An Abnormally High Closing Potential of the OMPF Porin Channel from Yersinia Ruckeri: The Role of Charged Residues and Intramolecular Bonds.

Electrophysiological experiments on bilayer lipid membranes showed that the isolated outer membrane major porin of Yersinia ruckeri (YrOmpF) exhibits activity typical of porins from Gram-negative bacteria, forming channels with a mean conductance of 230 pS (in 0.1 M KCl) and slight asymmetry with respect to the applied voltage. Under acidic conditions (up to pH = 3.0), there was no significant decrease in the total conductance of the YrOmpF channel reconstituted into the bilayer. The studied channel significantly differed from the porins of other bacteria by high values of its critical closing potential (Vc): Vc = 232 mV at pH = 7.0 and Vc = 164 mV at pH = 5.0. A theoretical model of the YrOmpF spatial structure was used for the analysis of the charge distribution in the mouth and inside the channel to explain these properties and quantitatively assess the bonds between the amino acid residues in the L3 loop and on the inner wall of the barrel. The parameters of YrOmpF were compared with those of the classical OmpF porin from E. coli. The results of electrophysiological experiments and theoretical analysis are discussed in terms of the mechanism for voltage-dependent closing of porin channels.

Non-Specific Porins of Yersinia pseudotuberculosis as Inductors of Experimental Hyperthyroidism in Mice.

In vivo experiments showed that antibodies to OmpC and OmpF porins of Yersinia pseudotuberculosis increased thyroxine (T4) level in the blood of experimental animals. The mice were immunized with different antigens: recombinant OmpF porin in a soluble monomeric form, trimers of OmpC and OmpF porins isolated from the outer membrane, or antibodies to them. The level of thyroxine in the blood of mice immunized with OmpF and OmpC porins increased by 5.47 and 22.3 times, respectively; after immunization with antibodies to these proteins, blood thyroxine increased by 9.28 and 14.29 times. Immunization with recombinant OmpF porin induced no reliable increase in thyroxine level. Hence, the serum to recombinant OmpF porin contains no antibodies specific to conformational antigenic determinants that are present in the protein trimer and, according to our previous findings from molecular docking studies, determine cross-reactions between OmpF porin of Y. pseudotuberculosis and thyroidstimulating hormone receptor.

Hct-a is a new actinoporin family from the heteractis crispa sea anemone.

Several new actinoporin isoforms with molecular weights of 18995.5 to 19398.7 Da exhibiting a high hemolytic activity were isolated from the tropical sea anemone Heteractis crispa using a combination of liquid chromatography techniques. The actinoporins were demonstrated to occur as mono-, di-, and trimers in aqueous solutions. The sequences of the genes encoding actinoporins were identified, and the amino acid sequences of the new polypeptides belonging to the Hct-A actinoporin family were obtained. The new acinoporins differ in their isoelectric points, the number and localization of charged amino acid residues at the functionally important N-terminal fragment of the molecule, as well as in the charge of a tetrapeptide (amino acid residues 74-77) involved in an electrostatic interaction with the cytoplasmic membrane. A recombinant actinoporin, rHct-A2, with a molecular weight of 19141 Da, pI of 9.64, and hemolytic activity of 4.0 × 104 HU/mg, was obtained. The conductivity of the ion channels formed by rHct-A2 in the BLM was demonstrated to be similar to that of the native actinoporin from H. crispa. The obtained data expand knowledge on the structural and functional relationships of actinoporins and contribute to our understanding of the functioning mechanism of these molecules, which is the basis for the development of compounds with a high biomedical potential. Currently, they are considered as models for obtaining antitumor, antibacterial, and cardiac-stimulating agents.

[Interaction of sea amemone Heteractis crispa Kunitz type polypeptides with pain vanilloid receptor TRPV1: in silico investigation].

Using methods of molecular biology we defined the structures of the 31 sea anemone Heteractis crispa genes encoding polypeptides which are structurally homologous to the Kunitz proteinase inhibitor family. Identified amino acid sequences have point residue substitutions, high degree of homology with sequences of known H. crispa Kunitz family members, and represent a combinatorial library of polypeptides. We generated their three-dimensional structures by homologous modeling methods. Analysis of their molecular electrostatic potential enabled us to divide given polypeptides into three clusters. One of them includes polypeptides APHC1, APHC2 and APHC3, which were earlier shown to possess a unique property of inhibiting of the pain vanilloid receptor TRPV1 in vitro and providing the analgesic effects in vivo in addition to their trypsin inhibitory activity. Molecular docking made possible establishing the spatial structure of the complexes, the nature of the polypeptides binding with TRPV1, as well as functionally important structural elements involved in the complex formation. Structural models have enabled us to propose a hypothesis contributing to understanding the APHC1-3 impact mechanism for the pain signals transduction by TRPV1: apparently, there is an increase of the receptor relaxation time resulted in binding of its two chains with the polypeptide molecule, which disrupt the functioning of the TRPV1 and leads to partial inhibition of signal transduction in electrophysiological experiments.

New Actinoporins from sea anemone Heteractis crispa: cloning and functional expression.

A new actinoporin Hct-S4 (molecular mass 19,414 ± 10 Da) belonging to the sphingomyelin-inhibited α-pore forming toxin (α-PFT) family was isolated from the tropical sea anemone Heteractis crispa (also called Radianthus macrodactylus) and purified by methods of protein chemistry. The N-terminal nucleotide sequence (encoding 20 amino acid residues) of actinoporin Hct-S4 was determined. Genes encoding 18 new isoforms of H. crispa actinoporins were cloned and sequenced. These genes form a multigene Hct-S family characterized by presence of N-terminal serine in the mature proteins. Highly conserved residues comprising the aromatic phosphorylcholine-binding site and significant structure-function changes in the N-terminal segment (10-27 amino acid residues) of actinoporins were established. Two expressed recombinant actinoporins (rHct-S5 and rHct-S6) were one order less hemolytically active than native actinoporins.

[Isolation of conjugates of albumin and ribonuclease]. / Vydelenie kon''iugatov al'bumina i ribonukleaz.

A method for separation of albumin-ribonuclease (RNase) conjugates has been proposed, based on the use of macroporous silicates. It was established that about 76% of ligand-free human serum albumin (LFHSA) formed complexes with enzymes. It was shown that most of the conjugates of albumin and pancreatic RNase contained up to 2 mol enzyme per 1 mol LFHSA. The conjugates of albumin and bacterial RNase, isolated from the cells of the strain Bacillus intermedius 7P, displayed higher specific activities, containing, on average, 2.3 mol RNase per 1 mol LFHSA (for the conjugates with molecular weights below 92 kDa) or 3.3 mol RNase per 1 mol protein carrier (for the conjugates with higher molecular weight).

[Conjugates of pancreatic ribonuclease and ligand-free human serum albumin]. / Kon''iugaty pankreaticheskoi ribonukleazy s bezligandnym syvorotochnym al'buminom cheloveka.

Conjugates of pancreatic RNase and ligand-free human serum albumin (LFHSA) have been obtained. The number of hydrophobic binding sites both for initial HSA and LFHSA has been determined by the polarised luminescence method. Interaction between RNase and HSA involves additional electrovalent linkage. Unlike initial enzyme, conjugates exhibit activity toward double-strand RNA. After intravenous injection, transferase activity of unmodified enzyme remains in the blood during 20 min., whereas 30-40% of this activity is detected at the fourth day after administration of RNase conjugates. A single dose administration of LFHSA-RNAse conjugates exhibited high antiviral activity in mice, infected with influenza A and influenza B viruses.

[Interaction of Bacillus intermedius 7P ribonuclease with ligand-free serum albumin]. / Vzaimodeistvie ribonukleazy Bacillus intermedius 7P s bezligandnym syvorotochnym al'buminom.

The efficiency of the binding of RNase 7P molecules to albumin on cocondensation with the aim of producing the prolonged action forms of the enzyme can be increased by using ligand-free human serum albumin (LFHSA). The CD method showed that LFHSA underwent changes of the cooperative character under the action of acid and urea. On potentiometric titration the number of titrated groups of LFHSA decreased with time. The GPC method demonstrated the RNase bound more efficiently to freshly dissolved LFHSA. In this case part of the enzymic activity was manifested only after proteolysis of the albumin carrier. Cocondensation with the aid of glutaraldehyde resulted in the formation of LFHSA-RNase conjugates composed of 1-2 moles of human serum albumin and 1-6 moles of RNase. More than 50% of transferase activity retained in the blood plasma for 2-3 days after intravenous injection of the conjugates with a molecular weight of 70-80 kD to rabbits.

[Permeation of pancreatic ATPase through the blood-brain barrier into the cerebral parenchyma]. / Proniknovenie pankreaticheskoi RNKazy cherez gematoéntsefalicheskii bar'er v parenkhimu mozga.

Penetration of exogenous RNAases, native and albumin polymer-bound, from circulation into the brain parenchyma was studied. Native pancreatic RNAase was found to extend through the hematoencephalic barrier; maximal enzymatic activity was detected within 15-30 min after administration into a rat tail vein. The enzyme activity was not observed within 90 min both in blood serum and brain tissue. The conjugate of RNAase and human albumin with molecular mass of 300,000 penetrated through the hematoencephalic barrier. Maximal enzymatic activity was estimated within 3-5 hrs after administration and maintained rather high within 24 hrs in blood and brain parenchyma. This suggests that the preparations of this kind are promising in experimental medicine.

[Nuclease conjugates with ligand-free serum albumin]. / Kon''iugaty nukleaz s bezligandnym syvorotochnym al'buminom.

In order to obtain nuclease and human serum albumin (HSA) conjugates with a high enzyme content it is proposed to use a ligand-free HSA. The ligands are removed with the help of a strong anion exchanger. A two-stage procedure of conjugate preparation is proposed. It consists in the complexation of ligand-free HSA and enzyme and subsequent co-condensation of protein molecules of the poly-complex with the aid of glutaric aldehyde. When the conjugates are administered to rabbits intravenously, the RNAase activity is manifested in blood for 3-5 days. Moreover, in the case of conjugates with a molecular weight of 80 kDa, the prolongation time is greater than for conjugate with a higher molecular weight.