[Telomeric oligonucleotide complexes with PGEk protein vector: internalization by target cells and antiproliferative properties].

The recombinant protein PGEk, containing residual of the human epidermal factor (hEGF) bearing DNA binding sequence, retains ability of hEGF to bind with hEGF receptor and to induce cell proliferation was shown. On an example of PGEk complexes with telomeric mimic-oligodeoxyribonucleotide d(TTAGGG)4 and with its thio-analogue we had found such systems can be effectively and selectively internalized by hEGF receptors super expressing cells. The association of this process with a protein/oligonucleotide ratio in complexes was investigated. The intracellular localization of oligonucleotides was explored. We had shown that PGEk not only promotes intensive delivery of oligonucleotides, but also protects them from degradation by nucleases. The oligonucleotides in composition of complexes have considerably more expressed cytotoxic activity in comparison with free oligonucleotides.

[Cyclic AMP-specific nucleotide phosphodiesterase from the insoluble fraction of the human brain]. / cAMP-spetsifichnaia fosfodiésteraza tsiklicheskikh nukleotidov nerastvorimoi fraktsii mozga cheloveka.

The cAMP-specific cyclic nucleotide phosphodiesterase (PDE) from pellet fraction of human brain has been identified. Two alternative methods for isolation and purification of the enzyme are presented. The first method consists of three chromatographic steps (separation on the DEAE-Toyopearl column followed by gel filtrations on AcA-34 and G3000 SW columns) and results in purification of several forms of PDE. One of those is weakly inhibited by cGMP and is insensitive to Ca(2+)-calmodulin or cyclic nucleotides. The second procedure consisting of the chromatographic separation on blue agarose and DEAE-TSK-5PW ion-exchange resin leads to an isolation of several peaks of cAMP-specific PDE. Both methods produce different cAMP-specific forms of PDE with molecular masses ranging from 36 to 47 kDa. These forms differ in kinetic properties (Km = 2.0-7.3 microM, Ki = 1480-5300 microM for cGMP). However, the KicGMP/KmcAMP ratio remains constant for all the peaks of the cAMP-specific enzyme.

[Ca2+-calmodulin-activated cyclic nucleotide phosphodiesterase from the soluble fraction of the human brain: Kinetic properties and the effect of the antidepressant pyrazidol and its nitro analog on the enzyme]. / Ca2+-kal'modulin-aktiviruemaia fosfodiésteraza tsiklicheskikh nukleotidov rastvorimoi fraktsii mozga cheloveka: Kineticheskie svoistva, deistvie antidepressanta pirazidola i ego nitroanaloga na ferment.

The kinetic characteristics of two forms of soluble Ca(2+)-calmodulin-activated phosphodiesterase (PDE I and PDE II) from human brain are presented. Both PDE forms display a higher affinity for cGMP than for cAMP: the differences in the Km values of PDE II for the both cyclic nucleotides are less pronounced. For the both enzyme forms cGMP competitively inhibits cAMP hydrolysis. Activation of PDE I by Ca(2+)-calmodulin is characterized by increasing Vmax without any changes in Km for both substrates. In case of PDE II the Ca(2+)-calmodulin-dependent increase in Vmax is accompanied by decreasing Km for cAMP and cGMP. Pyrazidol and nitropyrazidol inhibit Ca(2+)-calmodulin induced activity with no effect on basal PDE activity. The data obtained exclude competition of the both compounds with the substrate and the activator for the catalytic site and the allosteric Ca(2+)-calmodulin-specific site. It is suggested that the allosteric effect of these compounds on the activity of PDE involves a site in the enzyme which is distinct from the calmodulin-binding site.

[3',5'-cyclic nucleotide phosphodiesterase from human brain]. / Fosfodiésteraza 3', 5'-tsiklicheskikh nukleotidov iz mozga cheloveka.

3':5'-Cyclic nucleotide phosphodiesterase was isolated from human brain and characterized. After the first stage of purification on phenyl-Sepharose, the enzyme activity was stimulated by Ca2+ and micromolar concentrations of cGMP. High pressure liquid chromatography on a DEAE-TSK-3SW column permitted to identify three ranges of enzymatic activity designated as PDE I, PDE II and PDE III. Neither of the three enzymes possessed a high selectivity for cAMP and cGMP substrates. The catalytic activity of PDE I and PDE II increased in the presence of Ca2+-calmodulin (up to 6-fold); the degradation of cAMP was decreased by cGMP. The Ca2+-calmodulin stimulated PDE I and PDE II activity was decreased by W-7. PDE I and PDE II can thus be classified as Ca2+-calmodulin-dependent phosphodiesterases. With cAMP as substrate, the PDE III activity increased in the presence of micromolar concentrations of cGMP (up to 10-fold), Ca2+ and endogenous calmodulin (up to 2-3-fold). No additivity in the effects of saturating concentrations of these compounds on PDE III was observed. Ca2+ did not influence the rate of cGMP hydrolysis catalyzed by PDE III. In comparison with PDE I and PDE II, the inhibition of PDE III was observed at higher concentrations of W-7 and was not limited by the basal level of the enzyme. These results do not provide any evidence in favour of the existence of several forms of the enzyme in the PDE III fraction. The double regulation of PDE III creates some difficulties for its classification.