In vitro study of the antibacterial effect of the bacteriophage T5 thermostable endolysin on Escherichia coli cells.

AIMS: This study aimed to evaluate lysis of Escherichia coli stationary cell cultures induced by the combined action of bacteriophage T5 endolysin (l-alanyl-d-glutamate peptidase) and low doses of various cationic agents permeabilizing the outer membrane of Gram-negative bacteria (polymyxin B, gramicidin D, poly-l-lysine, chlorhexidine and miramistin). METHODS AND RESULTS: The enzyme activity was assayed with the turbidimetric method. Antimicrobial activity was assessed through the number of colony-forming units (CFUs); the results of calculation were represented as logarithmic units. The optical microscopy examination of bacterial cells was conducted in the phase-contrast mode. The use of bacteriophage T5 endolysin in combination with polymyxin B (0·4 µg ml-1 ) or chlorhexidine (0·5 µg ml-1 ) made it possible to reduce the number of CFUs by five orders of magnitude; and in combination with poly-l-lysine (80 µg ml-1 ) by four orders, as compared to control. The endolysin was found to be a thermostable protein: it retained ~65% of its initial activity after heating for 30 min at 90°C. Examining the curves of its thermal denaturation revealed the half-transition temperature to be 56·3 ± 1·0°C. Circular dichroism spectra showed that after recooling the protein restored up to 80% of its native structure. CONCLUSIONS: A substantial synergistic effect of the bacteriophage T5 endolysin and membrane-permeabilizing compounds was demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: The study of thermal stability of the bacteriophage T5 endolysin and the quantified assessment of its antimicrobial activity have been done for the first time. The approach examined lays foundations for designing a two-component preparation which would effectively lyse cells of Gram-negative pathogens from outside.

Method for isolation of intact titin (connectin) molecules from mammalian cardiac muscle.

Cardiac titin was isolated from rabbit and ground squirrel ventricular muscles by a method that was used earlier to obtain myofibrils with intact minor proteins located in A-bands of sarcomeres (Podlubnaya, Z. A., et al. (1989) J. Mol. Biol., 210, 655-658). Small pieces of cardiac muscle were incubated for 2-3 weeks at 4°C in Ca²âº-depleting solution before their homogenization to decrease activity of Ca²âº-dependent proteases. Then the muscle was homogenized, and titin was isolated by the method of Soteriou, A., et al. (1993) J. Cell Sci., 14, 119-123. In control experiments, titin was isolated from cardiac muscle without its preincubation in Ca²âº-depleting solution. Sometimes control titin preparations contained only T2-fragment, but generally they contained ~5-20% N2B-isoform of titin along with its T2-fragment. Preparations of titin obtained from rabbit cardiac muscle by our method contained ~30-50% of N2BA- and N2B-titin isoforms along with its T2-fragment. The content of α-structures in titin isolated by our method was increased. Actomyosin ATPase activity in vitro increased in the presence of titin preparations containing more intact molecules. This result confirms the significant role of titin in the regulation of actin-myosin interaction in muscles. The method used by us to preserve titin might be used for isolation of other proteins that are substrates of Ca²âº-dependent proteases.

Changes in isoform composition, structure, and functional properties of titin from Mongolian gerbil (Meriones unguiculatus) cardiac muscle after space flight.

Changes in isoform composition, secondary structure, and titin phosphorylation in Mongolian gerbil (Meriones unguiculatus) cardiac muscle were studied after 12-day-long space flight onboard the Russian spacecraft Foton-M3. The effect of titin on the actin-activated myosin ATPase activity at pCa 7.5 and 4.6 was also studied. Almost twofold increase in titin long N2BA isoform content relative to that of short N2B isoform was found on electrophoregrams of cardiac muscle left ventricle of the flight group gerbils. Differences in secondary structure of titin isolated from cardiac muscle of control and flight groups of gerbils were found. An increase in phosphorylation (1.30-1.35-fold) of titin of cardiac muscle of the flight group gerbils was found. A decrease in activating effect of titin of cardiac muscle of the flight group gerbils on actomyosin ATPase activity in vitro was also found. The observed changes are discussed in the context of M. unguiculatus cardiac muscle adaptation to conditions of weightlessness.

[Thermodynamic behaviour and conformational changes of alcohol oxidase of yeasts Hansenula polymorpha].

We studied influence of heating, ethanol and sodium azide on the structural and conformational changes of the alcohol oxidase from yeast Hansenula polymorpha. The increase of fluorescence of alcohol oxidase -cofactor, flavin adenine dinucleotide, was revealed when heated to 60 degrees C while the enzymatic activity of alcohol oxidase remained unchanged. Differential scanning microcalorimetry revealed that ethanol stabilized the protein structure and increased the temperature of melting, Based on the data of circular dichroism, we concluded that the percentage of helicities in the secondary structure of alcohol oxidase was not influenced by both ethanol and sodium azide, however ethanol significantly modified the circular dichroism spectrum associated with the tertiary structure of alcohol oxidase.

[Secondary structure of SsoII-like (cytosine-5)-DNA methyltransferases N-terminal region determined by circular dichroism spectroscopy].

(Cytosine-5)-DNA methyltransferase SsoII (M.SsoII) has a long N-terminal region (1-71 residues) preceding the sequence with conservative motifs, which are characteristic for all DNA methyltransferases of such kind. The presence of this region provides M.SsoII capability to act as a transcription regulator in SsoII restriction-modification system. To perform its regulatory function, M.SsoII binds specifically to a 15-mer inverted repeat in the promoter region of SsoII restriction-modification system genes. In the present work, properties of the protein delta(72-379)M.Ecl18kI are studied, which is a deletion mutant of the SsoII-like DNA-methyltransferase M.Ecl18kI and is homologous to M.SsoII N-terminal region. delta(72-379)M.Ecl18kI capability to bind specifically a DNA duplex containing the regulatory site is demonstrated. However, such a binding takes place only in the presence of high protein excess relative to DNA, which could indicate an altered structure in the deletion mutant in comparison with the full-length M.SsoII. Circular dichroism spectroscopy demonstrated that delta(72-379)M.Ecl18kI has a strongly pronounced secondary structure and contains 32% a-helices and 20% beta-sheets. Amino acid sequences alignment of M.SsoII N-terminal region and transcription factors of known spatial structure is made. An assumption is made how alpha-helices and beta-sheets are arranged in M.SsoII N-terminal region.

[2'-aldehyde oligonucleotides: synthesis and use for affinity modification of DNA-recognizing proteins].

Oligonucleotides with 1,2-diol grouping were prepared from 2'-O-[2-(2,3-dihydroxypropyl)amino-2-oxo-ethyl]uridine 3'-phosphoramidite. The thermal stability of modified DNA duplexes and their ability to form complexes with the p50 subunit of the NF-kappaB transcription factor and (cytosine-5)-DNA methyltransferase SsoII were studied. The periodate oxidation of the l,2-diol grouping of the oligonucleotides resulted in reactive 2'-aldehyde derivatives. The opportunity of their use for the affinity modification of DNA-recognizing proteins was studied.