The DNA-[N6-adenine] methyltransferase of T4 phage (T4 Dam MTase) catalyzes methyl group transfer from S-adenosyl-L-methionine (AdoMet) to the N6-position of adenine in the palindromic sequence, GATC. We have investigated the effect of eliminating different structural components of the recognition site on the ability of a substrate to be bound and methylated by T4 Dam. For this purpose, steady state binding (by gel shift assays) and kinetic parameters of methylation (using the methyl donor, [3H-CH3]-AdoMet, at 25 degrees C) were studied using various synthetic duplex oligonucleotides containing some defect in the DNA-target site; e.g., the absence of an internucleotide phosphate or a nucleotide(s) within the recognition site, or a single stranded region. The salient results are summarized as follows: (1) Addition of T4 Dam to a complete reaction mixture (with a 20-mer duplex as substrate) resulted in a 'burst' of 3H-methylated product, followed by a constant rate of product formation that reflected establishment of steady-state conditions. This suggests that the rate-limiting step is release of product methylated DNA from the enzyme [and not the transfer of the methyl group]. (2) A number of the defects in duplex structure had only a weak influence on the binding and Km values, but strongly reduced the kcat. At the same time, several poorly bound duplexes retained good substrate characteristics, especially duplexes having uninterrupted GAT-sequences in both strands. Whereas having only one half of the recognition site element intact was sufficient for stable complex formation, the catalytic turnover process had a strict requirement for an uninterrupted GAT-sequence on both strands. (3) There was no correlation between Km and binding capability; the apparent Kd for some duplexes was 5-70 times higher than Km. This indicates that the T4 Dam methylation reaction can not be explained by a simple Michaelian scheme.

The published data on the preparation and the The published data on the preparation and the dispersion-structural properties of nano-sized TiO dispersion-structural properties of nano-sized TiO 2 2 are are considered. Attention is focused on its sol ± gel synthesis considered. Attention is focused on its sol ± gel synthesis from different precursors. The possibilities for the purpose-from different precursors. The possibilities for the purposeful control and stabilization of properties of TiO ful control and stabilization of properties of TiO 2 2 nano-nanopowders and sols are analyzed. Information on powders and sols are analyzed. Information on physicochemical methods used in studies of the particle physicochemical methods used in studies of the particle size and the phase composition of nanodisperse TiO size and the phase composition of nanodisperse TiO 2 2 is is presented. The prospects of using nano-sized TiO presented. The prospects of using nano-sized TiO 2 2 in in medicine and nanobiotechnology are considered. The bib-medicine and nanobiotechnology are considered. The bibliography includes 95 references liography includes 95 references. . Synthesis and stabilization of nano-sized titanium dioxideZ R Ismagilov, L T Tsykoza, N V Shikina, V F Zarytova, V V Zinoviev (deceased), S N Zagrebelnyi

The fluorescence of 2-aminopurine ((2)A)-substituted duplexes (contained in the GATC target site) was investigated by titration with T4 Dam DNA-(N6-adenine)-methyltransferase. With an unmethylated target ((2)A/A duplex) or its methylated derivative ((2)A/(m)A duplex), T4 Dam produced up to a 50-fold increase in fluorescence, consistent with (2)A being flipped out of the DNA helix. Though neither S-adenosyl-L-homocysteine nor sinefungin had any significant effect, addition of substrate S-adenosyl-L-methionine (AdoMet) sharply reduced the Dam-induced fluorescence with these complexes. In contrast, AdoMet had no effect on the fluorescence increase produced with an (2)A/(2)A double-substituted duplex. Since the (2)A/(m)A duplex cannot be methylated, the AdoMet-induced decrease in fluorescence cannot be due to methylation per se. We propose that T4 Dam alone randomly binds to the asymmetric (2)A/A and (2)A/(m)A duplexes, and that AdoMet induces an allosteric T4 Dam conformational change that promotes reorientation of the enzyme to the strand containing the native base. Thus, AdoMet increases enzyme binding-specificity, in addition to serving as the methyl donor. The results of pre-steady-state methylation kinetics are consistent with this model.

. After AdoHcy release, the enzyme remains in the F conformational form and is able to preferentially bind AdoMet (unlike form E, which randomly binds AdoMet and DNA), and the AdoMet-F binary complex then binds DNA to start another methylation cycle. We also propose an alternative pathway in which the release of AdoHcy is coordinated with the binding of AdoMet in a single concerted event, while T4 Dam remains in the isomerized form F. The resulting AdoMet-F binary complex then binds DNA, and another methylation reaction ensues. This route is preferred at high AdoMet concentrations.

The small-angle X-ray scattering technique (SAXS) is proposed for the investigation of equilibrium macromolecular interactions of the enzyme-substrate type in solution. Experimental procedures and methods of analysing the data obtained from SAXS have been elaborated. The algorithm for the data analysis allows one to determine the stoichiometric, equilibrium and structural parameters of the enzyme-substrate complexes obtained. The thermodynamic characteristics for the formation of complexes of tRNAphe with phenylalanyl-tRNA synthetase have been determined and demonstrate negative cooperativity for binding of the two tRNAPhe molecules. The structural parameters (I$, &, semi-axes) have been determined for free phenylalanyl-tRNA synthetase and tRNAPhe from E. coli MRE-600 and of enzyme complexes possessing one and two tRNAPhe molecules, indicating structural rearrangements of the enzyme in the interaction with tRNAPhe.Small-angle X-ray scattering; Phenylalanyl-tRNA synthetase; Enzyme-substrate interaction; tRNAPhe