Thermal denaturation of class 1 eukaryotic translation termination factor eRF1. Relationship between stability and functional activity of eRF1 mutants

Differential scanning calorimetry (DSC) was used to study thermal denaturation of the human class 1 translation termination factor eRF1 and its mutants. Free energy changes caused by amino acid substitutions in the N domain were computed for eRF1. The melting of eRF1, consisting of three domains, proved to be cooperative. The thermostability of eRF1 was not affected by certain substitutions and was slightly increased by certain others. The corresponding residues were assumed to play no role in maintaining the eRF1 structure, which agreed with the published X-ray data. In these mutants (E55D, Y125F, N61S, E55R, E55A, N61S + S64D, C127A, and S64D), a selective loss of the capability to induce hydrolysis of peptidyl-tRNA in the ribosomal P site in the presence of a stop codon was not associated with destabilization of their spatial structure. Rather, the loss was due to local changes in the stereochemistry of the side groups of the corresponding residues in functionally important sites of the N domain. Two amino acid residues of the N domain, N129 and F131, proved to play an important role in the structural stability of eRF1 and to affect the selective recognition of mRNA stop codons in the ribosome. The recognition of the UAG and UAA stop codons in vitro was more tightly associated with the stability of the spatial structure of eRF1 as compared with that of the UGA stop codon.     

Temperature dependence of cytochrome photooxidation and conformational dynamics of Chromatium reaction center complexes

A temperature dependence of multiheme cytochrome c oxidation induced by a laser pulse was studied in photosynthetic reaction center preparations from Chromatium minutissimum. Absorbance changes and kinetic characteristics of the reaction were measured under redox conditions where one or all of the hemes of the cytochrome subunit are chemically reduced (E _h =+300 mV or E _h =–20 to -60 mV respectively). In the first case photooxidation is inhibited at temperatures lower than 190–200 K with the rate constant of the photooxidation reaction being practically independent on temperature over the range of 300 to 190 K (k=2.2×10⁵ s^-1). Under reductive conditions (E _h =–20 to -60 mV) lowering the temperature to 190–200 K causes the reaction to slow from k=8.3×10⁵ s^-1 to 2.1×10⁴ s^-1. Under further cooling down to the liquid nitrogen temperature, the reaction rate changes negligibly. The absorption amplitude decreases by 30–40% on lowering the temperature. A new physical mechanism of the observed critical effects of temperature on the rate and absorption amplitude of the multiheme cytochrome c oxidation reaction is proposed. The mechanism suggests a close interrelation between conformational mobility of the protein and elementary electron tunneling act. The effect of freezing conformational motion is described in terms of a local diffusion along a random rough potential.     

Photo-induced electron transport and water state in Rhodospirillum rubrum chromatophores

It is shown that in bacterial chromatophores the pronounced changes in the free water content with a proton spin-spin relaxation time (T₂) of 10^?3—10^?2 s does not influence the efficiency of electron transfer from the photosynthetic reaction centre to the membrane pool of secondary acceptors. An abrupt inhibition of this process occurs only after the loss of the water with faster proton spin-spin relaxation time (T₂ of 10^?4 s). The process is reversible. The water fraction in question is obviously bound to the chromatophore proteins and forms the primary hydration layer.     

Possible interaction between synaptic and pacemaker mechanisms of electrical activity in bursting neurons of Helix pomatia

Membrane hyperpolarization induced by short pulses of inward current, by stimulation of the anal nerve, which leads to the appearance of a long IPSP in the neuron, and developing during the appearance of spontaneous IPSPs in the neuron was investigated in neuron RPa1 ofHelix pomatia. Short-term hyperpolarization of the neuron membrane by an inward current (10 msec) led to the development of self-maintained (regenerative) membrane hyperpolarization lasting several seconds. The amplitude and duration of regenerative hyperpolarization increased with an increase in amplitude and duration of the pulse of inward current. The time course of IPSPs arising spontaneously in the neuron and in response to stimulation of the anal nerve was similar to that of regenerative hyperpolarization evoked by a pulse of inward current. It is suggested that regenerative hyperpolarization associated with activation of endogenous mechanisms of regulation of the bursting activity of the neuron may be due not only to short-term membrane hyperpolarization of the test neuron by the electric current, but also to hyperpolarization occurring during IPSP generation.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 13, No. 1, pp. 67–74, January–February, 1981.     

Functional role of soluble mitochondrial ATPase subunits

A preparation of soluble mitochondrial ATPase (coupling factor F₁) containing no and 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 and subunits are necessary to form specific contacts between the F₁ molecule and components of the mitochrondrial membrane.Abbreviations SMP submitochondrial particles - F₁ coupling factor (soluble mitochondrial ATPase) - PCB^– phenyl dicarbaundecaborane anions     

Interactions of endogenous lectin from theHelix pomatia reproductive system (HPA) with the surface of CNS neurons of the snail

The surface of unidentified cultured neurons and electrophysiologically identified units RPa1, RPa2, and LPa3 of the snailHelix pomatia was studied using a colloidal gold-labelled endogenous gonad lectin of this molluse (Helix pomatia agglutinin -HPA), which is specific to -anomer N-acetyl-D-galactosamine, and some other lectins. It was found that only two populations of cultured neurons with a specific ultrastructure typical of peptidergic cells and differing from the ultrastructure of other peptidergic and non-peptidergic neurons possessHPA receptors. These neurons average about 1% of cultured nerve cells. Using a variety of plant lectins withHPA-like specificity (LBA, VVA, SBA) showed that the surface of many peptidergic neurons contains -anomer-like structures, yet it does not bindHPA. These data were supported in the experiments on identified snall neurons with the use ofSBA. The results suggest a putative role of endogenous lectins in regulation of activity of neurons participating in processes of secretion in the gonads.Neirofiziologiya/Neurophysiology, Vol. 28, No. 1, pp. 17–29, January–February, 1996.     

Postsynaptic mechanisms initiating bursting activity in theHelix pomatia RPal neuron under the influence of an interneuron

Postsynaptic mechanisms of the connection between the interneuron in the visceral ganglion initiating bursting activity in RPal and B7 neurons and these neurons themselves were investigated in the snail (Helix pomatia). Using voltage clamping at the membrane of these cells, stimulation of the interneuron gave rise to a slow inward current with a 2 sec latency; it rose in amplitude as stimulation increased in duration. Reducing the temperature from 25 to 5°C diminished the rise and decay rate of this current with a temperature coefficient of about 10. The current-voltage relationship of the slow inward current was nonlinear, with a maximum of –65 mV. Reducing the concentration of sodium ions in the extracellular fluid increased the amplitude of the current. While hyperpolarization of the burster neuron membrane produced a burst of inward current prior to stimulation, this same hyperpolarization induced a pulse of outward current at the peak of the slow inward current. Stimulating the interneuron is thus thought to activate at least two types of ionic channel in the cell body of the burster neurons: a steady sodium and a voltage- and time-dependent channel for outward current. This process could well be mediated by a biochemical cytoplasmic chain reaction.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 19, No. 1, pp. 28–36, January–February, 1987.