Titration of aspartate-85 in bacteriorhodopsin: what it says about chromophore isomerization and proton release.

Titration of Asp-85, the proton acceptor and part of the counterion in bacteriorhodopsin, over a wide pH range (2-11) leads us to the following conclusions: 1) Asp-85 has a complex titration curve with two values of pKa; in addition to a main transition with pKa = 2.6 it shows a second inflection point at high pH (pKa = 9.7 in 150-mM KCl). This complex titration behavior of Asp-85 is explained by interaction of Asp-85 with an ionizable residue X'. As follows from the fit of the titration curve of Asp-85, deprotonation of X' increases the proton affinity of Asp-85 by shifting its pKa from 2.6 to 7.5. Conversely, protonation of Asp-85 decreases the pKa of X' by 4.9 units, from 9.7 to 4.8. The interaction between Asp-85 and X' has important implications for the mechanism of proton transfer. In the photocycle after the formation of M intermediate (and protonation of Asp-85) the group X' should release a proton. This deprotonated state of X' would stabilize the protonated state of Asp-85.2) Thermal isomerization of the chromophore (dark adaptation) occurs on transient protonation of Asp-85 and formation of the blue membrane. The latter conclusion is based on the observation that the rate constant of dark adaptation is directly proportional to the fraction of blue membrane (in which Asp-85 is protonated) between pH 2 and 11. The rate constant of isomerization is at least 10(4) times faster in the blue membrane than in the purple membrane. The protonated state of Asp-85 probably is important for the catalysis not only of all-trans <=> 13-cis thermal isomerization during dark adaptation but also of the reisomerization of the chromophore from 13-cis to all-trans configuration during N-->O-->bR transition in the photocycle. This would explain why Asp-85 stays protonated in the N and O intermediates.     

Sampling properties of DNA sequence data in phylogenetic analysis

We inferred phylogenetic trees from individual genes and random samples of nucleotides from the mitochondrial genomes of 10 vertebrates and compared the results to those obtained by analyzing the whole genomes. Individual genes are poor samples in that they infrequently lead to the whole-genome tree. A large number of nucleotide sites is needed to exactly determine the whole-genome tree. A relatively small number of sites, however, often results in a tree close to the whole-genome tree. We found that blocks of contiguous sites were less likely to lead to the whole-genome tree than samples composed of sites drawn individually from throughout the genome. Samples of contiguous sites are not representative of the entire genome, a condition that violates a basic assumption of the bootstrap method as it is applied in phylogenetic studies.      

Quantum efficiency of the photochemical cycle of bacteriorhodopsin

Values in the literature for the quantum efficiency of the photochemical cycle of bacteriorhodopsin (bR) range from 0.25 to 0.79 and the sum of the quantum yields of the forward and back photoreactions [Formula: see text] has been proposed to be 1. In the present work, low intensity laser flashes (532 nm) and kinetic spectroscopy were used to determine the quantum efficiency of bR photoconversion, [UNK]_bR, by measuring transient bleaching of bR at 610 nm in the millisecond time scale. Bovine rhodopsin (R) in 2% ammonyx LO was used as a photon counter. We find that the ratio of the quantum yields of bacteriorhodopsin photoconversion and bleaching of rhodopsin, [UNK]_bR/[UNK]_R, is 0.96 ± 0.04. Based on the quantum yield of the photobleaching of rhodopsin, 0.67, the quantum efficiency of bR photoconversion was determined to be 0.64 ± 0.04. The quantum yield of M formation was found to be 0.65 ± 0.06. From the transient bleaching of bR at 610 nm with a saturating laser flash (28 mJ/cm²) the maximum amount of bR cycling was estimated to be 47 ± 3%. From this value and the spectrum of K published in the literature, the ratio of the efficiencies of the forward and back light reactions, [UNK]₁/[UNK]₂, was estimated to be 0.67 ± 0.06 and so [UNK]₂ ≈ 1 (0.94 ± 0.06). The sum of [UNK]₁ + [UNK]₂ ≈ 1.6. It was found that repeated high-intensity laser flashes (>20 mJ/cm²) irreversibly transformed bR into two stable photoproducts. One has its absorption maximum at 605 nm and the other has a well-resolved vibronic spectrum with maxima at 342, 359 (main peak), and 379 nm. The quantum yield of the formation of the photoproducts is ≈ 10^-4.     

Effect of apomorphine on rat brain opiate receptors

Stereospecific binding of apomorphine to rat brain opiate receptors was shown by assaying the competition of 7,8(n)--3H--naloxone and D-ala2-tyrosyl-3,5-3H--enkephalin (5-D-leucine) for opiate receptor binding. EC-NaCl50, the concentration of apomorphine which inhibited 50% binding of the radioactive naloxone and D-ala2, D-leu5-enkephalin in the absence of NaCl were 20 and 42 microM, respectively. EC+NaCl 50, the concentration of apomorphine which inhibited 50% binding of the radioactive naloxone in the presence of 100 mM NaCl was 17 microM. From the ratio of EC+NaCl 50 to EC-NaCl the value of "sodium shift" of effective concentration can be calculated as 0.85. From the data obtained it is concluded that apomorphine, like naloxone, is a "pure" antagonist but it has much less affinity for enkephalin and opiate binding sites. The probable mechanisms of the pharmacological action of apomorphine are discussed.     

Ultrastructural study of the internal organs of ixodid ticks in the scanning electron microscope

The structure of the surfaces of midgut and salivary glands in hungry and engorged females of Hyalomma asiaticum was studied by means of scanning electron microscopy. Preparations were fixed in glutaraldehyde osmium and then dehydrated by the critical point method and gold or platinum coating Different periods of fixation at room temperature or at 4 degrees C did not affect the condition of surface structures of gut and salivary glands.     

The role of blood-sucking ticks and insects in natural foci of infections

During 60 years passed after the creation of the conception on natural focus of human infections by the academician E. N. Pavlovski? our knowledge on a role of blood-sucking insects and ticks in this phenomenon have been significantly enlarged. It has been recovered, that these arthropods serve not only as vectors of infection agents, but also as natural reservoirs and amplificators of these agents. In the process of the infection agent circulation in the natural focus there are several additional pathways of circulations without a participation of vertebrates. These pathways are as follows: the dispersion of infection agent in cases of simultaneous feeding of infected and "clean" ticks, and also the sexual, transovarial and transphase transmissions of pathogens. These mechanism of circulation are most often occur in the ixodid ticks, which can play the main role in supporting the infection agent in the focus.     

The endogenous allosteric regulators of receptors

The literature data devoted to endogenous allosteric regulators of membrane bound receptors are summarized in the present review. The allosteric processes are classified to (i) cooperative interaction, (ii) nonspecific, (iii) functional, and (iv) specific regulations according to target topography in a receptor. The specific endogenous allosteric regulators are described for GABAA, NMDA, muscarinic, nicotinic, serotonin, and opioid receptors. Substances of different chemical structure (peptides, lipids, and polycyclics) are able both to activate or inhibit binding and function of respective receptors. Some pathological processes appear to depend on endogenous receptor modulators. The role of the regulators is speculated in terms of receptor homeostasis, in particular, counteraction of receptor tolerance and/or sensitisation during physiological pulsation in a ligand' level in synaptic cleft.