A serine protease inhibitor with a molecular mass of 6106 +/- 2Da (designated as InhVJ) was isolated from the tropical anemone Radianthus macrodactylus by a combination of liquid chromatography methods. The molecule of InhVJ consists of 57 amino acid residues, has three disulfide bonds, and contains no Met or Trp residues. The N-terminal amino acid sequence of the inhibitor (19 aa residues) was established. It was shown that this fragment has a high degree of homology with the N-terminal amino acid sequences of serine protease inhibitors from other anemone species, reptiles, and mammals. The spatial organization of the inhibitor at the levels of tertiary and secondary structures was studied by the methods of UV and CD spectroscopy. The specific and molar absorption coefficients of InhVJ were determined. The percentage of canonical secondary structure elements in the polypeptide was calculated. The inhibitor has a highly ordered tertiary structure and belongs to mixed alpha/beta or alpha + beta polypeptides. It was established that InhVJ is highly specific toward trypsin (Ki 2.49 x 10(-9) M) and alpha-chymotrypsin (Ki 2.17 x 10(-8) M) and does not inhibit other proteases, such as thrombin, kallikrein, and papain. The inhibitor InhVJ was assigned to the family of the Kunitz inhibitor according to its physicochemical properties.
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.
A low-molecular-weight immunoglobulin-binding protein (IBP) bound with the cell envelope has been isolated from Yersinia pseudotuberculosis cells and partially characterized. This IBP is a hydrophilic protein with a high polarity index of 55.3%. The molecular weight of the protein has been determined by MALDI-TOF mass spectrometry as 14.3 kD. CD spectroscopy showed that the IBP has high contents of the beta-structure and random coil structure. The IBP contains glycine as the N-terminal amino acid. The protein can be stored for a long time at acidic pH values but aggregates and loses activity at alkaline and neutral pH. The IBP binds rabbit IgG with optimum at pH of 6.0-7.5. The IBP interacts with IgG molecule in the Fc-fragment region. The protein retains activity after heating at 100 degrees C in the presence of SDS.
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