage sensitivity resembles that seen with 50 [mu]M carbachol. Voltage- jump relaxations follow an exponential time-course; the rate constants are about twice as large as those seen with 50 μM carbachol and have the same voltage and temperature sensitivity. With reversible agonists, the rate of channel opening increases with the frequency of agonist-receptor collisions: with tethered trans-Qbr, this rate depends only on intramolecular events. In comparison to the conductance induced by reversible agonists, the QBr-induced conductance is at least 10-fold less sensitive to competitive blockade by tubocurarine and roughly as sensitive to “open-channel blockade” bu QX-222. Light-flash experiments with tethered QBr resemble those with the reversible photoisomerizable agonist, 3,3’,bis-[α-(trimethylammonium)methyl]azobenzene (Bis-Q): the conductance is increased by cis {arrow} trans photoisomerizations and decreased by trans {arrow} cis photoisomerizations. As with Bis-Q, ligh-flash relaxations have the same rate constant as voltage-jump relaxations. Receptors with tethered trans isomer. By comparing the agonist-induced conductance with the cis/tans ratio, we conclude that each channel’s activation is determined by the configuration of a single tethered QBr molecule. The QBr-induced conductance shows slow decreases (time constant, several hundred milliseconds), which can be partially reversed by flashes. The similarities suggest that the same rate-limiting step governs the opening and closing of channels for both reversible and tethered agonists. Therefore, this step is probably not the initial encounter between agonist and receptor molecules.     

Recombination reduction of photooxidized cytochrome c in reaction centers of Rhodopseudomonas viridis at low temperature

The temperature dependence of dark reduction of photooxidized cytochrome c was studied in isolated preparations of Rhodopseudomonas viridis reaction centers. Within the range from room temperature to 260 K this process was found to be mediated by thermal diffusion of exogenous donor molecules, whereas at lower temperatures photooxidized cytochrome is reduced as a result of indirect recombination with photoreduced primary quinone acceptor. Kinetic simulation allowed certain thermodynamic characteristics of this reaction to be calculated. To the first approximation, these characteristics correlate with the estimates obtained from the results of direct redox titration.     

Effect of Dipyridamole on Membrane Energization and Energy Transfer in Chromatophores of Rba. sphaeroides

Biochemistry (Moscow) - Effect of dipyridamole (DIP) at concentrations up to 1 mM on fluorescent characteristics of light-harvesting complexes LH2 and LH1, as well as on conditions of...     

A study of protein structure changes during hydration by diffuse X-ray scattering: II. Fourier transform analysis of diffuse X-ray scattering data

Radial distribution functions were deduced by Fourier transform analysis of the angular dependences of diffuse X-ray scattering intensities for the following proteins with different hydration degrees: water-soluble α-protein myoglobin, water-soluble (α + β) protein lysozyme, and transmembrane proteins from the photosynthetic reaction centers of purple bacteria Rhodobacter sphaeroides and Blastochlorii (Rhodopseudomonas) viridis. The results of Fourier transform analysis of X-ray scattering intensities give quantitative characteristics of the mechanism underlying the influence of water on the formation of biological macromolecules. On the one hand, water loosens the network of hydrogen bonds, which results in a considerable conformational mobility in the molecules of lysozyme and myoglobin and the reaction centers. On the other hand, water stabilizes and orders the protein globule. A strict correlation was found between the shift of the “first” maximum of the radial distribution function, loosening of the intraglobular hydrogen bonds, increase in the intramolecular mobility, and appearance of pronounced functional activity in macromolecules. The pattern of behavior of the first maximum in the transmembrane proteins of the reaction center was similar to that observed for the water-soluble proteins. However, the first maximum reached the limiting value of 2.9 Å at a considerably lower hydration degree compared with the water-soluble proteins. A quick transition of the protein complex of the reaction center to its native state is due to the fact that the dehydrated conformation of this complex is very close to the native conformation. Comparison of the radial distribution function for water, water-soluble proteins, and transmembrane proteins suggests a quantitative conclusion that water is the least densely packed and ordered system, the water-soluble proteins are more densely packed than water, and the transmembrane proteins are the most densely packed and ordered system.     

Reversible Redox-Dependent Inactivation of Photosystem II as Photosynthesis Regulation in Chlorella

Inactivation of photosystem II (PSII) in the alga Chlorella pyrenoidosa Chick induced by photoinhibition (high light illumination at an intensity 10 times higher than photosynthesis-saturating light) or by incubation at a supraoptimum temperature (41°C) in darkness, resulted in a decrease in the relative yield of variable fluorescence due to a selective suppression of the slow phase of its rise. This indicates that low-activity PSII complexes, with a low efficiency of Q_A ^– formation are inactivated first. We suppose that the transition of normal PSII complexes to a low-activity state precedes the complete loss of their photochemical activity. The existence of some common stages of PSII inactivation, when induced by photoinhibition or incubation at supraoptimum temperature in darkness, is discussed. We suggest a scheme of the sequential stages in the regulation of photosynthetic light reactions involving a reversible redox-dependent PSII inactivation.     

The influence of dipyridamole and its derivatives on the membrane energization state of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> bacterial chromatophores

The mechanisms of the specific influence of dipyridamole therapy remain as a subject of research. In the present work the effects of dipyridamole and some its derivatives on the energization state of photosynthetic membranes of purple bacteria chromatophores have been studied. It was shown that under illumination of chromatophores in the presence of dipyridamole the occurrence of its radical form was observed due to the presence of electron-proton donation properties. These properties are vital to trigger the energization of a functionally active photosynthetic membrane when it is illuminated in the presence of dipyridamole and its derivatives. It is obvious that these properties of dipyridamole should be taken into account while studying the effects of this agent as a medical preparation with specific action.     

Temperature dependence of protein fluorescence in <Emphasis Type="Italic">Rb. sphaeroides</Emphasis> reaction centers frozen to 80 K in the dark or on the actinic light as the indicator of protein conformational dynamics

The differences in the average fluorescence lifetime (τ_av) of tryptophanyls in photosynthetic reaction center (RC) of the purple bacteria Rb. sphaeroides frozen to 80 K in the dark or on the actinic light was found. This difference disappeared during subsequent heating at the temperatures above 250 K. The computer-based calculation of vibration spectra of the tryptophan molecule was performed. As a result, the normal vibrational modes associated with deformational vibrations of the aromatic ring of the tryptophan molecule were found. These deformational vibrations may be active during the nonradiative transition of the molecule from the excited to the ground state. We assume that the differences in τ_av may be associated with the change in the activity of these vibration modes due to local variations in the microenvironment of tryptophanyls during the light activation.     

Genome-wide differentially methylated genes in prostate cancer tissues from African-American and Caucasian men

Increasing evidence suggests that aberrant DNA methylation changes may contribute to prostate cancer (PCa) ethnic disparity. To comprehensively identify DNA methylation alterations in PCa disparity, we used the Illumina 450K methylation platform to interrogate the methylation status of 485,577 CpG sites focusing on gene-associated regions of the human genome. Genomic DNA from African-American (AA; 7 normal and 3 cancers) and Caucasian (Cau; 8 normal and 3 cancers) was used in the analysis. Hierarchical clustering analysis identified probe-sets unique to AA and Cau samples, as well as common to both. We selected 25 promoter-associated novel CpG sites most differentially methylated by race (fold change > 1.5-fold; adjusted P < 0.05) and compared the β-value of these sites provided by the Illumina, Inc. array with quantitative methylation obtained by pyrosequencing in 7 prostate cell lines. We found very good concordance of the methylation levels between β-value and pyrosequencing. Gene expression analysis using qRT-PCR in a subset of 8 genes after treatment with 5-aza-2′-deoxycytidine and/or trichostatin showed up-regulation of gene expression in PCa cells. Quantitative analysis of 4 genes, SNRPN, SHANK2, MST1R, and ABCG5, in matched normal and PCa tissues derived from AA and Cau PCa patients demonstrated differential promoter methylation and concomitant differences in mRNA expression in prostate tissues from AA vs. Cau. Regression analysis in normal and PCa tissues as a function of race showed significantly higher methylation prevalence for SNRPN (P = 0.012), MST1R (P = 0.038), and ABCG5 (P < 0.0002) for AA vs. Cau samples. We selected the ABCG5 and SNRPN genes and verified their biological functions by Western blot analysis and siRNA gene knockout effects on cell proliferation and invasion in 4 PCa cell lines (2 AA and 2 Cau patients-derived lines). Knockdown of either ABCG5 or SNRPN resulted in a significant decrease in both invasion and proliferation in Cau PCa cell lines but we did not observe these remarkable loss-of-function effects in AA PCa cell lines. Our study demonstrates how differential genome-wide DNA methylation levels influence gene expression and biological functions in AA and Cau PCa.