GTP-dependent structural rearrangement of the eRF1:eRF3 complex and eRF3 sequence motifs essential for PABP binding

Translation termination in eukaryotes is governed by the concerted action of eRF1 and eRF3 factors. eRF1 recognizes the stop codon in the A site of the ribosome and promotes nascent peptide chain release, and the GTPase eRF3 facilitates this peptide release via its interaction with eRF1. In addition to its role in termination, eRF3 is involved in normal and nonsense-mediated mRNA decay through its association with cytoplasmic poly(A)-binding protein (PABP) via PAM2-1 and PAM2-2 motifs in the N-terminal domain of eRF3. We have studied complex formation between full-length eRF3 and its ligands (GDP, GTP, eRF1 and PABP) using isothermal titration calorimetry, demonstrating formation of the eRF1:eRF3:PABP:GTP complex. Analysis of the temperature dependence of eRF3 interactions with G nucleotides reveals major structural rearrangements accompanying formation of the eRF1:eRF3:GTP complex. This is in contrast to eRF1:eRF3:GDP complex formation, where no such rearrangements were detected. Thus, our results agree with the established active role of GTP in promoting translation termination. Through point mutagenesis of PAM2-1 and PAM2-2 motifs in eRF3, we demonstrate that PAM2-2, but not PAM2-1 is indispensible for eRF3:PABP complex formation.     

The mobility of chromatophore membranes from Ectothiorhodospira Shaposhnikovii revealed by Rayleigh scattering of Mössbauer radiation (RSMR) experiments

Chromatophores from Ectothiorhodospira Shaposhnikovii in solvents of different viscosity were investigated by RSMR experiments in the temperature range between 112 K and room temperature. Additional RSMR-experiments were done on solvents only. The mobility of the molecules and within the molecules is the given by the Debye-Waller factor which yields the meansquare displacement, , averaged over the atoms in the system. The mobility of the atoms of the chromatophores roughly follows the mobility of the atoms of the solvents. At low temperatures the mobility of the chromatophores remains slightly larger than the mobility of the frozen solvent. At room temperature, however, of the chromatophores remains significantly smaller.Chromatophores in a glycerol-water mixture (0.001 M Tris-HCl buffer) and in water (0.05 M Tris-HCl buffer) show a different dynamic behaviour. A region with enhanced mobility near T=180 K was indicated for the chromatophores in the glycerolwater mixture.A correlation has been suggested between the rate of electron transfer from the primary to the secondary quinone and the increase of the conformational mobility of the chromatophores in glycerol-water mixture.Abbreviations Mb Myoglobin - Met-Mb Metmyoglobin     

Functional role of soluble mitochondrial ATPase subunits.

A preparation of soluble mitochondrial ATPase (coupling factor F1) containing no gamma and delta minor subunits has been isolated. The minor-subunits-deficient F1 was found to be competent in ATP hydrolysis. However, it did not demonstrate a "coupling" effect in EDTA-submitochondrial particles. A portion of the ATPase activity of EDTA particles, stimulated by the minor-subunits-deficient F1, was insensitive to oligomycin. ATPase activity of Na+-particles was changed only slightly by this F1. It is suggested that gamma and delta subunits are necessary to form specific contacts between the F1 molecule and components of the mitochrondrial membrane.     

Effect of temperature on the phototransformation of purple sulfur bacteria bacteriochlorophylls and isolated chromatophores]

Photobleaching of P890 was shown to be independent of temperature within the range of +20 to -160 degrees C in purple sulphur bacteria and isolated chromatophores under oxidative conditions; therefore changes in the absorption at 890 nm are due to the primary photoact. No changes were detected in the absorption at 850 nm upon a slight decrease of temperature, which suggested the absorption at 850 nm upon a slight decrease of temperature, which suggested the conformation nature of these changes. The effect of temperature, which suggested the conformation nature of these changes. The effect of temperature on the photoinduced changes of absorption under reductive conditions seems to be due to the electron transport and the accompanying processes being blocked. The effect of temperature on the kinetics of P890+ reduction in the darkness under conditions when the cytochromes are preliminarily oxidized is determined by the participation of the secondary electron acceptors in this process. A decrease in temperature leads to blocking the transport of electrons from the primary acceptor to the secondary acceptors, which is expressed by a gradual disappearance of a slow component in the kinetics of p890+ reduction in the disappearance of a slow component in the kinetics of P890+ reduction in the darkness and by the intensification of a fast component resulting from the darkness and by the intensification of a fast component resulting from the interaction between the primary acceptor and P890+. Methodical aspects of absorption differential spectrophotometry of photosynthesizing organisms at low temperatures are discussed.     

Modulation of electrical activity of neuron RPa2 ofHelix pomatia

Neuron RPa2 ofHelix pomatia can generate rhythmic (beating) or periodic (bursting) activity. A spontaneous switch from beating to bursting activity takes place in the course of tens of minutes. Similar changes in electrical activity can be induced by the addition of the water-soluble fraction obtained from a homogenate of snail ganglia to the experimental chamber. Artificial polarization of the membrane of neuron RPa2 by asteady inward current leads to an increase in the duration of intervals between bursts and to a decrease in the number of action potentials in the burst. With an increase in amplitude of the polarizing current, action potential generation ceases completely, but generation of waves of membrane potential persists. If the voltage on the neuron membrane is clamped, periodic fluctuations of membrane current disappear. It is suggested that action potential generation by neurons RPa2 is determined by the properties of the potential-dependent conductance of its membrane, i.e., that it is endogenous in origin and can be regulated by compounds acting on the membrane. These compounds, secreted by other neurons, resemble neurotransmitters or neurohormones.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 13, No. 4, pp. 406–412, July–August, 1981.     

Effect of theophylline on electrical activity of bursting type in an identified Helix pomatia neuron

The effect of theophylline, an inhibitor of cyclic nucleotide phosphodiesterase, on electrical activity of bursting neuron RPa1 ofHelix pomatia was investigated. In a concentration of 1 mM theophylline, when added to the external solution, increases the frequency and number of action potentials in the burst and also the duration of the inter-burst interval and the amplitude of membrane potential waves. In concentrations of 2.5 and 5.0 mM theophylline leads to reversible inhibition of bursting activity. During rinsing this activity rises to a higher level and then returns to the original value. The action of theophylline develops and disappears (as a result of rinsing) in the course of 1–5 min, depending on concentration of the inhibitor. It is suggested that electrical activity of the molluscan bursting neuron is controlled through the cyclic nucleotide system.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 13, No. 1, pp. 75–79, January–February, 1981.     

Mechanisms of hydration effects on the structural-dynamic and functional characteristics of photosynthetic membranes in various purple bacteria

NMR spectra and T₁, T₂ relaxation times for ¹H, ¹³C and ³¹P nuclei in membranes of R. rubrum and Rb. sphaeroides recorded at different relative humidity, as well as hydration curves and electron transfer efficiency of these membranes and membranes of E. shaposhnikovii, reveal complicated relations between structural-dynamic and functional characteristics. A number of sites of the electron transfer chain are shown to be under the control of structural-dynamic mechanisms. Different parameters characterizing these membranes at low humidity and during hydration have been established. These findings and analysis of the data from model systems reveal four different stages of hydration. Each of them is associated with specific changes in structure, dynamics, and function of photosynthetic membranes and their components. In the first stage the hydration of some polar groups leads to local changes in the dynamics of the protein component and this influences the recombination between photoactive pigment P and intermediate acceptor Q_A. The second stage is induced by incorporation of water molecules into the hydrogen bonds between the polar head groups of the lipids and within macromolecules. This results in changes of the dynamics of the membranes, the efficiency of the electron transfer between the quinones and the efficiency of photooxidation of cytochrome c. In the third stage all polar groups are hydrated owing to the appearance of free water with a high dielectric constant. This makes possible lateral mobility of membrane components and changes in distances between the interacting macromolecular components. Therefore, the regulation of photosynthetic processes can be mediated with the participation of mobile carriers. Finally, in the fourth stage, complete humidification provides conditions for regulation of photosynthesis at the cell level. The mechanisms influencing these processes and the efficiency and regulation of electron transfer in various parts of the photosynthetic chain are discussed. Received: 1 August 1996 / Accepted: 4 July 1997     

Energetic aspects of CO oxidation in desulfovibrio desulfuricans

In cell suspension of Desulfovibrio desulfuricans B-1388, oxidation of CO as the only energy source is associated with reduction of SO42-. After a 2-h incubation of cells in 8% CO, 81% of the gas is converted. Oxidation of 1 mole CO results in formation of 0.23 mole H2S. Intracellular ATP content increases from 2.5 (control) to 8.3 nmoles/mg (during CO conversion). Dinitrophenol inhibits sulfate reduction and CO oxidation. CO dehydrogenase was detected in cytoplasmic and membrane cell fractions (59 and 34%, respectively).     

Mechanisms of Generation of Pacemaker Activity by Identified Helix Neurons

We studied the mechanisms of generation of pacemaker activity in identified neurons of Helix pomatia. For this purpose, we isolated the PPa2 and PPa7 neurons generating spontaneous rhythmic monomodal activity and PPa1 neuron with bursting activity. It was demonstrated that isolated PPa2 and PPa7 cells produce endogenous rhythmic activity that was not considerably modified by external application of 1 mM CdCl₂. Sometimes, only low-amplitude dendritic action potentials (AP) were observed instead of generation of full-amplitude somatic AP. In contrast, isolation of the PPa1 neuron eliminated its bursting activity, but subsequent application of oxytocin on this neuron recovered such activity. This finding shows that the bursting activity of the PPa1 neuron is of an exogenous nature. Application of 1 mM CdCl₂ suppressed this bursting activity, but when Cd²⁺ was applied against the background of superfusion of the neuron with Ringer solution containing a bursting activity-initiating neuropeptide obtained from the molluscan CNS, this blocker was incapable of suppressing the bursting activity. A blocker of the hyperpolarization-activated current (I _h , H current), Cs⁺ (10 mM) exerted no noticeable effect on the activity of the studied neurons. Our findings allow us to conclude that the pacemaker activity is initiated within the dendritic tree of a cell and is then electrotonically spread to the soma, where full-amplitude AP are generated. It seems probable that Ca²⁺ ions and H current are not directly involved in generation of the pacemaker activity in the studied snail neurons.     

An enzyme immunoassay system for aflatoxin B1

Indirect enzyme immunoassay based on immobilized conjugate of aflatoxin B₁ carboxymethyloxime with bovine serum albumin and polyclonal rabbit antibodies allows determining aflatoxin B₁ with a low relative cross-reactivity against aflatoxin B₂, G₁, G₂, M₁ B_2a, and G_2a and sterigmatocystin (15.5, 15.5, 1.7, 1.0, 0.03, 0.03 and 0.01%, respectively) with a sensitivity of 0.04 ng per well or 4.0 ng per ml organic solvent.