SpkH (Sll0005) from Synechocystis sp. PCC 6803 is an active Mn2+-dependent Ser kinase.

Twelve genes for the potential serine-threonine protein kinases (STPKs) have been annotated in the genome of Synechocystis sp. PCC 6803. Based on similarities and distinctive domain organization, they were divided into two clusters: serine/threonine-protein N2-like kinases (PKN2-type) and "activity of bc1 complex" kinases (ABC1-type). While the activity of the PKN2-type kinases have been demonstrated, no ABC1-type kinases activity have hitherto been reported. In this study, a recombinant protein previously annotated as a potential STPK of ABC1-type (SpkH, Sll0005) was expressed and purified to homogeneity. We demonstrated SpkH phosphorylating activity and substrate preference for casein in in vitro assays using [γ-32P]ATP. Detailed analyses of activity showed that Mn2+ had the strongest activation effect. The activity of SpkH was significantly inhibited by heparin and spermine, but not by staurosporine. By means of semi-quantitative mass-spectrometric detection of phosphopeptides, we identified a consensus motif recognized by this kinase - X1X2pSX3E. Thus, we first report here that SpkH of Synechocystis represents a true active serine protein kinase, which shares the properties of casein kinases according to its substrate specificity and sensitivity to some activity effectors.

[Involvement of serine/threonine protein kinases in cold stress response in the cyanobacterium Synechocystis sp. PCC 6803: functional characterization of a protein kinase Spke].

Stress responses of the unicellular cyanobacterium Synechocystis are fulfilled via a number of regulatory systems, namely, two-component systems as well as through negative supercoiling of the genome DNA. We have studied an involvement of serine/threonine protein kinases (STPK) in the cyanobacterium Synechocystis cold stress response. A search for the STPK mutants allowed us to determine four protein kinases, SpkB, SpkD, SpkE and SpkG, which could regulate transcription under the low temperature. According to a proteome analysis, SpkE significantly affects the protein pattern in Synechocystis. Functional activity of the recombinant SpkE was confirmed in in vitro phosphorylation assay with a use of a set of potential protein kinase substrates. It have been demonstrated that the basic proteins are preferable substrates for the recombinant protein kinase SpkE.

[Protein sensors and transducers of cold, hyperosmotic and salt stresses in cyanobacteria and plants].

The genome-wide investigation of gene expression at the transcript level with use of DNA microarray has recently allowed to list almost all the genes that are induced by a distinct environmental stress in cyanobacterial and plant cells. Acclimation of living organisms to stress conditions begins with the perception and transduction of the stress signal. The combination of systematic mutagenesis of potential sensors and transducers with DNA microarray analysis in an attempt to identify these components led to significant progress in understanding the mechanisms for perception of environmental stresses in photosynthesizing cells. This review is focused on signaling systems that perceive and transduce the signals of cold, hyperosmotic, and salt stresses in cyanobacteria and plants.

Nucleoside diphosphate kinase is a possible component of the ethylene signal transduction pathway.

In etiolated seedlings of Pisum sativum and leaves of Arabidopsis thaliana, in vivo ethylene treatment resulted in an increase in in vitro phosphorylation of 17 kD (P. sativum) or 16 and 17 kD (A. thaliana) polypeptides. These polypeptides were identified as nucleoside diphosphate kinase (NDPK) based on both biochemical properties and interaction with antibodies against NDPK from P. sativum. Using the receptor-directed antagonist of ethylene action 2,5-norbornadiene and the ethylene-insensitive mutants of A. thaliana etr1-1 and eti5, ethylene specificity and receptor dependence of NDPK phosphorylation have been demonstrated. In pea epicotyls, ethylene treatment also led to increase in nucleoside transferase activity unlike in A. thaliana leaves. The increases in nucleoside transferase activity and NDPK phosphorylation were very rapid and transient. The results suggest a role for NDPK as a possible component of the ethylene signal transduction chain.

Ethylene signal perception and transduction: multiple paradigms?

Current progress on the mechanisms of ethylene signal perception and transduction are reviewed with an emphasis on reconciling data from molecular genetics and from biochemical approaches. It is proposed that there exist two or more interacting transduction pathways.

The effect of ethylene on MAPKinase-like activity in Arabidopsis thaliana.

Protein kinase activity was studied in cytosolic extracts from leaves of wild type Arabidopsis thaliana, the ethylene-insensitive mutant, etr1, and the constitutive triple-response mutant, ctr1. Treatment of wild type with ethylene resulted in increased myelin basic protein (MBP) phosphorylation. In etr1, constitutive protein kinase activity was lower than in wild type, but in ctr1, activity was enhanced. A protein of M(r) approximately 47 kDa associated with MBP-phosphorylating activity was detected using in gel protein kinase assays and phosphorylation of this protein was promoted by ethylene treatment in wild type while activity in the mutants reflected that of MBP phosphorylation. Both MAPKinase (ERK 1) and phosphotyrosine antibodies immunoprecipitated MBP-phosphorylating activity and detected a polypeptide band at M(r) approximately 47 kDa. Immunoprecipitated MBP-phosphorylating activity was again much lower in etr1 compared to wild type but much higher in ctr1. Antibodies to phosphorylated MAPKinase recognised proteins at approximately 47 kDa and the signal was upregulated in response to ethylene. The data obtained suggest that the detected protein(s) is a MAPKinase and provide further evidence confirming that a MAPKinase cascade(s) is involved in ethylene signal transduction.

The effect of ethylene and cytokinin on guanosine 5'-triphosphate binding and protein phosphorylation in leaves of Arabidopsis thaliana.

Binding of [alpha-32P]guanosine 5'-triphosphate ([alpha-32P]GTP) has been demonstrated in a Triton X-100-solubilised membrane fraction from leaves of Arabidopsis thaliana (L.) Heynh. Binding was stimulated by 1 h pre-treatment of leaves with ethylene and this effect was antagonised by the inclusion of N6-benzyladenine in the medium used for homogenisation. The ethylene-insensitive mutants eti 5 and etr showed contrasting responses. In eti 5 the constitutive level of GTP binding was higher than in the wild type whereas in etr the level was much lower. Neither ethylene nor cytokinin affected GTP binding in the mutants. The GTP-binding activity was localised in two bands at 22 and 25 kDa, both of which were immunoprecipitated by anti-pan-Ras antibodies, indicating that the activity is due to small GTP-binding proteins. In a similar membrane fraction, ethylene was shown to increase protein phosphorylation and benzyladenine antagonised this effect. In eti 5 the constitutive level of protein phosphorylation was higher than in the wild type, but benzyladenine increased activity substantially while ethylene was without effect. In etr, protein phosphorylation was lower than in the wild type, ethylene was without effect, but cytokinin increased activity. A protein of M(r) 17 kDa was detected on gels using antibodies to nucleoside diphosphate kinase. Phosphorylation of this protein was upregulated by ethylene but nucleoside diphosphate kinase activity was unaffected. The results are compared with the effect of the two hormones on the senescence of detached leaves and discussed in relation to pathways proposed for ethylene signal transduction.

[Neurotropic action of probon compared to amidopyrine]. / Neirotropnoe deistvie probona v sravnenii s amidopirinom.

In comparing the neurotropic action of probon and amidopyrine it became evident that both analgesics inhibit the behavioral reactions of rats in "open field" tests, fail to produce any protective action with respect to the reflex reaction of the heart in response to pain stimulation and attenuate such an effect of seduxen. By lengthening the hexobarbital-induced sleep probon and amidopyrine change the EEG nature typical of the effect produced by barbiturates. The difference between the study analgesics is rather a quantitative one, viz. in many rests probon is more potent and its action manifestes itself in a wider range of dosages.

[Pain-alleviating action of fentanyl and pentazocine in combination with droperdol and carbidine]. / Boleutoliaiushchee deistvie fentanila i pentazotsina v kombinatsii s droperidolom i karbidinom

Tests staged on rabbits showd that analgesic (phentanyl and pentasocine), neuroleptics (droperidol and carbidine) and their combinations produce a more pronounced and analgesic action with an electric irritation of the dental tissues than with that of the rabbit's paw. Neuroleptanalgesic mixtures containing pentasocine display a somewhat weaker analgesic activity, but their effect lasts longer and they have a less marked influence on the respiration. Mixtures containing cabidine in lieu of droperidol produce a somewhat greater analgesic effect and relax and skeletal muscles less.