[The phosphorylation state of transducin beta-subunits].

The supposition that nucleoside diphosphate kinase is the enzyme that phosphorylates transducin beta-subunits on one of the histidine residues (His-266) has been analyzed. It stands the reason that 1) this enzyme is multifunctional and plays in particular the role of protein histidine kinase; and 2) the phosphorylated beta-subunit of transducin may activate transducin via the mechanism of transphosphorylation. Nevertheless, in our experiments, in which different forms of transducin preparations were incubated with α- and ß-isoforms of recombinant rat NDP kinase in the presence of [γ32P]ATP or [γ32P]GTP (specific activity of about 1 Ci/mmol) followed by separation of proteins by electrophoresis and-gel radio-autography, the phosphorylation of the transducin beta-subunit wasn't succeeded to be found. The negative result of our experiments most likely implies that the major part of transducin beta-subunits in the preparations has already been phosphorylated via a process that takes place in vivo.

[Involvement of cyclic adenosine monophosphate in the control of motile behavior of Physarum polycephalum plasmodium].

Possible involvement of autocrine factors into the control of motile behavior via a receptor-mediated mechanism was investigated in Physarum polycephalum plasmodium, a multinuclear amoeboid cell with the auto-oscillatory mode of motility. Cyclic adenosine monophosphate (cAMP) and extracellular cAMP-specific phosphodiesterase, its involvement into the control of plasmodium motile behavior was proved by action of its strong inhibitor, were regarded as putative autocrine factors. It was shown that the plasmodium secreted cAMP. When it was introduced into agar support, 0,1-1 mM cAMP induced a delay of the plasmodium spreading and its transition to migration. When locally applied, cAMP at the same concentrations induced typical for attractant action the increase in oscillation frequency and the decrease of ectoplasm elasticity. The ability to exhibit positive chemotaxis in cAMP gradient and the dependence of its realization were shown to depend on the plasmodium state. Chemotaxis test specimens obtained from the migrating plasmodium, unlike those obtained from growing culture, generate alternative fronts which compete effectively with fronts oriented towards the attractant increment. The results obtained support our supposition stated earlier that advance of the Physarum polycephalum plasmodium leading edge is determined by local extracellular cAMP gradients arising from a time delay between secretion and hydrolysis of the nucleotide.

[Purification of transducin betagamma-subunit complex from isotonic extracts of bovine retinal rod outer segments].

A method for obtaining a free complex of transducin betagamma-subunits from bovine retinal rod outer segments in a highly purified state has been suggested.

[Involvement of extracellular cAMP-specific phosphodiesterase in control of motile activity of Physarum polycephalum plasmodium].

Possible involvement of extracellular cAMP-specific phosphodiesterase in the control of cell motile behavior has been investigated in Physarum polycephalum plasmodium, a multinuclear amoeboid cell with the autooscillatory mode of motility. It was found that the rate of the hydrolysis of 10 mM cAMP by a partially purified preparation of cAMP-specific phosphodiesterase secreted by the plasmodium in the course of migration decreases 20-30 times under the action of 1 mM dithiothreitol. In the presence of 1-5 mM of this strong reducing agent, the onset of the plasmodium spreading and the transition to the stage of migration were delayed in a concentration-dependent manner. In accordance with the morphological pattern of motile behavior, the duration of the maintenance of high frequency autooscillations, which normally precede the increase in the rate of the spreading and appear also in response to the application of attractants at spatially uniform concentrations, strongly increased by the action of dithiothreitol. The results obtained suggest that the autocrine production of cAMP and extracellular cAMP-specific phosphodiesterase is an important constituent of the mechanism controlling the motile behavior of the Physarum polycephalum plasmodium.

[Thermostable extracellular cyclic nucleotide phosphodiesterase from Physarum polycephalum plasmodium].

The cyclic nucleotide phosphodiesterase secreted by Physarum polycephalum plasmodium into extracellular medium has been partially purified by DEAE cellulose chromatography, ultrafiltration, and HPLC. The results obtained by gel filtration, HPLC, electrophoresis, and isoelectric focusing suggest that, the native enzyme in solution is a monomer with a molecular mass of about 90 kDa and pI in the range 3.6 - 4.0. The Km values were estimated to be about 0.9 mM and 7.7 mM, respectively, and Vm for both substrates were similar (up to several thousand micromoles of cAMP hydrolyzed/hour per mg of enzyme). The partially purified enzyme was shown to be extremely stable. It did not lose the activity after heat treatment at 100 degrees C during 30 min. The enzyme was active in the presence of 1% SDS, but it was fully inactivated under the same conditions in the presence of beta-mercaptoethanol. The properties of the phosphodiesterase from Physarum polycephalum are discussed.