Caldesmon-calmodulin interaction. Study by the method of protein intrinsic tryptophan fluorescence.

Addition of calmodulin to caldesmon causes a concentration-dependent shortwave shift and an increase of fluorescence intensity of caldesmon tryptophan residues. The existence of a protein complex is confirmed by the increase of the caldesmon sedimentation coefficient s0(20,w) from 3.0 S to 4.5 S in the presence of calmodulin. These findings allow application of the method of protein intrinsic tryptophan fluorescence for quantitative study of unmodified caldesmon and calmodulin in solution. The affinity of the caldesmon-calmodulin interaction (Kass = 1.8 x 10(6) M-1, in 0.1 M-KCl at 25 degrees C) and Ca2+ requirement (half-maximum binding at 0.8 microM-Ca2+) determined by means of the fluorescence technique are in agreement with previously reported values, thus confirming the validity of the method. The same approach was further used to provide information about the nature of interactions stabilizing the caldesmon-calmodulin complex. Association of the proteins and dissociation of the complex were studied in different physicochemical conditions, including variation of pH, temperature and ionic strength and the addition of quenchers, denaturants and anticalmodulin drugs. The results obtained suggest that caldesmon tryptophan residues, together with charged groups, are involved in calmodulin binding. Hydrophobic, electrostatic and hydrogen interactions contribute to the stability of the protein complex, making it insensitive to variations of physicochemical conditions within physiological limits.     

Elongation factor EF-Ts interacts with the aminoacyl-tRNA.EF-Tu.GTP complex]

The fluorescence polarization technique has been used to study the interaction of the EF-Ts dansyl derivative with EF-Tu after nucleotide exchange and binding of the aminoacyl-tRNA to EF-Tu.GTP. It is shown that the ternary complex formation results in the increase of EF-Ts affinity to EF-Tu and EF-Ts remains bound to EF-Tu up to the GTP hydrolysis stage on the ribosome.     

Expression of Bacillar Glutamyl Endopeptidase Genes in Bacillus subtilis by a New Mobilizable Single-Replicon Vector pLF

The pLF1311 natural plasmid from Lactobacillus fermentum 1311 was used to construct a single-replicon vector suitable for rapid cloning in a wide range of gram-positive hosts and Escherichia coli. The new vector is capable of conjugative mobilization from E. coli to various hosts by conjugal transfer. The final vector (3.4 kb) showed a high segregational and structural stability and a high copy number. Glutamyl endopeptidase genes from Bacillus licheniformis (gseBL) and B. intermedius (gseBI) were cloned in both pLF9 and pLF14 vectors and introduced to B. subtilis. The yield of enzymes in the pLF-derived producers was 6- to 30-fold more than in the natural producers and reached 100–150 mg/L of mature protease.     

The effect of pH on the conformation and stability of the structure of plant toxin-ricin

The effect of pH on the conformation of ricin and its A- and B-chains has been studied by measuring their intrinsic fluorescence. At pH 5.0 and 7.5, the structural stability of toxin and subunits was estimated according to the denaturing action of guanidine hydrochloride. It was demonstrated that the fluorescence of native toxin and catalytic A-subunit does not depend significantly on pH in the range pH 3-8, whereas ricin B-chain undergoes a structural transition at pH less than 5.0. The structural stability of ricin and isolated chains differs significantly at pH 7.5 and 5.0; the structural stability of ricin and the A-chain increases, whereas that of the B-chain decreases.     

Recombination of micellar complexes of apolipoprotein A1--phosphatidylcholine in the presence of complex components]

Interaction of micellar complexes apolipoprotein A1--phosphatidyl choline (apoA1--DMPC and apoA1--EPC) with complex components: apoA1 (dansyl-A1) and phosphatydil cholines (DMPC, EPC and spin labelled PC) was studied in the absence of lipoproteins and plasma components. Recombination of the complexes (changes in complex sizes and stoichiometry) was shown to occur in the presence of the complex components. Interaction of lipid-free apoA1 with the complex is a fast process; incorporation of PC molecule takes place more slowly. This recombination is considered to be a kinetikally complicated process, the rate of recombination depending on PC exchange and interconversion.     

Effect of lyophilization methods on the formation of free radicals, the survivability and makeup of a population of the lincomycin producer]

It was shown that a short-time exposure of the spores of Act. roseolus 981 to air (oxygen) on their lyophilization by the chamber method resulted in formation of increased numbers of the free radicals in the lyophilized spores suspension and decreased the survival of the spores on their further storage under vacuum. The phenomena were less pronounced when the exposure of the spores to the air was excluded by using the collector method of lyophilization. This testified to the advantages of the latter method as compared to the chamber one. No effect of the lyophilization methods on the population composition of the spontaneous variants of Act. roseolus 981 was observed.     

Inhibitor of inflammation,peptide fragment (65–76) of monocyte chemotactic protein-1 (MCP-1), inhibits binding of MCP-1 to heparin

Monocyte chemotactic protein-1 (MCP-1, CCL2) is one of the most important chemokines involved in inflammation. MCP-1 stimulates migration of monocytes and certain lymphocyte populations to the affected area, in particular to the sites of atherosclerotic plaque formation. Development of drugs inhibiting MCP-1 is now a topical task. We earlier designed and synthesized a dodecapeptide from C-terminal domain of MCP-1 (65–76, peptide X) that possessed an anti-inflammatory activity. The mechanism of action of chemokines (in particular, of MCP-1) in vivo is based on activation of CCR2 receptor on target cells and binding to glycosaminoglycans (GAGs) on the cell surface of and extracellular matrix. Peptide X did not affect the MCP-1-CCR2 interaction. Thus, we hypothesized that peptide X could impair MCP-1 binding to GAGs. Here we studied the effect of peptide X on the MCP-1 binding to heparin using the label-free biosensing device Picoscope®, enzyme-linked immunoassay (ELISA), and the intrinsic fluorescence method. According to the data obtained, peptide X interfered with the MCP-1-heparin binding, which may be due to the competition of peptide X with MCP-1 for heparin binding sites. Probably, this effect determines the anti-inflammatory activity of peptide X in vivo.